interactions of mast cells with the lymphatic system - dukespace
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Interactions of Mast Cells With the Lymphatic System:
Delivery of Peripheral Signals to Lymph Nodes by Mast Cell-Derived Particles
by
Christian Kunder
Department of Pathology Duke University
Date:_______________________ Approved:
___________________________
Soman Abraham, Ph.D., Supervisor
___________________________ Michael Dee Gunn, M.D.
___________________________
Laura Hale, M.D., Ph.D.
___________________________ Meta Kuehn, Ph.D.
___________________________
Salvatore Pizzo, M.D., Ph.D.
___________________________ Herman Staats, Ph.D.
Dissertation submitted in partial fulfillment of
the requirements for the degree of Doctor of Philosophy in the Department of Pathology in the Graduate School
of Duke University
2009
ABSTRACT
Interactions of Mast Cells With the Lymphatic System:
Delivery of Peripheral Signals to Lymph Nodes by Mast Cell-Derived Particles
by
Christian Kunder
Department of Pathology Duke University
Date:_______________________ Approved:
___________________________
Soman Abraham, Ph.D., Supervisor
___________________________ Michael Dee Gunn, M.D.
___________________________
Laura Hale, M.D., Ph.D.
___________________________ Meta Kuehn, Ph.D.
___________________________
Salvatore Pizzo, M.D., Ph.D.
___________________________ Herman Staats, Ph.D.
An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosphy in the Department of
Pathology in the Graduate School of Duke University
2009
Copyright by Christian Kunder
2009
iv
Abstract
Mast cells, best known for their pathologic role in allergy, have recently been
shown to have key roles in the initiation of adaptive immune responses. These cells are
located throughout the body just beneath barriers separating host from environment,
possess multiple pathogen recognition systems, and store large quantities of fully active
inflammatory mediators. These key features make them uniquely situated to act as
sentinels of immunity, releasing the very earliest alarm signals when a pathogen is
present. As a testament to the importance of these cells, mast cell-deficient mice have
suboptimal immune responses, and mast cell activators can act as potent adjuvants for
experimental immunizations. Specifically, mast cells have been shown to enhance the
number of naïve lymphocytes in infection site-draining lymph nodes, and to encourage
the migration of dendritic cells to responding lymph nodes.
Although infections usually occur at peripheral sites, adaptive immune
responses are initiated in distant lymph nodes. Despite the distance, signals from the
site of infection result in dramatic, rapid reorganization of the node, including massive
recruitment of naïve lymphocytes from the circulation and extensive vascular
restructuring to accommodate the increase in size. How such signals reach the lymph
node is not well understood.
When mast cells degranulate, in addition to releasing soluble mediators such as
histamine, they expel large, stable, insoluble particles composed primarily of heparin
and cationic proteins. The work presented herein demonstrates that these particles act
as extracellular chaperones for inflammatory mediators, protecting them from dilution
into the interstitial space, degradation, and interaction with non-target host cells and
molecules. The data show clearly that mast cells release such particles, that the particles
v
are highly stable, contain tumor necrosis factor (a critically important
immunomodulator), can traffic from peripheral sites to draining lymph nodes via
lymphatic vessels. Furthermore, extensive biochemical characterization of purified mast
cell-derived particles was performed. Finally, evidence is presented that such particles
can elicit lymph node enlargement, an infection-associated phenomenon that favors the
development of adaptive immunity, by delivering peripheral TNF to draining lymph
nodes.
This signaling concept, that particles may chaperone signals between distant
sites, also has important implications for adjuvant design. The evidence presented here
shows that encapsulation of TNF into synthetic particles similar to mast cell-derived
particles greatly enhances its potency for eliciting lymph node enlargement, an
indication that adaptive immunity may be improved. This delivery system should
ensure that more adjuvant arrives in the draining lymph node intact, where it would
lead to changes favorable to the development of the immune response. Such a system
would also facilitate the delivery of multi-component adjuvants that would act
synergistically at the level of the lymph node when gradually released from
microparticle carriers. An additional advantage of microparticle encapsulation is that
vaccine formulations of this type may require much lower doses of expensive antigen
and adjuvants.
The delivery of inflammatory mediators to lymph nodes during immune
responses may be an important general feature of host defense. Although the action of
mediators of peripheral origin on draining lymph nodes has been described before, this
is the first demonstration of a specific adaptation to deliver such mediators. Not only is
vi
the characterization of mast cell-derived particles important to basic immunology, but
mimicking this adaptation may also lead to improved therapeutics.
vii
Dedication
This dissertation is dedicated to my dear wife Rebecca.
viii
Contents
Abstract........................................................................................................................................... iv
List of Tables ................................................................................................................................. xii
List of Figures............................................................................................................................... xiii
List of Abbreviations .................................................................................................................... xvi
Acknowledgements .................................................................................................................... xxiii
1. Introduction ..................................................................................................................................1
1.1 Mast Cells ...........................................................................................................................1
1.2 Mast Cell Specializations Critical to Sentinel Function ...............................................4
1.2.1 Mast Cells Can Be Activated by a Variety of Endogenous and Exogenous Stimuli.....................................................................................................................................4
1.2.2 Mast Cells Store Large Quantities of Inflammatory Mediators in Highly Specialized Granules ............................................................................................................5
1.2.3 Some Mediators Remain Associated with the Granule After Exocytosis............8
1.3 Roles for Mast Cells in Early Inflammation ................................................................10
1.3 Critical Effector Pathways in Early Inflammation .....................................................14
1.4 Mechanistic Basis for Mast Cell Control of Early Inflammation..............................17
1.4.1 Interactions Between Mast Cells and the Blood Endothelium ............................17
1.4.1.1 Near-Instantaneous Vascular Effects of Preformed Mast Cell Mediators .18
1.4.1.2 Rapid Vascular Effects of Newly Synthesized Lipid Mediators..................29
1.4.1.3 Slower Vascular Effects of Newly Synthesized Protein Mediators.............32
1.4.2 Interactions Between Mast Cells and the Lymphatic Endothelium ...................33
1.4.3 Summary of Mast Cell Interaction with the Vasculature.....................................36
1.5 Innate Versus Adaptive Immunity...............................................................................39
1.6 Roles for Mast Cells in the Induction of Adaptive Immune Responses .................41
ix
1.6.1 Mechanisms of Mast Cell Control of Lymph Node Enlargement ......................41
1.7 Hypothesis: Extracellular Mast Cell Granules Transport Tumor Necrosis Factor to Draining Lymph Nodes .......................................................................................................43
2. Studies on the Composition and Biochemical Features of Extracellular Mast Cell Granules ...45
2.1 Introduction .....................................................................................................................45
2.1.1 Previous Work on the Composition and Characteristics of Mast Cell Particles................................................................................................................................................45
2.1.2 Previous Work on Mast Cell Particle Function......................................................47
2.1.2.1 Proinflammatory Effects of Purified Mast Cell Particles ..............................47
2.1.2.2 Effects of Mast Cell Particles Related to Their Phagocytosis .......................48
2.1.2.3 A Role in Atherosclerosis: The Work of Petri Kovanen................................49
2.2 Materials and Methods...................................................................................................50
2.2.1 Purification of Mast Cell Granules ..........................................................................50
2.2.2 Purification of Mast Cells from Rat Peritoneal Lavage ........................................52
2.2.3 Cell Culture .................................................................................................................53
2.2.4 L929 TNF Cytotoxicity Assay...................................................................................53
2.2.5 SDS-PAGE and Immunoblotting .............................................................................54
2.2.6 Sample Preparation for Mass Spectrometry...........................................................54
2.2.6 Microscopy..................................................................................................................55
2.2.7 Generation of TNF-GFP Fusion Protein-Expressing Mast Cells .........................56
2.2.8 Heparin-Based Microparticle Fabrication ..............................................................56
2.3 Results...............................................................................................................................57
2.3.1 Mast Cells Release Insoluble Particles in vitro and in vivo and These Can Be Isolated..................................................................................................................................57
2.3.2 Quantification of Mast Cell-Derived Particles .......................................................63
2.3.3 Initial Characterization of Mast Cell-Derived Particles........................................68
x
2.3.4 Analysis of the Mast Cell Secretome .......................................................................73
2.3.5 Mast Cell-Derived Particles Contain Tumor Necrosis Factor .............................82
2.3.6 Development of a System for Modeling Mast Cell-Derived Particles ...............90
2.4 Discussion ........................................................................................................................94
3. Studies Investigating the Role of Mast Cell-Derived Particle-Mediated Transport of
Inflammatory Mediators in Draining Lymph Node Responses......................................................97
3.1 Introduction .....................................................................................................................97
3.1.1 Studies on the Fate of Mast Cell-Derived Particles ...............................................97
3.1.2 The Structure of the Initial Lymphatic Endothelium and Its Permeability to Particulate Material.............................................................................................................98
3.1.3 The Movement of Peripheral Signals to Draining Lymph Nodes: Remote Control ................................................................................................................................100
3.1.4 Mechanisms Underlying Lymph Node Enlargement During Immune Responses ...........................................................................................................................102
3.2 Materials and Methods.................................................................................................104
3.2.1 Animals......................................................................................................................104
3.2.1.1 Intraperitoneal Injections.................................................................................104
3.2.1.2 Footpad Injections ............................................................................................104
3.2.1.3 Tail Injections ....................................................................................................105
3.2.1.4 Epicutaneous PMA application ......................................................................105
3.2.1.5 Mast Cell Reconstitution..................................................................................105
3.2.2 Flow Cytometry........................................................................................................105
3.2.3 Microscopy................................................................................................................106
3.2.4 Encapsulation of TNF in Heparin/Chitosan Microparticles .............................107
3.3 Results.............................................................................................................................107
3.3.1 Local Fates of Mast Cell-Derived Particles...........................................................107
xi
3.3.2 Mast Cell-Derived Particles Can Enter Initial Lymphatics ................................112
3.3.3 Mast Cell-Derived Particles Can Traffic to Local Lymph Nodes......................118
3.3.4 Mast Cell-Derived Particles and Their Associated TNF Mediate Lymph Node Enlargement.......................................................................................................................124
3.4 Discussion ......................................................................................................................127
4. Conclusions and Discussion .....................................................................................................131
References ...................................................................................................................................134
Biography .....................................................................................................................................163
xii
List of Tables
Table 1. Vasoactive mast cell-derived mediators ....................................................................19
Table 2: Protein identifications made in six mass spectrometry experiments with five individual granule preparations excluding likely contaminants .........................................75
Table 3: Quantitative proteomic analysis of granule-associated proteins...........................77
Table 4: Granule-associated proteins between 14 and 19 kDa identified by MS ...............78
Table 5: Semiquantitative analysis of soluble proteins released during compound 48/80-induced degranulation................................................................................................................80
Table 6: Proteins identified in control supernatants that were reduced after 48/80-induced degranulation................................................................................................................81
xiii
List of Figures
Figure 1: Transmission EM of a mast cell in mouse ear dermis..............................................1
Figure 2: Proximity of mast cells to vascular structures. .........................................................3
Figure 3: Structure of mast cell granules. ...................................................................................7
Figure 4: Release of particles by rat peritoneal mast cells in response to compound 48/80 in vitro. ...........................................................................................................................................58
Figure 5: Mast cells release heparin-containing particles in vivo ..........................................60
Figure 6: Quantitation of released particles by mast cells of rat mesentery. ......................61
Figure 7: Fibroblast-cocultured BMMCs release particles in response to compound 48/80..............................................................................................................................................62
Figure 8: Absorbance spectra of purified mast cell-derived particles .................................65
Figure 9: Absorbance of particle suspensions at 280 and 600 nm are linear with concentration. ...............................................................................................................................65
Figure 10: Azura A metachromasia is a quantifiable measure of heparin concentration .67
Figure 11: Mast cell-derived particles are highly stable.........................................................68
Figure 12: Purified rat PMCs upon exposure to 0.05% Triton X-100 ...................................70
Figure 13: Effect of pH on isolated mast cell granules ...........................................................71
Figure 14: SYPRO Ruby-stained SDS-PAGE separated mast cell granule-associated proteins..........................................................................................................................................74
Figure 15: Detection of TNF in granule-associated, but not soluble, proteins released by mast cells after activation ...........................................................................................................83
Figure 16: Presence of TNF in granule-associated proteins demonstrated with a second polyclonal anti-human TNF antibody ......................................................................................84
Figure 17: Detection of TNF in granule-associated proteins using a monoclonal anti-TNF antibody ........................................................................................................................................85
Figure 18: Granule-induced cytotoxicity is not neutralized by anti-TNF. ..........................86
Figure 19: Release of soluble TNF by rat peritoneal lavage cells treated with compound 48/80 measured by L929 cytotoxicity assay ............................................................................87
xiv
Figure 20: Release of soluble TNF by rat peritoneal lavage cells treated with compound 48/80 measured by ELISA .........................................................................................................88
Figure 21: TNF-GFP fusion protein remains with the granule after exocytosis.................89
Figure 22: Particles formed upon addition of 0.1% polylysine to 2% heparin ...................91
Figure 23: Linear relationship between heparin/polylysine particle concentration and turbidity ........................................................................................................................................92
Figure 24: Encapsulation of TNF in heparin/polylysine particles .......................................93
Figure 25: Toluidine blue-stained mouse peritoneal lavage 1 hour after IP injection of compound 48/80........................................................................................................................108
Figure 26: CD11bhi peritoneal macrophages take up released mast cell granules ...........108
Figure 27: Kinetics of mast cell granule uptake and degradation by peritoneal macrophages...............................................................................................................................110
Figure 28: Phagocytosis of mast cell-derived particles in tissues .......................................111
Figure 29: Fluorescent microspheres inside superficial lymphatic vessels of mouse tail......................................................................................................................................................112
Figure 30: Network of initial lymphatic vessels in rat mesentery windows.....................113
Figure 31: Colocalization between mast cell-derived particles and initial lymphatics after intraperitoneal compound 48/80 instillation ........................................................................114
Figure 32: Mast cell-derived particles inside an initial lymphatic vessel in rat mesentery......................................................................................................................................................115
Figure 33: Cross section of rat mesenteric lymphatic vessel demonstrated by LYVE-1 IHC counterstained with toluidine blue ................................................................................116
Figure 34: A mast cell-derived particle in the lumen of an initial lymphatic vessel........117
Figure 35: Peripherally injected fluorescent microspheres rapidly appear in the draining lymph node.................................................................................................................................119
Figure 36: Mast cell-derived particles in the subcapsular sinus of the murine popliteal lymph node after footpad injection of compound 48/80 ....................................................120
Figure 37: Mast cell-derived particles in popliteal lymph node after epicutaneous footpad PMA application .........................................................................................................121
Figure 38: Popliteal lymph node of a mast cell deficient mouse footpad-reconstituted with mMCP5-GFP expressing BMMCs and injected with PBS ..........................................122
xv
Figure 39: Presence of purified mouse mast cell-derived particles in popliteal lymph node after footpad injection .....................................................................................................123
Figure 40: Heparin/chitosan microparticles in the popliteal lymph node 30 minutes after footpad injection ........................................................................................................................124
Figure 41: Popliteal lymph node enlargement twenty-four hours after footpad injection of purified rat mast cell-derived particles..............................................................................125
Figure 42: Mast cell-derived particles purified from wild-type mice elicit popliteal lymph node enlargement after footpad injection while those from TNF knockout mice do not......................................................................................................................................................126
Figure 43: TNF delivered inside heparin/chitosan microparticles elicits more lymph node hypertrophy than soluble TNF ......................................................................................127
xvi
List of Abbreviations
ACTH Adrenocorticotropic Hormone
BCA Bicinchoninic Acid
BMMC Bone Marrow-Derived Mast Cell
BSA Bovine Serum Albumin
BTEE N-Benzoyl-L-Tyrosine Ethyl Ester
Ca++ Calcium Ion
Cdc42 Cell Division Cycle 42
cDNA Complementary Deoxyribonucleic Acid
CE Common Era
CFA/KLH Complete Freund's Adjuvant/Keyhole Limpet Hemocyanin
cGMP Cyclic Guanosine Monophosphate
CGRP Calcitonin Gene-Related Peptide
CNS Central Nervous System
CO2 Carbon Dioxide
COX-1 Cyclooxygenase 1
COX-2 Cyclooxygenase 2
CysLT1 Cysteinyl Leukotriene Receptor 1
DAB 3,3'-Diaminobenzidine
DAG Diacylglycerol
dH2O Distilled Water
DIC Differential Interference Contrast
DLN Draining Lymph Node
DMEM Dulbecco's Modified Eagle's Medium
xvii
DMF Dimethyl Formamide
DNA Deoxyribonucleic Acid
DNase-2 Deoxyribonuclease 2
DTT Dithiothreitol
EC Endothelial Cell
ELISA Enzyme-Linked Immunosorbent Assay
EM Electron Microscopy
eNOS Endothelial Nitric Oxide Synthase
ESAM Endothelial Cell-Selective Adhesion Molecule
ESEM-FEG Field Emission Environmental Scanning Electron Microscope
FBS Fetal Bovine Serum
Fc Fc
Fc
GFP Green Fluorescent Protein
H2O2 Hydrogen Peroxide
3H-DFP Tritiated Diisopropyl Fluorophosphate
HEPES 4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid
HEV High Endothelial Venule
HRP Horseradish Peroxidase
ICAM-1 Intercellular Adhesion Molecule-1
xviii
IEJ Intraendothelial Junction
IFN- Interferon
IgE Immunoglobulin E
IgG Immunoglobulin G
IHC Immunohistochemistry
IL-1 Interleukin 1
IL-1 Interleukin 1
IL-1 Interleukin 1
IL-2 Interleukin 2
IL-3 Interleukin 3
IL-4 Interleukin 4
IL-5 Interleukin 5
IL-6 Interleukin 6
IL-8 Interleukin 8
IL-9 Interleukin 9
IL-10 Interleukin 10
IL-13 Interleukin 13
IP Intraperitoneal
IP3 Inositol Triphosphate
JAM-A Junctional Adhesion Molecule A
K+ Potassium Ion
LDL Low Density Lipoprotein
LEC Lymphatic Endothelial Cell
LFA-1 Leukocyte Function-Associated Antigen 1
xix
LN Lymph Node
LTA4 Leukotriene A4
LTB4 Leukotriene B4
LTC4 Leukotriene C4
LTC4S Leukotriene C4 Synthase
LTD4 Leukotriene D4
LTE4 Leukotriene E4
LPS Lipopolysaccharide
LYVE-1 Lymphatic Vessel Endothelial Hyaluronan Receptor 1
MC Mast Cell
MC-CPA Mast Cell Carboxypeptidase A
MCD Mast Cell-Degranulating Peptide
MCGC Mast Cell Granule Core
MCP-1 Monocyte Chemotactic Protein 1
MEM Minimal Essential Medium
Mg++ Magnesium Ion
MHC Major Histocompatibility Complex
MIP-1 Macrophage Inflammatory Protein 1
MIP-1 Macrophage Inflammatory Protein 1
MIP-2 Macrophage Inflammatory Protein 2
MLCK Myosin Light Chain Kinase
mMCP5 Murine Mast Cell Protease 5
mMCP6 Murine Mast Cell Protease 6
MS Mass Spectrometry
xx
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide
Na+ Sodium Ion
NaCl Sodium Chloride
NF- B Nuclear Factor B
NH4HCO3 Ammonium Bicarbonate
NO Nitric Oxide
OsO4 Osmium Tetroxide
pAb Polyclonal Antibody
PAF Platelet-Activating Factor
PAMP Pathogen-Associated Molecular Pattern
PAR-2 Protease-Activated Receptor 2
PBS Phosphate-Buffered Saline
PCR Polymerase Chain Reaction
PGD2 Prostaglandin D2
PGF2 Prostaglandin F2
PGI2 Prostacyclin
PGN Peptidoglycan
PKC Protein Kinase C
PKG Protein Kinase G
PLA2 Phospholipase A2
PLC Phospholipase C
PLL Poly-L-Lysine
PMA Phorbol 12-Myristate 13-Acetate
PMC Peritoneal Mast Cell
xxi
PNAd Peripheral Node Addressin
PVDF Polyvinylidene Fluoride
RANTES Regulated Upon Activation, Normal T-Cell Expressed and Secreted
rIL-3 Recombinant Interleukin 3
rmTNF Recombinant Murine Tumor Necrosis Factor
rrTNF Recombinant Rat Tumor Necrosis Factor
rTNF Recombinant Tumor Necrosis Factor
rSCF Recombinant Stem Cell Factor
RMCP-1 Rat Mast Cell Protease 1
RMCP-5 Rat Mast Cell Protease 5
RNA Ribonucleic Acid
RPMI 1640 Roswell Park Memorial Institute Medium 1640
S1P Sphingosine-1-Phosphate
S1P1 Sphingosine-1-Phosphate Receptor 1
SCF Stem Cell Factor
SDS Sodium Dodecyl Sulfate
SEM Scanning Electron Microscopy
TBS Tris-Buffered Saline
TBST Tris-Buffered Saline With 0.5% Tween 20
SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
TEM Transmission Electron Micrograph
TFA Trifluoroacetic Acid
TGF-
TLR Toll-Like Receptor
xxii
TLR2 Toll-Like Receptor 2
TLR4 Toll-Like Receptor 4
TNF Tumor Necrosis Factor
TNFR1 Tumor Necrosis Factor Receptor 1
tPA Tissue Plasminogen Activator
TRITC Tetramethyl Rhodamine Isothiocyanate
UV Ultraviolet
UVB Ultraviolet B
VCAM-1 Vascular Cell Adhesion Molecule 1
VE-cadherin Vascular Endothelial Cadherin
VEGF Vascular Endothelial Growth Factor
VEGF-A Vascular Endothelial Growth Factor A
VEGF-C Vascular Endothelial Growth Factor C
VIP Vasoactive Intestinal Peptide
VLA-4 Very Late Antigen 4
vWF von Willebrand Factor
WPB Weibel-Palade Body
xxiii
Acknowledgements
I would like to acknowledge my family, for the wonderful support they have
given me, without which I think I would not be writing this. Rebecca, my wife, has been
there for me whenever I needed her. Thanks to my mother, Patricia, who has been a
strong voice of encouragement and a source of love during this sometimes trying period
in my life. Also I’d like to thank my siblings, aunts, uncles, grandmother, cousins, and
niece and nephew, each of whom has enriched my life over these years.
Great thanks to my friends, Sue, Rusty, Richard, Priya, Monica, Mickey, Melissa,
Martin, Liz, Laura, Kim, Jesse, Jess, Javi, Heather, Graham, Gabe, Erin, Erin, Ellen, Chris,
Chad, Brian, Bonnie, Bart, and Al, for their support, empathy, and entertainment.
I wish to acknowledge especially Soman Abraham, for his guidance and advice
on the practice of science, and for never giving up on me.
I am grateful to the members of my committee, for their fine suggestions and
insights concerning my project, and for their patience. Also thank you to the members
of my lab, for their criticism, skills, and good humor, especially Ashley St. John, without
whose skills the impact of this work would be greatly lessened.
Thanks to Brent Berwin, Ph.D., for the beautiful scanning electron micrographs,
and to Kam Leong, Ph.D., for advice on polyelectrolyte complexation and
microparticles.
Finally, I acknowledge my faithful dog, Stella, for always being excited to see me.
And my cats, Q and Mojo, although their happiness to see me is more variable.
1
1. Introduction
1.1 Mast Cells
Mast cells are granulated cells of hematopoietic origin (Figure 1). They were
discovered in 1877 by Paul Ehrlich, by virtue of their metachromasia upon staining with
aniline dyes (1). Although he and many others have speculated as to the functions of
these ubiquitous cells, only recently has any real insight into their true physiologic
purposes been gained. That mast cells play an important role is suggested by their
conservation throughout (at least) the chordates. Similar cells (containing histamine and
heparin) have been found in animals as distantly related to mammals as sea squirts (2).
Figure 1: Transmission EM of a mast cell in mouse ear dermis.
Mast cells are endowed with an impressive capacity to elicit inflammation. The
prominent cytoplasmic granules in these cells contain large quantities of potent
2
inflammatory mediators, which are available for rapid release into the surrounding
extracellular environment upon stimulation. Mast cells are also capable of elaborating de
novo mediators, including arachidonic acid metabolites and inflammatory cytokines.
This, in combination with the fact that mast cells can respond to a wide variety of
stimuli, suggest that they may play a primary role in the early stages of inflammation.
Another feature that is critical to mast cell control of inflammation is their
proximity to vascular structures. As will be discussed in greater detail below, both the
blood and lymphatic vasculatures are vital to innate and adaptive immune responses.
Mast cells are often found in intimate association with blood vessels, a fact observed first
by Ehrlich himself (1). In some areas, this relationship is so close that they appear to be a
structural element of the vessel (Figure 2A). Figure 2B shows a mast cell associated with
a venule/arteriole pair. This image illustrates, when mast cells are activated, the
underlying endothelium (especially the venular endothelium, which has no supporting
cells) will be exposed to high concentrations of mast cell mediators in the event of the
cell's activation. Lymphatic vessels are also an important element of connective tissues,
and mast cells are in prime locations to interact with these as well (see Figures 2C and
2D).
Mast cells are best known as major effector cells of allergic inflammation, because
of their high affinity IgE receptors (Fc RI) and large quantities of stored histamine.
Indeed, Fc RI crosslinking by allergens is among the most potent physiologic activating
stimuli known for mast cells. Although there is substantial evidence to show that
allergic-type inflammation can be beneficial, such as in the expulsion of certain types of
ticks from skin (3) and nematodes from gut (4), it seems likely that mast cells have more
important functions.
3
Figure 2: Proximity of mast cells to vascular structures.
(A) Confocal micrograph of whole mounted mouse ear; red: blood endothelium (CD31), blue: mast cell heparin. (B) TEM of a venule-associated mast cell. V indicates venule lumen (5)1. (C) Confocal micrograph of mouse footpad section; green: lymphatic endothelium (LYVE-1), red: CD31, blue: mast cell heparin. (D) Confocal micrograph of whole mounted mouse ear. Same markers as C). It is rapidly becoming clear that mast cells have a role to play in most, if not all,
forms of inflammation. However, in contrast to allergy, in which mast cells are one of
the primary effector cells, they appear to have a more immunostimulatory role in other
kinds of inflammation, in which other cell types carry out the effector functions. Their
1 Used with permission from R.C. Wagner, imaged accessed on 5/12/09 at http://www.udel.edu/biology/Wags/histopage/empage/ect/ect13.gif
4
special features and location in peripheral tissues make them ideal candidates for the
role of "immune sentinel," as they are capable of detecting pathogens and other insults
and rapidly soundly the alarm, inducing a host response. This early period, including
the "recognition event" and the inflammatory events that immediately follow, has not
been extensively studied.
1.2 Mast Cell Specializations Critical to Sentinel Function
This section will discuss in greater depth some of the structural and functional
features of mast cells that enable them to effectively perform an early warning function
in immunity.
1.2.1 Mast Cells Can Be Activated by a Variety of Endogenous and Exogenous Stimuli
Mast cells are capable of responding to a variety of stimuli, making them
versatile detectors of tissue injury and incipient infection. It is widely known that mast
cells undergo dramatic degranulation with release of mediators after cross-linking of
high affinity IgE receptors with antigen. Mast cells also have receptors for non-IgE
antibody isotypes, such as Fc Rs, which when cross-linked can cause degranulation and
cytokine production (6, 7). This may be relevant to pathogen detection by natural
antibodies or during memory responses. Degranulation also occurs in response to the
complement components C3a, C4a, and C5a, which are generated early in encounters
with pathogens (8-10).
Many other endogenous inflammatory signals can elicit mast cell activation,
including some cytokines, especially IL-1 (13) and SCF (12). A number of peptides also
elicit responses; these include bradykinin (14), substance P (15), somatostatin (16),
adrenocorticotropic hormone (ACTH) (14), calcitonin gene-related peptide (CGRP) (17),
vasoactive intestinal peptide (VIP) (18), and neurotensin (19), although the significance
5
of these findings remains unclear. These peptides share structural motifs with several
exogenous peptides that also cause mast cell degranulation, such as mastoparan (20) and
mast cell-degranulating peptide (MCD) (14), found in wasp and bee venom,
respectively. All of these fall ultimately into a category of mast cell activators generally
referred to as polybasic compounds, that operate by a common mechanism involving
insertion directly into the membrane and interaction with cytoplasmic G proteins (21,
22). Several synthetic mast cell activating compounds also are thought to work through
this mechanism (compound 48/80 (23), poly-lysine (24), polymyxin B, protamine (14),
and a number of drugs).
Mast cells respond directly to pathogen-associated molecular patterns (PAMPs)
via Toll-like receptors (TLRs), with some stimuli causing outright degranulation and
others secretion of de novo products without degranulation (25, 26). Mast cell CD48 has
been shown to detect Gram-negative fimbrial proteins (27). Other pathogen-derived
products, such as ion-conducting cytotoxins, can also elicit significant responses (28).
Although most studies of mast cell activation has focused on a single stimulus, in an
actual infection or other injury, many exogenous and endogenous signals are
simultaneously elaborated, which are certain to have synergistic or at least additive
effects. This implies that mast cell responses are likely to be even more profound in vivo.
1.2.2 Mast Cells Store Large Quantities of Inflammatory Mediators in Highly Specialized Granules
The most morphologically distinctive feature of mast cells is certainly their
cytoplasmic granules (see Figure 1). These are more complex than just sacs of soluble
chemicals, however. The main structural determinants of mast cell granule organization
appear to be heparin proteoglycans. Heparin was discovered in liver extracts as an
anticoagulant activity and has since been used very widely as such. The basis of this
6
activity is heparin's interaction with the anticoagulant enzyme antithrombin III. It is
produced exclusively by mast cells. Heparin is a highly sulfated, unbranched
glycosaminoglycan consisting of repeating disaccharides of glucosamine and glucuronic
acid. This basic structure is adorned by slight modifications to the sugar residues and
by varying quantities of sulfate substitution, making the molecule quite heterogeneous.
In secretory cells, long heparin chains (up to 100 kDa) are attached via an O-linkage to
serine residues on the proteoglycan core protein serglycin, so named because a section of
the protein is composed of alternating serine and glycine residues. Serglycin is the core
protein for most proteoglycans involved in granule storage and destined for secretion.
The high level of sulfation on heparin molecules gives it one of the highest
negative charge densities of any known macromolecule, and this feature is key to its
storage role in mast cells. The reason for this is that nearly every other molecule stored
in mast cell granules has a net positive charge, especially given the fact that the pH of
the resting granule is maintained at a low 5-6 (29). Many of the most abundant proteins,
the mast cell proteases, have very high isoelectric points, so these are highly cationic to
begin with, but even neutral molecules will be positively charged at this pH. It should
be noted that the proteases, while fully processed, are not catalytically active at this pH,
presumably protecting other granule contents from proteolysis. The sulfate side chains,
on the other hand, have a pKa of around 0.5, and these retain their negative charge.
Thus, mast cell mediators are stored in electrostatic complexes with heparin (see Figure
3). Because of this mechanism, the granule has a very low water content, and
consequently very large quantities of inflammatory mediators can be loaded into the
granules. It also results in extensive cross-linking between individual heparin
proteoglycan molecules, as many mediators are multivalent cations in the acidic granule.
7
This compresses the granular contents, in a sense, giving the organelle its characteristic
electron dense appearance in micrographs.
Figure 3: Structure of mast cell granules.
(A) SEM of a released mast cell granule. (B) Schematic showing relationship between granule contents in the intact granule. Histamine is a divalent cation at the acidic pH of the granule and binds to
heparin electrostatically. It is stored at very high concentrations, estimated to be on the
order of 0.6 M (30), necessitating a counterion with very high charge density like heparin
to maintain electroneutrality. At this concentration, histamine is the most abundant
cation present, whereas in the vast majority of aqueous biological systems, it is a mineral
electrolyte, either K+ or Na+. When degranulation occurs, the heparin matrix is bathed in
the extracellular fluid, where sodium is the most abundant cation and where the pH is
usually approximately 7.4. At this pH, histamine is a monovalent cation, and is rapidly
replaced by sodium on the heparin matrix, which acts essentially as an ion exchange
resin. This results in the rapid solubilization of histamine and other loosely bound
mediators (31, 32).
8
This unique mechanism is crucial for the storage of mast cell granule contents.
Knockout mice for the enzyme catalyzing the final step in heparin synthesis have highly
abnormal mast cells with large, empty-appearing granules and much lower levels of
stored mediators (33, 34).
1.2.3 Some Mediators Remain Associated with the Granule After Exocytosis
That mast cells release insoluble particles has been evident for some time from
electron microscopic studies. The evidence determining the nature of these came from
observations that, of the known mast cell granule components, not all are released in the
same relative quantities after activation. For example, when purified rat peritoneal mast
cells are activated, they release a much smaller percentage of their total cellular heparin
(another mast cell product stored in granules) than they do histamine (31, 35). The
mechanism underlying this finding requires some correlation with μltrastructural
studies of mast cell degranulation. During degranulation, granule membranes fuse both
with each other and with the plasma membrane. This creates a labyrinth of spaces
within the cell that is continuous with the extracellular environment. Every granule
within these spaces becomes visibly altered because they are now functionally
extracellular. Although some of these particles are actually released entirely into the
extracellular medium, others are prevented from leaving, being retained within the
system of spaces created by granule-granule fusion (36). Thus, a portion of the cell's
heparin, which remains associated with the granule core even after functional
exteriorization, ends up remaining intracellular at the conclusion of the response.
Histamine, on the other hand, is entirely soluble, so every granule in communication
with the external space during the response has released its histamine, which rapidly
dissolves away into the extracellular fluid.
9
Whereas histamine and some other granule contents dissociate from the heparin
proteoglycan matrix rapidly after degranulation, others remain associated. Examples of
these include the highly cationic proteases rat mast cell protease I (RMCP-1) and mast
cell carboxypeptidase A (MC-CPA) are known examples (31). The large number of
electrostatic interactions between these cationic proteins and heparin preserve the
granule core’s structural integrity after release (see Figure 3). The effect of this
differential solubility on various mast cell mediators has not been explored in any depth,
but naturally such changes are likely to alter the disposition of granule contents.
Histamine, for example, being completely soluble, rapidly diffuses away from the
releasing cell and therefore exerts its effects very early in the response. The diffusion of
the insoluble proteases, on the other hand, is restricted by their attachment to the
extracellular granule.
Thus far, only mast cell proteases have been examined in any detail with regard
to their stable association with mast cell granules after exocytosis. This focus on
proteases is understandable, because these proteins are by far the most abundant stored
in granules, and they have easily measurable enzymatic activities that can be probed
using type-specific inhibitors. One important unanswered question, however, is
whether other molecules, including the critical immunomodulatory cytokine tumor
necrosis factor (TNF), remain associated, as insoluble mediators likely have different
fates and functions than soluble ones. Central to this question is whether or not such
mediators bind to heparin under physiologic conditions. A large number of
inflammatory molecules and growth factors are known to bind to heparin in vitro
including many cytokines (37), fibroblast growth factor (FGF) family members (38),
vascular endothelial growth factor (VEGF) (39), and essentially all chemokines (40).
Some of these heparin binding proteins, including basic fibroblast growth factor (bFGF)
10
and vascular endothelial growth factor (VEGF), are known to be produced and stored by
mast cells (41, 42). There is also evidence to suggest that TNF can bind to heparin (43,
44), so it is probable that, when released, it remains in associated with the extracellular
granule.
The heparin-based granule has been shown to stabilize mast cell proteases and to
provide barriers preventing large soluble molecules from entering the complex (45).
Also, many heparin-binding proteins are protected from proteolytic degradation by
heparin (46-49). Thus, granules may also act as extracellular chaperones for mast cell
mediators. This would mitigate many of the factors that limit the duration of action of
soluble mediators after release from mast cells. Specifically, this could protect mediators
from degradation, prevent their loss of activity by dilution, and inhibit their interactions
with other host molecules.
The fate of these particles after their release has not been investigated in great
detail. The earliest reports describe their phagocytosis by adjacent connective tissue
resident fibroblasts (50, 51). Studies of mast cell degranulation in the rodent peritoneal
cavity have documented their uptake by macrophages (52) and eosinophils in rat (53).
Phagocytosis of extracellular granules has also been documented for endothelial cells
(54), neutrophils (55), and reticular cells and macrophages in lymph nodes (56, 57).
After internalization, the particles are eventually degraded (50, 52). In all of these cases,
the studies only investigated the local environment surrounding the activated cells (the
lymph node studies were focused on lymph node-resident mast cells). The notion that
some particles may travel any significant distance has not been explored.
1.3 Roles for Mast Cells in Early Inflammation
Studies examining the role of mast cells in complex biological events like
inflammatory responses were very difficult until the discovery of mast cell-deficient
11
animals, such as the kitW/W=v mouse, which can be specifically reconstituted with bone
marrow-derived mast cells from their wild type counterparts (58). Using mast cell-
deficient mouse models, many have since provided convincing evidence for the roles of
mast cells in a variety of inflammatory situations.
Several studies of nonspecific or irritant inflammation suggest an important role
for mast cells. Mast cell-deficient mice, for example, have delayed neutrophil influx in a
thioglycollate peritonitis model (59). The same is true in a subcutaneous pouch model of
polyacrylamide gel-induced inflammation (60). Inflammation in UVB irradiation
(sunburn) also involves mast cell activation, which is associated with TNF release and
endothelial expression of E-selectin (61, 62). TNF release by mast cells has also been
implicated in initiating inflammation during myocardial ischemia/reperfusion injury
(63). Ear thickening and associated neutrophil recruitment after epicutaneous
application of phorbol 12-myristate 13-acetate requires mast cells (64), as does
inflammatory cell recruitment after peritonitis induced by the calcium ionophore
A23187 (65). Mast cells were also required for the initiation of inflammation in a murine
model of silicosis (66). Each of these models involves complex inflammation in response
to a variety of relatively nonspecific injurious stimuli. The requirement for mast cells in
these processes supports a general role in inflammation.
Inflammation induced by host-derived mediators also often involves the
participation of mast cells. For example, injection of substance P leads to rapid
eosinophil and neutrophil recruitment which is largely mast cell-dependent (67, 68).
Early eosinophil recruitment also requires mast cells after intraperitoneal administration
of eotaxin (69). Depletion of mast cells (by repeated intraperitoneal injection of the mast
cell activator compound 48/80, which does not so much deplete mast cells as exhaust
their preformed mediators) reduced neutrophil influx after the injection of LTB4,
12
although it did not affect that induced by fMLP or C5a des arg, all three of which are
canonical neutrophil chemoattractants (70). The mechanisms behind these responses
remain to be elucidated.
Mast cells appear to be involved in almost every type of immunologic
hypersensitivity reaction. Their role in allergic diseases (type I hypersensitivity) is well
known. However, there is now a good deal of evidence showing their impact on other
inflammatory processes. One study of mast cell-deficient rats showed a defective
neutrophil response to serum-induced pleural inflammation (71). Furthermore, immune
complex peritonitis (a form of type III hypersensitivity) shows mast cell dependence
(72), which is partially leukotriene-dependent (73). Neutrophil elicitation in this model
was also shown to require TNF (74). In immune complex-mediated skin inflammation
(the cutaneous Arthus reaction), mast cell deficiency leads to diminished neutrophil
recruitment involving complement and Fc receptors. A similar defect is seen in
immune complex-mediated lung injury (75). Delayed-type (type IV) hypersensitivity is
also defective in mast cell-deficient mice, and this was shown to be related to the release
of vasoactive amines by mast cells (76, 77). Experimental hypersensitivity pneumonitis is
severely diminished in the absence of mast cells (78).
Inflammation incited by microbial products appears to involve mast cells.
Zymosan, a yeast cell wall component, elicits rapid peritonitis. In this model also, mast
cell-deficient mice show defective neutrophil recruitment. However, the investigators
also document reduced levels of proinflammatory cytokines well into the response,
suggesting that mast cells initiate a cascade of effects. Here they demonstrate that early
neutrophil recruitment is partially histamine-dependent, but do not examine other
mediators (79). Another study examined peptidoglycan-induced peritonitis (a
component of bacterial membranes). This work shows that neutrophil influx is
13
dependent on complement, Toll-like receptor 2 (TLR2), and mast cells (80).
Inflammation associated with the injection of lipopolysaccharide (LPS, a Gram negative
cell wall component) in rat skin also involves mast cell activation and mediator release
(81). This is not to suggest that all of these agents directly act on mast cells to cause
activation, as it is known that, at least, in vitro, LPS stimulates cytokine production but
not preformed mediator release (82), suggesting that in vivo responses may involve more
complex combinations of signals.
Finally, substantial evidence now exists to the effect that mast cells are intimately
involved in the inflammation that accompanies active infections. In some of the earliest
reports demonstrating a clear link between mast cells and host defense during bacterial
infection, the presence of mast cells was shown to be required for survival in models of
acute bacterial infection of the peritoneal cavity, requiring TNF (83, 84) and early
recruitment of neutrophils to the site of infection (83). Neutrophil recruitment and
bacterial clearance was later shown to be also related to mast cell-dependent leukotriene
production in the same model (85). Using the cecal ligation and puncture (CLP)
polymicrobial peritonitis model, work from another group suggests that macrophage
inflammatory protein-2 (MIP-2; also known as CXCL2)-mediated mast cell activation
was crucial for subsequent TNF accumulation and partially P-selectin-dependent
neutrophil influx, although the source of MIP-2 was not identified (86). Mast cell
dependent neutrophil recruitment is important for host defense in experimental
Pseudomonas aeruginosa skin infection (87). Klebsiella pneumoniae peritonitis also requires
mast cells for an optimal host response, and again the key feature is neutrophil
elicitation, which in this study was shown to require the tryptase mMCP6 (88). The
response to murine CNS Sindbis virus infection also involves mast cells (89).
14
From this discussion, it is clear that mast cells are involved in many types of
inflammation in addition to allergic reactions. Although these cells are not required for
every kind of inflammation (90), mast cell activation does appear to be a critical factor in
many. Although the studies above were performed in a wide variety of specific, often
contrived models, a few general insights may be gleaned.
First, mast cells can mediate very early inflammatory cell recruitment. Second,
mast cells can be activated by a variety of stimuli. Most of the studies cited above do not
attempt to identify the mechanism of mast cell activation, and the ones that do suggest
various pathways. Finally, and perhaps most importantly, mast cell-dependent
inflammation, while often leading to similar outcomes (especially neutrophil infiltration
and changes in vascular permeability), is not the result of any single mast cell mediator.
The studies that have attempted to identify the responsible mediators suggest that TNF,
tryptase, leukotrienes, and/or other mediators are required.
The next step in understanding this critical physiology will be to integrate all of
these findings. Mast cells release many mediators with overlapping roles, and one
challenge for future research in this area will be to develop a synthesis of these various
effects. Inflammation involves a large number of cell types and molecules operating in
temporally and spatially complex ways, and research should attempt to more
completely reflect this complexity. Investigating the contributory roles of combinations
of mast cell-derived mediators in simple models of inflammation will lead to a better
understanding of the truly crucial molecules in the process, and will allow great
experimental control over it.
1.3 Critical Effector Pathways in Early Inflammation
A relatively small number of cells specialized for defense reside in peripheral
tissues, where pathogens are first encountered and immune responses initiated. These
15
include tissue macrophages, dendritic cells, and mast cells. In essence, what is known
about acute inflammation is that tissue damage and/or the presence of pathogen-
associated molecules stimulates the production of inflammatory signals leading to
endothelial activation and initiating the innate cellular response (the recruitment of
circulating effector immune cells). Simultaneously these signals are eliciting responses
from local cells as well as being amplified by the responses of other cells to the initial
signals. At the other “end” of the tissue lies the lymphatic vasculature, which at rest
collects excess fluid entering the tissue from the blood. Cells exiting the tissue (such as
dendritic cells) must cross the lymphatic endothelium to reach the draining secondary
lymphoid tissue, where antigen presentation occurs and the ensuing adaptive immune
response is initiated. Recent evidence suggests that lymphatic endothelium also
undergoes activation that regulates cellular exit from tissues and may have other
significant roles in inflammation, but this process is far less well understood.
Although local responses by resident cell types have significant effector functions
during inflammation, the participation of the circulatory system is truly crucial. The
cardinal signs of inflammation are calor (heat), tumor (swelling), rubor (redness), and
dolor (pain), as described by Celsus in the first century CE. The first three of these
clinical indications of inflammation are direct consequences of vascular changes. The
main processes that underlie these signs are vasodilation and edema. Vasodilation
results in the increased perfusion of the inflamed tissue, making the skin warmer and
contributing to redness. Edema results from increased tissue fluid, which occurs
primarily because of increased permeability of the blood vessels. At rest, the blood
endothelium is highly impermeable to molecules larger than about 3 nm due largely to
the presence of highly structured interendothelial junctions (IEJs) (91). This feature is
critical for vascular homeostasis, as it ensures the retention of plasma proteins in the
16
intravascular space. During inflammation, cytoskeletal rearrangements result in the
formation of gaps at IEJs in the microvasculature, permitting large increases in hydraulic
conductivity and greatly reduced size selectivity.
The physiologic significance of these changes is not entirely clear. It has been
hypothesized that the extra tissue fluid allows "flushing" of the tissue, which might
contribute to the removal of pathogens or debris. Another theory is that the changes
allow humoral factors from the blood (including large proteins like complement
components and immunoglobulins) to enter the tissue and perform effector or signaling
functions, as ordinarily the vascular endothelium is highly impermeable to large solutes.
Although less obvious, another function of the vasculature appears to be of even
greater importance in the resolution of infections and other damage. The tissue-resident
population of defense cells cannot control invading pathogens without assistance from
large numbers of circulating inflammatory cells, which enter the tissue when they detect
activated endothelium lining blood vessels at the site of injury. At the earliest time
points, neutrophils enter the tissue. These critical cells are avid phagocytes with a
significant arsenal of destructive chemicals that are required for the initial containment
of many types of pathogens. As they are rather indiscriminant, they are also major
contributors to tissue damage that occurs in inflammatory disorders. Later, other
important cell types are recruited for more specific purposes.
The lymphatic system is also of great importance during inflammation, although
its activities have received far less attention than the blood vasculature. Still, as
discussed in greater detail below, there is evidence suggesting that the lymphatic
vasculature also has a role in regulating tissue fluid dynamics. Whereas the blood
endothelium controls the entrance of circulating cells into inflamed tissues, the lymphatic
endothelium controls the exit of inflammatory cells from the tissue, and this process is
17
regulated by some of the same mediators that regulate blood vessels. These features are
likely to be of fundamental importance, and should receive more attention in the near
future, as improved models for their study are now available.
1.4 Mechanistic Basis for Mast Cell Control of Early Inflammation
Mast cell signaling during inflammation proceeds through several overlapping
stages. The first stage, which can begin to have functional effects within minutes of mast
cell activation, is mediated by the action of preformed granular mediators. This can be
further subdivided into the effects of the completely soluble mediators (e.g., histamine)
and those of partially insoluble ones remaining associated with the heparin matrix after
exocytosis. This latter group likely begins acting more slowly but the effects are
potentially long-lasting, as these particles (described in greater depth below) can remain
extracellular for hours. Mast cells begin generating de novo mediators quickly after
activation, and the first to be elaborated are the arachidonic acid metabolites. With these
come a second phase of mast cell-dependent effects beginning only minutes after the
initiation of the response. The production of new protein mediators takes the longest
(an hour or longer) and contributes to the “late phase” of allergic inflammation. The
following discussion of mast cell mediators and their effects is structured around this
progression.
1.4.1 Interactions Between Mast Cells and the Blood Endothelium
This section will describe the effects of various mast cell mediators on the
microvascular endothelium. Each mediator is described separately, and they are
grouped according to the kinetics of their release. A basic summary of this information
can be seen in Table 1.
18
1.4.1.1 Near-Instantaneous Vascular Effects of Preformed Mast Cell Mediators
1.4.1.1.1 Histamine
Acute changes in vascular permeability result in greater loss of fluid and plasma
proteins from the intravascular space into the interstitium near the affected blood vessel,
leading to edema. At rest, the blood endothelium is highly impermeable to molecules
larger than about 3 nm due largely to the presence of highly structured IEJs (91). This
feature is critical for vascular homeostasis, as it ensures the retention of plasma proteins
in the intravascular space. During inflammation, cytoskeletal rearrangement result in
the formation of gaps at IEJs in the microvasculature, permitting large increases in
hydraulic conductivity and greatly reduced size selectivity. One likely purpose for this
is to increase the delivery of defensive humoral factors (such as immunoglobulin and
complement) to the tissue, as ordinaily the endothelium is highly impermeable to large
proteins. It may also make it easier for leukocytes to enter the tissue. These effects are
initiated during MC-dependent responses by histamine, which was first localized to
mast cells in 1953 (92). Histamine is stored in very high concentrations in mast cell
granules and is immediately solubilized upon release. It exerts its effects very quickly,
and these effects also resolve quickly, as histamine is rapidly metabolized in the
interstitial space (half life ~1 min) (93, 94).
Early probings into histamine receptors were pharmacologic. The insight that
individual drugs inhibited only some actions of histamine led to idea that there must
exist multiple receptors and also to the naming of the two primary receptors, H1 and H2
(95). The H1 receptor is expressed most strongly on venular endothelial cells and is
responsible for most of histamine's acute vascular effects (96). Its activation greatly
increases the permeability of the venular endothelium via the formation of gaps between
endothelial cells, resulting in extravasation of fluid as well as plasma proteins (97). H1, a
19
Table 1. Vasoactive mast cell-derived mediators2
vascular
permeability WPB
exocytosis chemoattractant
adhesion molecule
upregulation Preformed
Histamine + + - * Tryptase + + - - Chymase * - - -
Cathepsin G + + - - Serglycin-heparin
proteoglycan - - ? - TNF + ? - + IL-8 - - + -
VEGF + + - ?/* bFGF - - - ?/*
Eicosanoids LTB4 - - + - LTC4 + + - - LTD4 + + - - LTE4 + + - - PGD2 ?/* - - - PAF + + + -
de novo Cytokines TNF + - - + IL-1 ?/* - - + IL-6 ? - - ?
GM-CSF - - - - CCL1 - - + -
MCP-1 (CCL2) - - + - MIP-1 (CCL3) - - + - MIP-1 (CCL4) - - + - MIP-2 (CXCL2) - - + -
seven transmembrane-spanning receptor, is coupled to the G q/11 family of G proteins
leading to phospholipase C (PLC) activation and production of inositol triphosphate
(IP3) and diacylglycerol (DAG). This results in increased cytoplasmic Ca++ levels
(probably via both store- and receptor-operated mechanisms) and activation of protein
2 +: clear evidence for a role in this process. -: no evidence. ?: possible role or conflicting evidence. *: potentiates the effect of another mediator. WPB stands for Weibel-Palade body.
20
kinase C (PKC) (98). The calcium signaling cascade has several downstream targets,
including most importantly endothelial nitric oxide synthase (eNOS) (99), myosin light
chain kinase (MLCK) (100, 101), and phospholipase A2 (PLA2) (102).
The activation of eNOS leads to the generation of nitric oxide (NO), a gaseous
signaling molecule that is lipid permeable and exerts its influence on nearby cells. One
major effect of endothelial NO is the induction of vascular smooth muscle relaxation
(thus it was originally called endothelium-derived relaxing factor) (103). This relaxation
is an important determinant of the vasodilation and edema observed during acute
inflammation (104). Endothelial NO also has autocrine effects, increasing cGMP levels
via activation of guanylyl cyclase (105). This opposes Ca++ accumulation, providing
protein kinase G (PKG)-dependent feedback inhibition for agonist-stimulated Ca++
increases (106). The effects of NO are primarily hemodynamic and do not appear to be
involved in the regulation of adhesion molecule expression on endothelial cells (107).
The phosphorylation of the myosin light chain by Ca++-calmodulin activated
MLCK is a crucial determinant of increased vascular permeability in response to Gq-
coupled agonists such as histamine or thrombin (108). This phenomenon correlates with
the appearance of "stress fibers" (bundles of actin and myosin reflecting actin
polymerization), cell contraction, and increased tension in vitro (109). This tension
contributes to the disruption of IEJs, resulting in gap formation.
Histamine also induces the rapid generation of the lipid metabolites prostacyclin
(PGI2) and platelet-activating factor (PAF) by endothelial cells via the action of
cytoplasmic calcium-dependent phospholipase A2 (110, 111) and several downstream
enzymes. Prostacyclin production is dependent on the constitutively expressed enzyme
cyclooxygenase-1 (COX-1). It is a vasodilator and inhibits platelet activation, but it has a
very short half-life and apparently is only produced for a few minutes after histamine
21
activation (111). PAF, which is also produced by mast cells (see below), contributes to
increased vascular permeability (112), and also activates integrins on circulating
neutrophils, enhancing their recruitment to affected endothelium (113).
The influx of inflammatory cells, which is so important for host defense, begins
when circulating leukocytes encounter "activated endothelium". This non-specific
phrase encompasses a significant repertoire of possible programs executed by
endothelial cells in various circumstances, but in general it indicates a state of enhanced
production of factors that encourage specific circulating cell types to stop and enter the
local tissue. The earliest event in this process involves the preformed products of
microvascular endothelial cells, which, like mast cell products, are also stored in
granules, called Weibel-Palade bodies (WPBs) (114). The calcium-dependent exocytosis
of these elements and the mediators they contain is stimulated by several agonists,
including histamine (115). These mediators include von Willebrand factor (vWF) (116),
P-selectin (117, McEver, 1989, #378) and interleukin-8 (although constitutive storage of
IL-8 may not occur in all endothelial cells without previous stimulation) (118, 119).
Endothelial presentation of P-selectin mediates early neutrophil rolling and tethering,
but it is rapidly endocytosed within about twenty minutes (120). Despite the transient
nature of its action, P-selectin is important for leukocyte recruitment as mice without it
have impaired neutrophil recruitment during thioglycollate-induced peritonitis (121)
and mice without WPBs (due to genetic vWF deficiency) have a similar phenotype upon
histamine challenge (122). Histamine-induced WPB exocytosis can cause leukocyte
rolling on postcapillary venules in vivo in a P-selectin-dependent fashion (123, 124). The
physiologic significance of the preformed pool of IL-8 in WPBs has not been
investigated, but it is reasonable to assume that, as in other contexts, this IL-8
contributes to neutrophil activation and diapedesis as well. Although the amounts of
22
leukocyte-recruiting products stored within WPBs are not likely to support sustained
influx into the tissue, it is interesting that all of the necessary elements appear to be
present (a selectin for initial tethering and rolling and inducers of integrin activation). It
is likely that WPB exocytosis, in concert with early PAF generation, acts as a critical
bridge, allowing the process to begin before the de novo products required for full-scale
endothelial activation have been generated.
In addition to these rapid changes evoked by histamine, the mediator also
upregulates the synthesis of several genes relevant to inflammation. The physiologic
significance of these observations is unclear, however, as in general these studies were
done in vitro with prolonged exposure to high levels of histamine. Histamine is rapidly
metabolized in vivo, but it is likely that it continues to be produced by mast cells (and
potentially other cell types) throughout the response. The upregulated genes include
endothelial pattern recognition receptors (125) and cytokines such as IL-6 (126) and IL-8
(127).
Acting alone, histamine has rapid but largely transient proinflammatory effects
on the local vasculature. Some data suggest that it may also synergize with other
mediators, which is to be expected since multiple mediators are acting simultaneously
during actual inflammation in vivo. Histamine enhances the expression of E-selectin and
intercellular adhesion molecule 1 (ICAM-1) on endothelial cells responding to TNF
(128). It also increases TNF- or LPS-stimulated production of IL-6, IL-8 and monocyte
chemotactic protein-1 (MCP-1, also known as CCL-2) (129).
Many of the most rapid effects of mast cell activation are referable to histamine.
Most importantly, increased vascular permeability and early endothelial activation
occur, leading to edema and the initial stages of neutrophil recruitment. Each of the
signaling steps between recognition (of the pathogen or other stimulus) and endothelial
23
cell response (including presentation of the first adhesion molecules on the endothelial
surface) is very rapid. Remarkably, then, edema and influx of circulating leukocytes to
the site can begin within seconds of the initial encounter, presumably enhancing the
ability of the host to resolve the infection or other insult quickly. Histamine signaling
likely continues to be important throughout the response, but further study is needed to
better define its role in this process.
1.4.1.1.2 Tumor Necrosis Factor
Tumor necrosis factor, for which data is presented below, is apparently not fully
soluble after granule exocytosis, but remains associated with the heparin matrix of the
granule. Thus, its local effects are likely occur with delayed, but more sustained kinetics
than if it were entirely soluble, as histamine is. Although it is generally considered an
inducer of transcription, TNF also has rapid effects on endothelial cells. It, like
histamine, can increase permeability by causing cytoskeletal rearrangements in
endothelial cells (130, 131). The finding that infusion of recombinant TNF can cause
hypotension and death within minutes suggests that these effects may occur quite
rapidly in vivo (130). TNF, like histamine, induces stress fiber formation (131).
However, the mechanism of activation is different, as TNF, unlike histamine does not
induce intracellular Ca++ accumulation. Instead, it appears to stimulate the production
of DAG (and consequent activation of PKC) through a phosphatidylcholine-specific
PLC, which does not generate any IP3 (132). The molecular identity of this enzyme
remains unknown. PKC activation appears to be required for TNF-dependent
endothelial contraction (133) as well as Rho, Cdc42, Rac, and MLCK (134). As one might
predict, some evidence suggests that the combination of TNF and histamine has at least
an additive effect on permeability (135).
24
Still, it is the more long-term effects on endothelial cell transcription that have
received the most attention. Several mast cell mediators can participate in
transcriptional endothelial activation, but thus far the evidence suggests that TNF is
among the most important. It has been known for some time now that TNF alone is
capable of eliciting the upregulation of a wide variety of inflammation-related genes in
endothelial cells, leading initially to neutrophil recruitment. The upregulated products
includes the crucial adhesion molecules E-selectin (136), ICAM-1 (137), and vascular cell
adhesion molecule 1 (VCAM-1) (138). Additionally, endothelial production of many
chemokines is upregulated, including IL-8 (139), MCP-1/CCL-2 (140), KC/CXCL1 (141),
and macrophage inflammatory protein 2 (MIP-2/CXCL2) (142). Finally, production of
cyclooxygenase 2 (COX-2), the inducible inflammatory version of the constitutive
enzyme, is increased after TNF exposure, increasing the production of prostaglandins
(143). These effects are mediated by TNFR1, which is coupled to activation of the
inflammation-associated transcription factor NF-KB. These products fill the roles that P-
selectin, PAF, and possibly preformed IL-8 play in the earliest stage of the response once
endothelial inflammatory transcription has commenced. Interestingly, the
transcriptional program induced by TNF treatment is very similar to that induced by
lipopolysaccharide, a bacterial product that signals through TLR4 and ultimately also
activates NF-KB (144). The fact that TNF is prestored within mast cell granules means
that its potent effects on endothelial cells can be exerted from the very beginning of the
response.
1.4.1.1.3 Mast Cell Proteases
Proteases are the most abundant proteins stored in mast cell granules. It is likely
that they play a storage role, as genetic deficiency of one protease can also affect the
storage of other granule products (145). Although the true physiologic functions of the
25
highly abundant mast cell granule proteases remain murky, a role is being carved out
for tryptase in the early recruitment of neutrophils. The injection of recombinant
mMCP6 (the predominant mouse tryptase) into the peritoneal cavity leads to rapid
neutrophil influx. The same study showed production of the neutrophil
chemoattractant IL-8 in response to mMCP6 in vitro, suggesting a possible mechanism
(146). The same effect on IL-8 production has also been demonstrated with human
tryptase (147). Finally, mMCP6-deficient mice show reduced survival in a Klebsiella
peritonitis model of infection, and this defect is associated with impaired early
neutrophil recruitment (88).
The link between tryptase and endothelial activation has not been entirely
elucidated, but it seems probable that it involves the activation of one of the family of
protease-activated receptors, PAR-2. These G-protein coupled receptors have a unique
mechanism of activation; protease cleavage of part of the extracellular domain allows a
tethered “ligand” to interact with the rest of the receptor, leading to signal transduction
through the membrane (148). Human tryptase is able to activate PAR-2 signaling, and
PAR-2 is expressed on endothelial cells among other cell types (149, 150). There are
numerous indications that PAR-2 activation in endothelial cells is relevant to
inflammation. PAR-2 agonists can elicit leukocyte rolling and adhesion (151), and PAR-
2 deficient animals have defective early leukocyte rolling after surgical trauma (152).
Like histamine, PAR-2 activation stimulates an increase in cytoplasmic Ca++ via
activation of Gq that results in exocytosis of Weibel-Palade bodies, which is important
for early leukocyte recruitment (153, 154). A similar effect is seen when tryptase is used
as the stimulus (155). Therefore, like histamine and TNF, tryptase can stimulate PAF
production by endothelial cells. Likewise, it has been shown in vitro that tryptase can
effect reduced barrier function in endothelial cells (156). PAR-2 activators upregulate
26
endothelial COX-2 (157). Perhaps most interestingly from the perspective of mast cell
responses is the finding that endothelial IL-6 production in response to TNF is
augmented by PAR-2 activation (158). This finding provides further evidence in
support of the notion that inflammatory mediators in various combinations result in
qualitatively or quantitatively different responses. Because mast cells release a number
of mediators simultaneously (including TNF and tryptase), these kinds of additive
effects must be investigated more rigorously.
It is also worth mentioning that a few studies have shown that tryptases (perhaps
in concert with other enzymes) can generate kinins, which would also affect vascular
permeability, but the significance of these findings is unknown (159-161).
Less is known about the role of chymases in inflammation, although there is
some evidence suggesting they are important regulators of vascular tone. One problem
is that it is very difficult to make generalizations concerning the chymases, as a) there
are very significant differences between species (humans have only one chymase gene,
for example, while rodents have several), and b) chymases have a variety of specificities.
One study showed that intradermal injection of chymase greatly potentiated the size of
histamine-induced wheals in allergic dogs, while not eliciting wheals when injected
alone (162). Such a finding further supports the idea that the effects of mast cell-derived
mediators cannot properly be considered in isolation. Some chymases (including
human chymase) are able to convert angiotensin I into angiotensin II, an extremely
potent vasoconstrictor (and in fact chymases appear to be the main source of angiotensin
II aside from angiotensin converting enzyme) (163). It is unclear how this activity is
related to the vasodilatory and other effects of mast cell-derived mediators, but one
study showed that suffusion of E. coli LPS onto a surgically exposed hamster muscle
27
caused vasoconstriction followed by vasodilation in a manner that was inhibitable by
angiotensin receptor blockers as well as chymase inhibitors (164).
Mast cells also store significant quantities of cathepsin G (primarily considered a
neutrophil enzyme) in their granules (165). The physiologic function of mast cell
cathepsin G has not been determined, but some insight may be gleaned from the
neutrophil literature. This protease, like tryptase, is also capable of eliciting inositol
phosphate metabolism and calcium flux in endothelial cells (166). Interestingly, this
effect was entirely abolished in the presence of soluble heparin. It is difficult to predict
what the effect of mast cell granule heparin would be, however, as it is largely insoluble.
Because cathepsin G is highly cationic (isoelectric point pH 11.69 for the human protein),
it may remain associated with the granule after exocytosis, although there is no
published evidence to demonstrate this, and we have not detected it in purified granule
cores from rat peritoneal mast cells (unpublished observations). The signaling elicited
by cathepsin G results in the formation of intraendothelial gaps (167) as well as PAF
production (168).
The other major protease is mast cell carboxypeptidase A. Although this protein
is extremely abundant, little is known about its functions. With reference to the
vasculature, one report suggests it may participate (along with chymase) in angiotensin
II generation in mice (169). Many other potential substrates of potential relevance to
vascular function have been identified in vitro for various mast cell proteases, but the
physiologic significance of these findings is uncertain.
1.4.1.1.4 Serglycin-Heparin Proteoglycans
Heparin is clearly a structural element of the mast cell granule, and in fact mast
cells that cannot produce heparin also have significantly reduced levels of many other
preformed mediators (33, 34). It is also required for the maintenance of the granule
28
"core” -- the electrostatic complex of highly anionic heparin and cationic proteases that
remains insoluble in the extracellular space after exocytosis (31). Still, whether heparin
has specific effects on the inflammatory process in general or on the vasculature’s role in
that process is still largely a matter of speculation. A few reports exist describing in vitro
interactions of mast cell heparin with endothelial cells -- it can induce the formation of
lacunae in endothelial monolayers and stimulate endothelial cell migration. These
events might be relevant to the role of mast cells in angiogenesis (170, 171).
A few other speculative possibilities are worth mentioning. A great number of
biological molecules, including important extracellular signals like growth factors and
cytokines, bind to heparin due in large part to its incredibly high negative charge
density (37-40). These molecules also tend to bind to heparan sulfate (a less sulfated, but
otherwise very similar, relative of heparin), which is ubiquitous in the extracellular
environment. The interaction with heparin (which is almost always of higher affinity
that with heparan sulfate) has generally been dismissed as irrelevant, as heparin is
apparently only found in mast cell granules. As heparan sulfate is an important
constituent of the endothelial basement membrane (172), it seems logical that if large
quantities of heparin are suddenly available in this area (by virtue of the degranulation
of immediately adjacent mast cells), that this could alter the disposition of those growth
factors and other molecules that are interacting with this structure. Such an effect could
be purely regulatory, as many exocytosed granule matrices are rapidly removed by
phagocytes (55) (and can, interestingly, be phagocytosed by endothelial cells (54)). It
might also be relevant in the formation of gradients of chemokines that are critical for
the guidance of leukocytes from the vessel into the tissue. Finally, such particles, by
moving through edematous tissues, might deliver signals to sites that would ordinarily
29
be too distant to be influenced by small quantities of preformed mediators. These
potential roles of mast cell heparin should be explored in the near future.
A highly specific (i.e. not simply charge-based) and very well studied interaction
of heparin is with antithrombin III. This is the basis for the use of heparin clinically as
an anticoagulant, which is certainly its most well-known activity. While a similar role
may be played in vivo inside the vasculature by heparan sulfate on the luminal surface of
endothelial cells, the interaction of antithrombin III with heparin may be important
during inflammatory events to deter the activation of the coagulation cascade in the
interstitial space. This is normally not a problem, since the proteins involved in
coagulation are large and unlikely to cross the intact endothelium. However, during
states of increased vascular permeability (or of tissue destruction) these proteins
extravasate to a much greater degree. The anticoagulant properties of heparin may
become relevant in such states to keep large amounts of fluid moving through
edematous tissues.
1.4.1.2 Rapid Vascular Effects of Newly Synthesized Lipid Mediators
Many inflammatory mediators are generated de novo after mast cell stimulation,
and the most rapidly acting of these mediators are the eicosanoids. Meaningful
quantities of these mediators can be released within minutes of stimulation, as no
transcription is required, only enzymatic activity (173). As one indication of the
diversity of potential responses mediated by mast cells, not all degranulating stimuli
also result in the production of large amounts of eicosanoids (174). However, when they
are generated, the process begins with the cleavage of membrane phospholipids by the
enzyme phospholipase A2 (PLA2). Then the resulting arachidonic acid is converted to
leukotrienes (via 5-lipoxygenase) or prostaglandins (via cyclooxygenase). Lipolysis of
30
other membrane phospholipids results in the precursor molecules for platelet activating
factor and related mediators.
1.4.1.2.1 Leukotrienes
Previously known as the slow-reacting substance of anaphylaxis (because it
cannot be detected in unstimulated tissues and its release lags behind that of histamine
and other preformed mediators), the leukotrienes are potent inflammatory mediators.
For the production of leukotrienes, arachidonic acid is first converted into LTA4 by 5-
lipoxygenase (175). The production of all three cysteinyl leukotrienes (LTC4, LTD4, and
LTE4) is dependent on the initial conversion of LTA4 to LTC4 by LTC4 synthase (LTC4S)
(176). Activated mast cells produce LTC4, which can be further converted to LTD4 and
LTE4 in the extracellular environment (177). All three of these species produce potent,
long-lasting wheal-and-flare responses upon injection into human skin, suggesting they
may enhance and prolong the immediate vascular changes evoked by histamine (178).
In a hamster cheek pouch model, it was shown that LTC4 and LTD4 are 100 times more
potent than histamine in eliciting increased vascular permeability (179). These mediators
act primarily through the leukotriene receptor CysLT1, yet another Gq-coupled receptor
expressed on endothelium among several other cell types (180, 181). Like histamine or
protease-activated receptors, leukotrienes elicit Ca++ mobilization in endothelial cells,
leading to secretion of Weibel-Palade bodies and P-selectin externalization (182). As
would be expected, this results in greater neutrophil adhesion, which is also likely
related to leukotriene-stimulated endothelial production of platelet-activating factor
(PAF) (183). In LTC4S or CysLT1 knockout animals, the increased vascular permeability
seen during experimental allergic inflammation or zymosan peritonitis is greatly
reduced (184, 185). All in all, the effects of the cysteinyl leukotrienes on the endothelium
appear to be similar to those of histamine and tryptase.
31
There is also evidence to suggest that mast cells may emit smaller quantities of
LTB4 (186), which is a neutrophil chemoattractant (187, 188). In animals deficient for
leukotriene A4 hydrolase (the enzyme that generates LTB4), there was deficient early
neutrophil recruitment in a zymosan peritonitis model of inflammation (189), a process
which is known to be at least partially mast cell dependent (79). This suggests that mast
cell-derived LTB4 may be physiologically significant, but further study is necessary.
1.4.1.2.2 Prostaglandins
PGD2 is the major prostaglandin generated by mast cells (190, 191). PGD2
injection in human skin causes erythema without substantial edema (probably due to
vasodilation) and increased vascular permeability in rats (192). Like prostacyclin, it
inhibits platelet aggregation (193). Intriguingly, while intranasal application of PGD2
did not cause any allergic-type symptoms in rats, when applied simultaneously with
histamine, it increased the potency of histamine for causing these effects by 1000 fold
(194). However, as many of its vascular effects are subtle or vary according to species
and vascular bed, it is difficult to generalize about its physiology.
1.4.1.2.3 Platelet Activating Factor
Finally, platelet activating factor (PAF) is also produced by activated mast cells
(195, 196). However, the most relevant source of PAF during inflammatory responses
may be the endothelial cell itself, as the endothelium produces PAF in response to
histamine (111), tryptase (155), LTC4 and LTD4 (183), cathepsin G (197), TNF (198), and
IL-1 (199). PAF was discovered by virtue of its platelet-activating activity, but it has
since been shown to have important roles in many other processes. Distinct from the
arachidonic acid metabolites, its synthesis requires PLA2, which converts membrane
phospholipids to 2-lysophospholipids. Then another enzyme, an acetyltransferase,
catalyzes the final reaction, generating PAF and other closely related compounds. The
32
PAF receptor is a pertussis toxin-insensitive G-protein coupled receptor whose
activation results in increased intracellular Ca++ (200). Endothelial cells express PAF
receptors linked to the same signaling pathway (201) and respond with cytoskeletal
rearrangements (199). In guinea pig and rat skin, PAF was shown to increase vascular
permeability with much greater potency than histamine (112, 202). Another study
showed leukocyte recruitment as well as increased permeability after intradermal
application of PAF (203), and, like histamine, this is mediated through gap formation at
IEJs (204). In support of the idea that PAF is an important part of inflammatory
signaling resulting in increased vascular leakiness, PAF receptor-deficient animals were
resistant to death from experimental passive systemic anaphylaxis, and they had no
evidence of increased pulmonary vascular permeability, whereas wild type animals did
(205). Similarly, intravenous PAF induces a rapid hypotensive anaphylactoid condition
in mice, which is unrelated to platelet activation (206). In another example of interaction
between multiple mast cell mediators, combined PAF and H1 inhibition almost
completely abolished the hypotension seen in passive systemic anaphylaxis (207).
1.4.1.3 Slower Vascular Effects of Newly Synthesized Protein Mediators
It is becoming better appreciated that mast cells are a very important source of
cytokines during inflammatory responses, and there are reports documenting the
production of a large variety of cytokines, as discussed in the introduction.
Unfortunately, the physiologic effects of this mélange (and also which are produced
when and under what circumstances) has not been much explored, and it is likely that
not all of these have general relevance to inflammatory responses. Nevertheless, a few
are deserving of additional attention.
In addition to releasing preformed TNF as has already been discussed, mast cells
also respond to activation by actively producing and secreting it (208). In various in
33
vitro settings, they also have the capacity to release IL-1 (25, 209, 210), IL-3 (211-213), IL-4
(25, 209, 211), IL-5 (25, 212, 214, 215), IL-6 (25, 26, 209, 211, 212), IL-8 (214), IL-9 (212, 213),
IL-10 (215), IL-13 (25, 214, 215), GM-CSF (214), TGF- (216), CCL1 (217), MCP-1/CCL2
(217), MIP-1 /CCL3 (26, 214), MIP-1 /CCL4 (217), RANTES/CCL5 (26), CXCL2 (MIP-2,
a mouse IL-8 homolog) (26). At this point, to assign physiologic functions to these (as
mast cell-derived mediators) is difficult, since none of them are made exclusively by
mast cells. Many of these do have effects on the microvasculature, however. IL-1,
another important proinflammatory cytokine, has very similar effects to TNF, in that it
enhances vascular permeability and upregulates the expression of adhesion molecules,
chemokines, and other effectors (218, 219). IL-1 also enhances TNF-dependent
hyperpermeability (220). IL-6 can also affect endothelial barrier function (221, 222), and
may be involved in the induction of lymphocyte adhesion in some circumstances (223,
224). However, several recent works have taken the approach of reconstituting mast
cell-deficient animals with mast cells lacking mediators of interest, such as TNF (225) or
mast cell carboxypeptidase A (226). This kind of study, which essentially generates a
mast cell-specific knockout animal, will be valuable to investigation of the roles of mast
cell-derived molecules that are also produced by other cell types.
1.4.2 Interactions Between Mast Cells and the Lymphatic Endothelium
Comparatively little is known about the activities of the lymphatic endothelium
during inflammation, and because there is no literature on mast cell/lymphatic
interactions, this discussion is restricted to studies testing the effects of specific
mediators on these vessels. Until quite recently, most of the work that had been done on
inflammatory mediators and the lymphatic system was done ex vivo with isolated large
lymphatic vessels. As lymphatic trunks do tend to follow the course of blood vessels, it
is likely that there are mast cells in close proximity to these, but the physiologic
34
significance of this is unknown. Many vasoactive compounds (including histamine,
serotonin, and PGF2 ) can elicit lymphatic smooth muscle contraction, which may act to
increase lymph flow (227).
In the past few years, improved in vitro models of lymphatic endothelium have
been developed, resulting in many informative studies as to lymphatic endothelial
biology. These are perhaps more relevant to inflammation than ex vivo large lymphatic
studies, as they model the initial lymphatics, or lymphatic capillaries. These vessels form
the interstitium-lymph boundary and perform a barrier function in regulating the
movement of substances into lymph. Unlike the vascular endothelium, the initial
lymphatic endothelium is highly permeable. Junctions between adjacent cells are in
some areas completely patent, although apposition between the membranes of each cell
prevents large objects from entering the lumen in uninflamed tissues (228). When the
tissue becomes edematous, however, these junctions are pulled open by connections
between the outer wall of the vessel and the surrounding connective tissue, and the
diameter of such openings can reach several microns (229). This process has generally
been assumed to be purely mechanical, but there are a few hints that it is an active
process. First, electron microscopy of initial lymphatics during inflammation (induced
both by thermal injury and histamine injection) shows changes suggestive of cellular
contraction, similar to what is seen in venular endothelial cells in response to histamine
(230, 231). Such contraction would also serve to pull open the junctions between
lymphatic endothelial cells (LECs), just as it does in the blood vessel endothelium. Also,
a recent study demonstrated that the permeability of LEC tubes in vitro is reduced by
increased cAMP, and the same is known to be true for blood endothelial cells (232).
Another showed that LEC tube permeability can be regulated by VEGF-C (233). It
should be noted that if initial lymphatic permeability is as highly regulated as venular
35
and capillary permeability, then this regulation is likely to be of importance in
determining tissue fluid accumulation in altered states as well as at baseline. This factor
is generally not considered in current paradigms of fluid dynamics in tissues.
Aside from a few well-characterized molecular differences, LECs are more like
their better-known counterparts than they are distinct. In a recent proteomic analysis of
arterial, venous, and lymphatic endothelial cells, there were fewer differences between
venous ECs and lymphatic ECs than between venous and arterial ECs (234). In fact,
before specific markers for lymphatic endothelium were found, such as LYVE-1 (235)
and podoplanin (236), differentiation of the two cell types was very difficult. Both are
derived from the same lineage (237). Both contain Weibel-Palade bodies (238), although
their function and the pathways leading to their release have not been investigated in
lymphatic endothelium. LECs also express eNOS, and its activity is induced by Ca++
ionophores, histamine, and TNF just as it is in the microvascular endothelium (239).
Two recent studies have also documented eNOS activation in human dermal LECs after
stimulation with VEGF-C or VEGF-A (240) or IL-20 (241), although in these cases it was
considered in the context of lymphangiogenesis and not inflammation. Histamine can
also upregulate the production of tissue plasminogen activator (tPA) in LECs (242).
Given the high level of similarity between blood and lymphatic endothelial cells, it is
reasonable to expect that many of the same rapid responses to mast cell-derived
mediators occur in both, but this needs to be confirmed with careful study.
Finally, it is rapidly becoming clear that lymphatic endothelial cells undergo
significant transcriptional changes during inflammatory events. One recent landmark
study showed that TNF induces the production of the adhesion molecules VCAM-1 and
ICAM-1 in dermal LECs, both in vitro and in vivo (243). These changes were shown to be
physiologically relevant for endothelial transmigration, as the movement of dendritic
36
cells to draining lymph nodes was impaired by VCAM-1 or ICAM-1 inhibition, and
these cells could be seen lining up on the outside of peripheral lymphatics. This report
initially used a microarray approach on TNF-treated human dermal LECs in vitro,
revealing the upregulation of a number of relevant inflammatory genes, including in
addition to the two just mentioned E-selectin, several chemokines, IL-6, and Toll-like
receptors. Interestingly, also upregulated was the gene for MLCK, suggesting that TNF
may regulate cytoskeletal activity in lymphatic endothelium through transcriptional
mechanisms as well as by direct signaling as discussed above for the blood endothelium.
Another group has since confirmed these findings and shown the upregulation of
cytokines and adhesion molecules in response to IL-1 as well as TNF and various TLR
ligands (244).
These and many other studies are rapidly establishing that the lymphatic
vasculature is anything but a passive player in inflammatory processes. The direct,
specific role of mast cells in inducing such changes has not yet been investigated, but the
fact that the lymphatic endothelium responds to many of the mediators mast cells
produce, as well as the close proximity of mast cells to lymphatic vessels, argues
strongly for more specific research in this direction.
1.4.3 Summary of Mast Cell Interaction with the Vasculature
The complexity of nearly all biological processes is daunting, and this is
especially true for inflammation. The quest to understand inflammatory processes in
particular resists attempts at reductionism, as inflammation involves the participation of
multiple cell types and many signals in a structurally complex environment. This is not
to say that there is nothing to be gained by experimental attempts to avoid these
intricacies; all of the work cited above engages in necessary oversimplification to greater
37
or lesser degrees. Still, a critical consideration is that the physiologic significance of each
piece of information must be confirmed in in vivo models of inflammation.
That said, a great deal of progress has been made in our understanding of the
interaction between mast cells and endothelial cells, which appears to be relevant to a
greater or lesser degree in many types of inflammation. One general principle that
emerges is that mast cell-dependent inflammation is highly temporally organized. This
organization results in a great deal of potential regulatory control at each step. The
phases include immediately acting soluble preformed mediators, less soluble granule-
associated preformed mediators, products of arachidonic acid metabolism, and finally
the secretion of de novo proteins. The actual sequence of events is likely much more
complicated than this, as transcriptional changes and enzymatic cascades can be
programmed at different rates. In addition to temporal organization, the mast cell
activation program will be influenced by autocrine and paracrine signaling from the
surrounding environment. For each of these steps, most of the inflammatory sequelae
are mediated directly through the vasculature.
The vascular response is equally complex, and although here we have only
considered the effects of mast cell-derived mediators, it is just as true that the
vasculature is responding to autocrine and paracrine signals from other cell types as
well. It is interesting that so many mast cell products are interpreted by Gq-coupled
receptors (resulting in increased intracellular calcium as a second messenger) on
endothelial cells, and this is true of many non-mast cell inflammatory mediators also.
The orderly temporal progression of Gq-coupled mast cell mediators, however, suggests
that it is important to maintain this general "alarm" signal in endothelial cells
continuously from the very beginning of the response. The centrality of Gq-mediated
signaling in inflammation was demonstrated recently by Korhonen, et. al., who showed
38
that mice with an endothelium-specific deficiency in Gq (and the closely related G11)
have greatly reduced responses to specific mediators and are protected against
anaphylaxis-induced hypotension and vascular leakiness (245).
Inflamed blood vessels also appear to have developed methods for supporting
several phases of response. Like mast cells, they contain preformed mediators, in
Weibel-Palade bodies, which are structurally and functionally similar to mast cell
granules. The contents of these granules, von Willebrand factor, P-selectin, and perhaps
other inflammatory molecules such as IL-8, support the early inflammatory response so
that cellular recruitment can begin as early as possible without waiting for a
transcriptional response. In addition to mast cell-derived mediators, other signals can
support these events as well, such as kinin activation or complement (246, 247). Finally,
the long-term response to inflammatory signals involves the sustained production of
new adhesion molecules and chemokines, supporting extensive recruitment of
inflammatory cells. These processes are crucial to host defense, as such circulating cells
are required to control many infections.
One area that has received far too little attention is the interaction of
inflammatory mediators with the lymphatic microvasculature. It has generally been
assumed that the lymphatic capillaries are mostly mechanical, responding passively to
changes in the condition of the tissues. However, recent literature suggests that initial
lymphatic vessels do actively respond to inflammatory signals in the tissue. This may
regulate the local inflammatory environment, but other changes, such as the expression
of adhesion molecules, seem directed at facilitating the development of adaptive
immunity (by enabling the entrance of dendritic cells into the lymphatics, for example).
Thus, regulation of the lymphatic endothelium by mast cells is likely to be an important
determinant of host defense.
39
The large number of pharmacologic agents targeting mast cell products is
testament to their importance, although most are aimed at preventing mast cell-
dependent inflammation. Because the mast cell-endothelial axis is so central to
inflammation, however, the events determined by it will remain a fruitful source of new
therapies. Indeed, as pharmacologic science moves away from the old paradigm of
receptor antagonists to more immunomodulatory agents, we will see more
sophisticated, targeted therapies. Some of these may even attempt to promote or
support mast cell activation, such as vaccine adjuvants.
The close association of mast cells with the vasculature is remarkable to nearly
anyone who has examined a toluidine blue-stained tissue section. This relationship is no
accident, and it merits great attention. It would appear that this relationship exists, at
least partially, to ensure the minimum time between encountering an inflammogen
(especially a pathogen) within protected space and mounting a response to reversing the
situation. This moment of contact and the very earliest events of innate immunity that
ensue have only been investigated to a limited extent. Nevertheless, it is clear in many
cases that the interaction between the mast cell and the endothelial cell is one of the
central elements of this process.
1.5 Innate Versus Adaptive Immunity
Many of the regulatory actions of mast cells during inflammation discussed thus
far have been specifically related to innate immunity, the more nonspecific, but faster-
acting arm of the immune system. In contrast, adaptive immunity is much slower (at
least on the first exposure), but ultimately leads to long-lasting, specific memory
responses that can eradicate primary infections and greatly increase the clearance speed
of recurrent infections.
40
Part of the innate bias in this discussion is due to the anatomy of immune
responses. Infections are generally encountered in peripheral tissues, near the
boundaries, such as skin, that form the first major line of defense against
microorganisms. Thus, mast cell-mediated changes in the local vasculature that cause
the signs of inflammation and the recruitment of (initially) nonspecific effector immune
cells, result in a potent, local innate immune response, which in many cases may be
adequate to clear the infection.
Adaptive immune responses, on the other hand, are initiated, not in the
peripheral tissues where the inciting infection occurs, but in secondary lymphoid tissues
draining the site of infection. The two are connected by the peripheral lymphatic
vasculature, a one-way system that begins in tiny, blind-ended vessels in peripheral
tissues and delivers excess tissue fluid and cells to the local lymphoid tissue. For most
tissues, this means a lymph node. During the early immune response, pathogen-derived
molecules are carried by antigen-presenting cells from the site of infection to the
draining lymph node, where they are displayed on MHC molecules to naive
lymphocytes, which are traveling continuously between the various secondary
lymphoid tissues and the circulation. When a naive lymphocyte carrying an antigen
receptor specific for the displayed pathogen-derived molecules encounters it (its cognate
antigen), it is stimulated to divide and differentiate. The end result of this greatly over-
simplified story is that large numbers of effector cells specific to the infection in question
are generated, and these attack the pathogen with far greater precision and efficacy than
the innate immune system.
In general, robust peripheral inflammation is required for a highly effective
adaptive immune response. This explains why vaccines, which otherwise would only
contain the foreign protein against which a specific response is desirable, also must
41
include in their formulation a supporting substance, or adjuvant, that causes nonspecific
inflammation at the site where the vaccine is administered. This suggests that there are
important connections between the innate and adaptive immune systems, and that
signals from the former are required for the correct operation of the latter. The nature of
these signals is only beginning to be elucidated.
1.6 Roles for Mast Cells in the Induction of Adaptive Immune Responses
Mast cells have been shown to participate in adaptive immune responses. As
mentioned, one critical event in the adaptive immune response is the migration of
dendritic cells via afferent lymphatic vessels to the lymph node, carrying antigens
acquired at the site of infection. This process is mast cell-dependent in some models
(248, 249). In addition, these cells are required for lymph node enlargement during
bacterial infection (225) and also in other models of inflammation (248, 250).
Enlargement of lymph nodes draining sites of infection is a common hallmark of
infection. It reflects massively increased recruitment and retention of naïve lymphocytes
from the circulation. By sequestering a larger proportion of the body’s pool of naive
lymphocytes in the draining lymph node (DLN), the chances that those rare cells
expressing receptors for infection-specific antigens will be present are maximized.
Enhancing the probability of this antigen presentation interaction increases the speed of
the immune response, which, of course, can be a critical factor determining the outcome
of the infection.
1.6.1 Mechanisms of Mast Cell Control of Lymph Node Enlargement
How mast cells perform these immunoregulatory functions is poorly
understood. In many cases, however, mast cell-derived tumor necrosis factor (TNF)
appears to be involved. This multifunctional cytokine is, like histamine, stored
42
preformed inside mast cell granules (208, 251). This unique feature of mast cells is
critical to their role as early responders, as TNF plays a central role in a variety of
inflammatory events (252, 253). When mast cell-deficient animals were reconstituted
with bone marrow-derived mast cells from TNF knockout mice, lymph node
enlargement was the same as in the unreconstituted animals, while wild-type mast cell-
reconstituted animals had robust lymph node hypertrophy. Recent studies have shown
that as rapidly as 1 hour after mast cell activation, increased levels of TNF are detectable
in prenodal lymph (63) and in the DLNs (225). The lack of any corresponding change in
DLN TNF message suggested that it was not newly synthesized locally, but was derived
from peripheral sources (225). Since a significant period of time is required for the
secretion of de novo cytokine after stimulation, this peripheral source appears to be
preformed mast cell TNF, a conclusion supported by the finding that most mast cells in
the vicinity of infection were degranulated (225). Similar findings have since been
reported in another inflammatory model examining lymph node enlargement after
mosquito bite. Here, also, lymph node changes were mast cell-dependent, and an early
increase in lymph node TNF was documented (250). A more recent report showed that
IgE-mediated lymph node hypertrophy was mast cell- and TNF-dependent, but that the
same effect elicited by peptidoglycan required mast cells but not TNF, suggesting that
these cells may mediate downstream lymph node changes in a number of ways (248).
One might presume that TNF, after its secretion by peripheral MCs into the
extracellular fluid, enters lymphatic vessels and then is carried to DLNs as a soluble
molecule in the lymph. However, considering the route of trafficking and the significant
distances involved, it is unclear how effective quantities of peripherally-derived TNF
could reach the DLNs. This scenario is especially difficult to rationalize early in the
response when it is expected that only small amounts of TNF are elaborated (each rat
43
peritoneal mast cell contains approximately 1.4 x 104 TNF molecules, according to one
analysis (254)), and as TNF has a very short half life in vivo (255). This uncertainty is
furthered by the observation that although peripherally injected chemokines can be
detected in DLNs, it typically requires the application of large quantities of recombinant
protein (256, 257).
That signals originating in peripheral tissues may influence events in the DLN is
not a new concept. It has been suggested that the periphery signals the DLN by “remote
control” (256, 258, 259). For example, a chemokine, MCP-1, has been suggested to drain
via afferent lymphatics into the DLN (259). The signals in these studies, however, were
all produced in large quantities by responding cells after transcriptional upregulation.
The movement of a preformed cytokine, on the other hand, over so great a distance is
surprising for a number of reasons. First, TNF, like many cytokines, has a very short
half-life in vivo, on the order of less than half an hour (260, 261). Second, soluble
molecules secreted into the tissue are rapidly diluted. Finally, the architecture of the
extracellular space is complex, with many adjacent cells with their receptors and
extracellular matrix molecules to potentially interact with before a cytokine finds itself in
a lymphatic vessel. All of these factors make it very unlikely that preformed soluble
mediators released in small quantities would be able to traverse such a large distance
and reach active concentrations in the target organ.
1.7 Hypothesis: Extracellular Mast Cell Granules Transport Tumor Necrosis Factor to Draining Lymph Nodes
One conceivable explanation for the long-distance action of mast cell-derived
TNF is its trafficking in a form resistant to dilution and degradation. As discussed
before, there is a theoretical basis for the notion that mast cell TNF is released in
44
association with heparin proteoglycans, and that it remains attached to these particles
after exocytosis.
Several criteria must be fulfilled in order to demonstrate that this mechanism is
operational. In addition to showing that mast cell-derived TNF is indeed released in
these complexes (a novel finding), it must also be shown that the particles can enter
lymphatic vessels and reach the lymph node. Finally, evidence must be presented to the
effect that the transport of TNF to lymph nodes in this form is required for it to mediate
its effects in the target organ.
Chapter 2 of this document will detail efforts to develop systems for describing
and studying these particles. Data will also be presented on their general characteristics,
including their makeup and techniques used to manipulate them experimentally.
Chapter 3 will then examine the role of these particles in the transportation of
inflammatory signals from the periphery to the draining lymph node and the
physiologic significance of this finding. Ultimately, Chapter 4 will present a synthesis of
the findings in this dissertation and will discuss their significance in the context of
immunology, mast cell biology, and therapeutic applications.
45
2. Studies on the Composition and Biochemical Features of
Extracellular Mast Cell Granules
2.1 Introduction
As discussed in Chapter 1, only a few studies have discussed the fate of
extracellular mast cell granules. Several more have investigated their composition and
functions. Those will be briefly reviewed here, with emphasis on the methods
employed, as they are not widely used and impact the significance of the findings.
Notably, the functional studies performed thus far have only investigated local functions
of these particles.
2.1.1 Previous Work on the Composition and Characteristics of Mast Cell Particles
For many years, the basic descriptive studies of mast cells were done using
primarily rat serosal mast cells (from the peritoneal and pleural cavities). The main
reason for this was that this was one of the only ways to get large quantities of pure
material before better techniques were developed for the isolation of mast cells from
complex tissues. The presence of free mast cells in serosal cavities is a feature
apparently unique to rodents, making the rat the initial object of study. Although much
has been learned from these studies, the caveat must be kept in mind that the mediator
stores in rat and human mast cells differ significantly.
One of the earliest methods for isolating mast cell granules was developed by
Lagunoff and Benditt and first published in 1963. This protocol involved the sonication
of unseparated rat peritoneal lavage cells (in isotonic 0.34 M sucrose) and the subsequent
separation of granules from debris by differential centrifugation. This yielded granules
without membranes, and because they were in an inorganic electrolyte-free (but
isotonic) buffer, they retained their histamine, which was releasable by adding low
46
concentrations of NaCl. This study measured the activity of granule-associated
chymotryptic activity (in normal salt-containing buffer), which was named for the first
time in this publication "chymase." The name "tryptase" was proposed for a trypsin-like
activity detected in dog mast cell tumor homogenates. This work was the first
demonstration that chymase was present in the extracellular granule (262). Later work
by the same authors determined that the dry weight of the intact granule was
approximately 30% heparin, 30% protein, 10% histamine, and 30% other (263).
The release by mast cells of insoluble ionic protein-heparin complexes was
described biochemically by Schwartz and Austen. They reproduced earlier findings that
the percentage release of various granular mediators, while linear over different degrees
of degranulation, was not the same, suggesting the retention of some contents. The
mediators showing lower levels of release, they found, could be eluted if the ionic
strength was high enough. If the concentration of inorganic ions is high enough, these
can dissociate even the interactions between the proteases and heparin, which involve
large numbers of ionic linkages. This is also the basis for the elution of bound
substances from ion exchange columns. They found that the release of chymase could
be increased by washing the degranulating cells with 1 M NaCl, and this was correlated
with the disruption of granule residue retained within the labyrinth of fused granules
(31). Surprisingly, even 1 M NaCl was not adequate to completely solubilize these
complexes, and the same group later found that carboxypeptidase A could be
solubilized if 3 M NaCl was used instead (264).
Dean Befus and his group conducted the first proteomic studies of mast cells and
their granules in the early 1990s. Two dimensional gel electrophoresis revealed a
number of basic proteins contained within mast cells and their purified granules (again
by sonication), several of which bound 3H-DFP, indicating the presence of serine
47
proteases. RMCP-1, the most abundant protease, was identified by immunoblotting
(265). A later study combining 2D electrophoresis with protein sequencing also
identified RMCP-5 (this paper discovered RMCP-5 and referred to it as such due to its
homology to mouse MCP-5) and carboxypeptidase A in rat peritoneal mast cells (PMCs)
(266). An analysis of human skin mast cell proteins revealed the presence of large
quantities of tryptase (the most abundant human mast cell protease) and cathepsin G,
again by immunoblotting (267). Chymase and mast cell-carboxypeptidase A were likely
also present, as their activities have also been demonstrated in human skin mast cells,
but antibodies to these were not used (268, 269). Still, this comparison should
underscore the large differences between human and rodent mast cells.
2.1.2 Previous Work on Mast Cell Particle Function
2.1.2.1 Proinflammatory Effects of Purified Mast Cell Particles
A series of studies in the early 1980s was performed on the biological activity of
mast cell granules. Granules in these studies were isolated from purified rat peritoneal
mast cells by initial sonication followed by sucrose gradient sedimentation to obtain a
preparation in which 80% of granules still possessed their membranes. These were then
subjected to water lysis, which resulted in membrane-free granules. Each of these
preparations (granules with and without membranes) elicited neutrophil infiltration
upon injection into rat skin. As would be expected, the intact granules were more
effective (as they still contained histamine, which as discussed in Chapter 1 contributes
to inflammatory cell recruitment), but both preparations resulted in neutrophilic
inflammation (270). One limitation of with this study is the use of cellular disruption to
purify granules. As discussed below in the Results section of this chapter, membrane-
free granules tend to attract cationic molecules, such as histones and other cellular
proteins. This can complicate efforts to assign biological consequences to the effects of
48
the granules themselves, as contaminants may actually be responsible for the observed
effects.
2.1.2.2 Effects of Mast Cell Particles Related to Their Phagocytosis
The first studies in this field were largely descriptive, as of course are the first
studies in all fields. The work of Higginbotham in the 1950s focused on the uptake of
these particles by fibroblasts (50, 271). In these articles it was hypothesized that the
purpose of this phenomenon was to protect the connective tissue from the possibly
injurious influence of the biologically active molecules stored within the granules.
Higginbotham later extended this concept to include the attractive idea that the
phagocytosis of mast cell granules may also serve to remove exogenous noxious agents,
such as hemolytic components of bee venom (272). Although heparin is known to
neutralize or at least inhibit a number of toxins (273, 274), not much work has been done
to explore the role of mast cells in this phenomenon. In keeping with the idea that mast
cell-derived particles may act as vehicles to enhance the uptake of cationic substances, a
recent study showed that purified granules could greatly enhance the uptake by
macrophages or smooth muscle cells of the cationic antibiotic gentamicin and the
chemotherapy agent doxorubicin (275). In another demonstration that binding of
compounds of non-mast cell origin might have physiologic importance, Henderson, et al.
showed that eosinophil peroxidase, as well as causing mast cell degranulation under
some conditions, binds to extracellular mast cell granules, and this binds enhances its
activity (276).
It was later shown that phagocytosis of mast cell granule by fibroblasts in vitro
enhanced their secretion of collagenase, suggesting a role in connective tissue
homeostasis and/or turnover (277). Interestingly, much more recent work has
demonstrated that tryptase induces fibroblast proliferation (278) and type I collagen and
49
collagenase synthesis (279), supporting this notion. An anti-inflammatory effect of
purified mast cell granules on macrophages was suggested by two studies, which
demonstrated reductions in superoxide production and NF-KB signaling after in vitro
exposure to the particles (280, 281).
In summary, mast cell-derived particles do appear to have the ability redirect the
movement of cationic compounds from the extracellular space into phagosomes. This
could be important for defense against envenomation, and it may also play a regulatory
role for endogenous molecules. Although some studies have suggested direct effects of
the particles on cells, it is difficult to generalize given the small quantity of data and the
general lack of demonstrated mechanism.
2.1.2.3 A Role in Atherosclerosis: The Work of Petri Kovanen
An extensive series of studies into the possible role of mast cells and their
granules in the development of atherosclerotic lesions has been conducted by Kovanen's
group in Finland. Their earlier studies, done with lysis-purified granules (as above),
showed that granular proteases degraded LDL and that granules enhanced their uptake
into macrophages (282, 283). As the accumulation by LDL by intimal macrophages in an
important event in the pathogenesis of atherosclerosis, and mast cells are also found in
these lesions, the general hypothesis was that mast cells may accelerate this process.
When these macrophages become packed with lipids, they are called "foam cells." Using
peritoneal macrophages, LDL, and mast cell granules, foam cells were generated in vitro
in a later study (284).
These findings further support the local role of mast cell granules in altering the
extracellular fate of various substances, but this series of studies is also interesting in
that a new technique for the purification of mast cell granules was elaborated. This
technique, also using rat peritoneal mast cells as the starting material, uses a
50
pharmacologic approach. By treating live cells with the activator compound 48/80, the
cells release granules in vitro, which can then be easily separated by differential
centrifugation (285). The clear advantage of this technique is that the noncytoxic
degranulation of cells by compound 48/80 is closer to physiologic than processes
involving the destruction of the cells. It is also much faster and can be done in normal
buffers without many changes in conditions. As such, modifications of their protocols
have been used in the studies below.
2.2 Materials and Methods
2.2.1 Purification of Mast Cell Granules
For the isolation of rat peritoneal mast cells, Sprague-Dawley rats (obtained from
Taconic) were first killed by CO2 asphyxiation. The abdomen was then sprayed with
70% ethanol and a midline incision made from the low abdomen up to the level of the
xiphoid process. Lateral incisions were then made at either end and the abdominal
musculature exposed by separating it from the skin using a razor blade. Then the
peritoneal cavity was opened by carefully cutting through the musculature and
membranes at the midline, and then extending the opening to approximately two
inches. Using forceps to hold one side of the incision up, 20 ml of ice cold RPMI 1640
cell culture medium containing 2% FBS was introduced into the cavity using a bulb
pipette. Once the whole volume was in, the abdomen was shaken for about 15 seconds,
and then as much lavage as possible was removed from the cavity, primarily from the
pools on either side near the spleen and liver (usually about 17.5 ml). This was returned
to a 50 ml conical tube on ice. Then, the falciform ligament was severed so that the
abdominal diaphragm could be visualized. Using sharp scissors, a small hole was made
in the diaphragm near its most ventral aspect, ensuring that air entered the pleural
space. Then 10 ml of the same media was introduced through this hole into the pleural
51
cavity. After shaking the cavity as well as possible, as much fluid was removed as
possible, usually about 7 ml. This procedure generated approximately 4 x 107 cells, on
average, containing about 2% mast cells. The cells were kept on ice, as it was found that
they become refractory to compound 48/80 more rapidly if allowed to warm. If the cells
are not collected in the presence of some protein, they also will not respond (responses
were noticeably less complete with less than 2% FBS, but BSA or gelatin may also be
used).
The cells were then centrifuged at 300 x g for 5 minutes at 4° C. After decanting
the supernatant, they were immediately treated by resuspension in 3 ml of 37° C RPMI
1640 containing 2% FBS and 5 μg/ml compound 48/80 (Sigma) and transferred to a well
in a 6 well ultra-low binding plate and incubated at 37° C for 5 minutes. One major
problem that was encountered was inconsistent response to the cells to the stimulus. If
the compound 48/80 was added in a small volume to the well (to the same
concentration), it was often noted that only cells in the immediate vicinity of the
addition would respond. This also occurred with ionomycin. Also, the effective dose
depends critically on the density of cells in the treatment volume. It is likely that the
drug interacts with cells rapidly, and that the concentration of free drug falls quickly in a
volume containing large numbers of cells. Resuspending the cells in the treatment
volume permits all the cells to see the initial concentration for at least a few seconds
(which is all that is required). Degranulation was confirmed visually by observing the
cells in the plate with a standard cell culture microscope -- the mast cells being larger
and more refractile than other cells -- a dramatic change of morphology and the
shedding of granules is immediately apparent.
After 5 minutes incubation, the cells were transferred to a 15 ml conical tube and
centrifuged at 450 x g at 4° C for 5 minutes. After this spin, the supernatant (containing
52
free granules) was carefully transferred to 1.5 ml microcentrifuge tubes on ice.
Approximately 100 μl was left on the cell pellet to reduce the contamination of the
granule preparation with cells. The supernatant-containing tubes were then centrifuged
at 12,000 x g at 4° C for 10 minutes. Generally, the granule pellet was visible, whitish-
tan with hazy borders. The supernatant was then removed and the granules
resuspended in various media depending on the application.
For the isolation of mouse PMC-derived particles, peritoneal lavage was
performed on 2-3 mice using DMEM and isolated cell suspensions were pooled and
stimulated by 1 μM ionomycin (Sigma). Ionomycin was used instead of compound
48/80 for these studies because the responsiveness of mPMCs to the latter drug ex vivo
was inconsistent, and also to reduce artifacts resulting from interactions between
compound 48/80 and the particles (discussed below). Treatment with ionomycin was
followed by incubation for 15 minutes in a cell culture incubator, after which the cellular
fraction was removed by two rounds of centrifugation at 500xg for 5 minutes.
Exocytosed particles were then pelleted by spinning at 12,000xg for 10 minutes at 4˚C.
2.2.2 Purification of Mast Cells from Rat Peritoneal Lavage
Peritoneal mast cells were purified from other lavage cells by discontinuous
density gradient sedimentation, as they are much denser than the other cells. 6 ml of
22.9% nycodenz was layered atop a 5 ml 40% nycodenz cushion in a 50 ml conical tube.
Peritoneal lavage cells in culture media were layered over the 22.9% nycodenz. The tube
was centrifuged at 600 x g for 20 minutes at 25° C with the brake off to minimize
interface disruptions. Mast cells were collected at the 22.9%/40% interface using a bulb
pipet. This procedure yielded preparations above 90% purity, and the cells were still
able to respond to stimuli such as compound 48/80.
53
2.2.3 Cell Culture
For experiments involving the culture of rat peritoneal lavage cells, the
peritoneum was initially lavaged with 20 ml of RPMI 1640 cell culture media
supplemented with 2% FBS. Cells were centrifuged at 300 x g for 5 minutes at 25°C and
the resuspended in 2.5 ml of the same media. These were then maintained in a 5% CO2
incubator at 37°C for up to 24 hours.
Bone marrow-derived mast cell cultures were initiated by washing femurs of
mice with RPMI 1640 supplemented with 10% FBS, HEPES, penicillin/streptomycin,
essential and nonessential amino acids, and beta-mercaptoethanol. Cells were cultured
in this medium with 5 ng/ml IL-3 and 10 ng/ml SCF for five weeks before use,
periodically removing the adherent cells.
RBL-2H3 rat basophilic leukemia cells were maintained in MEM cell culture
media supplemented with 10% FBS. L929 cells were maintained in DMEM with 10%
equine serum.
2.2.4 L929 TNF Cytotoxicity Assay
The night before the assay, L929 cells were seeded into a 96 well plate at 3.5 x 104
cells/well in 0.1 ml RPMI 1640 + 2% FBS. After overnight incubation, the medium was
removed and replaced with 50 μl RPMI 1640 + 2% FBS containing 4 μg/ml actinomycin
D. Then, 50 μl of each standard or sample was added to each well and the plate was
incubated for 24 hours. After this, 10 μl of 5 mg/ml MTT was added to each well, and
the plate was incubated at 37°C until adequate color had developed (2 - 4 hours). 50 μl
of 20% SDS/50% DMF was added to lyse the cells and solubilize the formazan reaction
product. Finally the plate is read at 492 nm, and levels of toxicity calculated from these
results.
54
2.2.5 SDS-PAGE and Immunoblotting
Purified mast cell-derived particles were placed in Laemmli buffer under
reducing conditions and boiled for 5 minutes. This treatment (specifically, boiling in an
anionic detergent) was found adequate for solubilizing granule-associated proteins.
This was then subjected to polyacrylamide gel electrophoresis to separate the proteins.
This was usually done on a 4-15% or 4-20% gradient gel to permit the maximum
separation over the full range of molecular weights. Gels were then stained with SYPRO
Ruby (from Molecular Probes, according to their instructions) and photographed under
UV illumination.
If used for immunoblotting, gels were transferred to PVDF membranes, blocked
with 5% milk in TBST, 5% BSA in TBST, or 1% fish gelatin in TBST, depending on the
antibody. Incubations with primary antibodies were performed overnight in blocking
buffer at 4° C. Membranes were washed 3 times for 10 minutes each in TBST, then
incubated an HRP-conjugated secondary antibody in the blocking buffer for 1 hour at
room temperature. Finally, membranes were washed 3 times for 10 minutes and a
fourth wash of 20 minutes before HRP detection using Femto West ultra-high sensitivity
substrate (Pierce).
2.2.6 Sample Preparation for Mass Spectrometry
Rat peritoneal mast cell-derived particles were purified as described above and
sedimented by centrifugation at 12,000 x g for 10 minutes at 4°C. They were then
resuspended in 0.2 ml 50 mM NH4HCO3, recentrifuged, and resuspended in 50 mM
NH4HCO3 with 5 mM DTT and 0.1% RapiGest SF (Waters). The sample was boiled for
five minutes in this solution, which dissolved the particles. To this, iodoacetamide was
added to a final concentration of 15 mM to block cysteine residues, and the solution was
incubated at room temperature for 30 minutes in the dark. Finally, 1 μg of mass
55
spectrometry-grade trypsin was added to the solution (Promega V5280) and the solution
was incubated overnight at 37°C. In the last step prior to analysis, TFA was added to a
final concentration of 0.5%, and the mixture incubated at 37° C for 30 minutes.
2.2.6 Microscopy
For cytospin preparations, approximately 250 μl of cells suspended in RPMI 1640
with at least 2% FBS was spun out onto slides using a Shandon Cytospin 3
cytocentrifuge. Cells were fixed in methanol and stained with eosin and methylene blue
or acidic toluidine blue.
For sections, 10 μm frozen sections were made and fixed in cold acetone before
being blocked in PBS with 1% BSA and subsequently labeled with a 1:2000 dilution of
AlexaFluor 488-labeled avidin (Molecular Probes) in PBS with 1% BSA. Slides were
washed 4 times for 5 minutes, and then coverslipped in ProLong Gold antifade solution
(Molecular Probes). For toluidine blue staining, frozen sections were rapidly fixed in
75% methanol, 20% formaldehyde, and 5% acetic acid and then stained in 0.1% toluidine
blue at pH 2 for 2-3 minutes, then washed and dehydrated before mounting and
coverslipping in Permount.
Whole mount rat mesentery preparations were made by stretching a loop of
bowel over a slide so that the transparent windows were spread across them, waiting for
these to dry, and then cutting away unwanted tissue. Then they were fixed with cold
acetone, permeabilized and blocked overnight in PBS with 0.3% Triton X-100 and 5%
goat serum, then labeled fluorophore (AlexaFluor-488 or TRITC, depending on the
experiment)-labeled avidin (Sigma) before imaging using a laser scanning confocal
microscope. Some images were made under epifluorescence illumination.
For scanning electron microscopy, rat peritoneal lavage cells were seeded onto
polylysine coated coverslips in RPMI 1640 media with 10% FBS and incubated for 15
56
minutes at 37˚C. They were then treated with compound 48/80 at 5 μg/ml for 15
minutes. Finally, the cells were fixed in 3% glutaraldehyde and processed for SEM.
Coverslips were postfixed for one hour in 1% OsO4 (in water) before being dehydrated
into ethanol and hexamethyldisilazane and finally dried. Then they were mounted and
coated with osmium for imaging on a XL-30 ESEM-FEG microscope (FEI Company).
2.2.7 Generation of TNF-GFP Fusion Protein-Expressing Mast Cells
For the generation of TNF-GFP expressing cells, total RNA was isolated from
bone marrow-derived mast cells (BMMCs) using an RNeasy kit (Qiagen). cDNA was
made using the iScript cDNA synthesis kit (Bio-Rad). The tnf gene was PCR amplified
using the primers tnfF: GAT CTC GAG ATG AGC ACA GAA AGC ATG ATC CG, tnfR:
GGT GGA TCC CGC AGA GCA ATG ACT CCA AAG TAG. The PCR product was
digested with XhoI/BamHI, and then ligated with XhoI/BamHI digested pLEGFP-N1
(BD Biosciences) to generate pTNF-GFP. Sequence accuracy and whether TNF and GFP
genes are in frame were confirmed by sequencing. The production of infectious
retroviruses and infection of RBL-2H3 cell line and BMMCs were done as recommended
by the vendor (BD Biosciences). BMMCs were cultured in the presence of rIL-3 (5
ng/ml, R&D Systems) and rSCF (5 ng/ml, R&D Systems) for four weeks followed by ten
days of culture in 5 ng/ml rIL-3 on a monolayer of 3T3 fibroblasts before treatment.
Both TNF-GFP expressing cell types were observed after activation with 1 μM
ionomycin.
2.2.8 Heparin-Based Microparticle Fabrication
Heparin/polylysine particles were made by combining 1-2% solutions of heparin
with 0.1% polylysine. For the encapsulation of TNF in heparin/polylysine particles, 1
μg recombinant murine TNF was added to a solution of 1% heparin, and this was
57
vortexed at 4° C for 30 minutes. Then two volumes of 0.1% polylysine were added. This
resulted in significant encapsulation of TNF in the particles
Synthetic heparin/chitosan particles were generated by gradually combining 1%
heparin (Calbiochem, 375095) and 1% chitosan (Vanson) (both in dH2O) in a 1:1 ratio at
approximately pH 5. To produce particles, one volume of 1% chitosan was added to five
volumes of 1% heparin and vortexed for 30 seconds. This was repeated to achieve a 1:1
ratio of 1% chitosan to 1% heparin. After ten minutes at room temperature, the pH was
then adjusted to neutrality to prevent further aggregation. Particles were centrifuged at
14,000xg for 10 minutes at 4˚C to form a pellet and washed with water before
resuspension in PBS for injections or water for visualization on coverslips.
2.3 Results
2.3.1 Mast Cells Release Insoluble Particles in vitro and in vivo and These Can Be Isolated
Isolated rat peritoneal mast cells degranulate within seconds in response to
compound 48/80, and release granules over a minute (see Figure 4). This process is so
dramatic that it can easily be observed in real time at low magnification. Whereas the
intact granule is very tight and darkly staining, upon exocytosis the granule expands
greatly in size and exhibits much fainter staining (Fig. 4B). This has been appreciated in
many electron microscopic studies and is apparently the result of water entering the
granule and fewer cationic substances being available to crosslink heparin
proteoglycans. As the particles separate entirely from the cells, it becomes possible to
separate them from cells in a suspension of each, as the particles are much smaller than
cells. As such, they take longer to sediment. This is the basis for the protocol used in
these studies to obtain purified mast cell granules (see Methods above).
58
Figure 4: Release of particles by rat peritoneal mast cells in response to compound 48/80 in vitro.
(A) and (C) Cytospin and SEM image, respectively, of a nondegranulated mast cell. (B) and (D) Cytospin and SEM image, respectively, of a mast cell 15 minutes after treatment with 5 μg/ml compound 48/80. (E) DIC micrograph of purified mast cells seconds after the addition of compound 48/80. Note the appearance of the unactivated cell (arrow). (F) Unactivated mouse PMC. (G) Mouse PMC 15 mins after compound 48/80.
59
With the electron microscope, intact mast cell granule contours could be seen
through the plasma membrane of some, but not all unactivated peritoneal mast cells (Fig
4C). 15 minutes after stimulation, a great number of extracellular granules can be seen
surrounding the cell (Fig 4D). Upon measurement, the granules averaged 917 nm in
diameter, and they were relatively homogeneous, being composed of smaller, mostly
spherical subunits that averaged 59.4 nm (see Figure 3A).
The homogeneity in appearance of purified mast cells imaged in these various
modalities suggests that all of the particles seen are identical. That they are in fact the
heparin-protein complexes described previously is further supported by their continued
binding to a basic dye (methylene blue) after their exocytosis.
Peritoneal mast cells from rats were used for the majority of in vitro studies, as
these can be harvested in the greatest amounts. A procedure for purifying mast cel-
derived particles from murine PMCs was developed also (described above), but the
yields were much lower, less predictable, and required greater numbers of animals.
Still, these cells release particles that can be purified (see Figure 5).
It is perhaps unsurprising that these particles can so easily separate themselves
from their parent cells in a suspension of free cells, but whether or not this occurs in vivo,
in complex connective tissues, is another question. In fact, when compound 48/80 is
injected into the mouse rear footpad, extensive degranulation is observed, and the cells
are surrounded by particles that appear to have the capacity to travel some distance
away from their cell of origin (Fig 5B). These cells are labeled with fluorescent avidin, a
highly basic protein that binds strongly to mast cell granules, both inside and after their
release. This interaction with mast cell heparin, generally considered a "nonspecific"
reaction, explains why streptavidin, a largely neutral protein, and various other altered
60
avidin derivatives have largely replaced avidin for the detection of biotinylated targets.
The similar labeling of intra- and extracellular granules by avidin is further proof that
these are indeed heparin-based particles.
Figure 5: Mast cells release heparin-containing particles in vivo
(A) Confocal micrograph of footpad section 30 minutes after PBS injection. (B) Footpad section 30 minutes after the injection of 5 μg compound 48/80. As will become clear, quantitation was a persistent challenge.
For example, it was difficult to determine how many particles had been purified
in each preparation. Quantifying the number of such extracellular particles in vivo was
also quite difficult, as sections do not adequately capture what is a three-dimensional
phenomenon, and one that occurs differently in different directions (see Fig. 5B). One
anatomical site, however, did prove amenable to this sort of analysis, and that was rat
mesentery. Rat mesentery can be observed in whole mount and it is very thin, and it is
by virtue of these facts that it is such an attractive model for connective tissue events.
Here we could count extracellular granules. Images of avidin-labeled mast cells were
converted to binary images with the subtraction of cells so that individual particles
could be automatically counted (see Fig. 6). In this system, the ratio of free granules to
mast cells was 27.4 (20.5-34.2 95% confidence interval) after the analysis of 17 such
images. This number is certainly an underestimate as: a) rat mesentery is probably not a
61
typical connective tissue as it is so spatially restrictive, b) this is only 30 minutes into the
response, and c) particles so close to the cells that they cannot be separated from the
parent cell's fluorescence are not counted.
Figure 6: Quantitation of released particles by mast cells of rat mesentery.
(A) Avidin-TRITC labeled whole-mounted rat mesentery window 30 minutes after the IP injection of 0.5 mg compound 48/80. (B) Binarized version with cells removed for automatic particle counting.
It would have been ideal to have a sustainable in vitro source of material --
relying exclusively on primary cells that lose crucial abilities within an hour of their
harvest is undesirable, for obvious reasons. Part of the problem is that most cell culture
models of mast cells do not produce highly mature, connective tissue-type mast cells like
PMCs. This is likely because the mast cell is so highly differentiated, and while tissue
resident mast cells do apparently have proliferative potential (286), ordinarily they are
quiescent. As proliferation is required for cell culture, whether cell lines or with
untransformed primary cells, these cells are less differentiated than in vivo. The
synthesis of heparin, in particular, is a feature of highly differentiated mast cells.
Because heparin is required for the structure of the extracellular granule, cells that do
not produce it presumably do not release truly stable granules.
62
Bone marrow-derived mast cells (BMMCs), while ordinarily fairly immature, can
be induced to differentiate in the presence of the cytokine stem cell factor and if
coculture with fibroblasts. Therefore, the purification of particles from BMMCs was
attempted. Fibroblast-matured BMMCs did degranulate in response to compound
48/80, although requiring a higher concentration (20 μg/ml). In addition, particles were
visibly shed from these cells upon treatment, although only by a small minority (Fig. 7).
However, the material recovered using the isolation protocol described above for rat
PMCs yielded a great deal of cellular debris as well as particles the size of the ones shed
during activation. This is likely a consequence of imperfect separation of fibroblasts
from BMMCs (as they are plated on a monolayer, there is debris). So, it would appear
that BMMCs are not a useful source of purified granules without a great deal more
optimization.
Figure 7: Fibroblast-cocultured BMMCs release particles in response to compound 48/80.
(A) through (D) Stills from a DIC video made of a BMMC degranulating and shedding visible particles in response to 20 μg/ml compound 48/80 over the course of about 30 seconds.
63
The purification of particles from the rat basophilic leukemia cell line RBL-2H3
was also attempted, but this was likewise unsuccessful (see Section 2.3.4). Although
most transformed mast cells (including RBL-2H3 cells) do not produce heparin, there are
some cell lines that do (287). Heparin-producing cell lines might also be tried in this
regard, but here it was observed that there are significant differences in the particles of
different mast cell sources, implying that extensive optimization would likely be
required to develop these sources. Finally, mast cell tumors, mastocytomas, are the
most common tumor of dogs, and these have been used to establish cell lines as well as
raw material for the purification of mast cell-associated factors. The more aggressive of
these tumors are quite anaplastic, but some remain highly differentiated. It is probable
that cells dissociated from such a tumor might be a source of large quantities of mast
cell-derived particles for studies requiring their isolation.
In summary, it was found that mast cells release discrete particles into the
extracellular environment upon degranulation. This was true for free mast cells isolated
from rodent serosal cavities and activated ex vivo, as well as for mast cells residing in
connective tissues in vivo. These particles were purified from mature, freshly isolated
peritoneal lavage cells of rats and mice, and possibilities for using other sources were
presented.
2.3.2 Quantification of Mast Cell-Derived Particles
Quantitation of purified granules is important for dosing in functional studies, as
well as for performing more controlled basic studies. There are several potential
approaches to this problem. The simplest, perhaps, would be to count them, and from
there express and calculate doses, etc. in terms of number of granules. The use of a
hemocytometer was attempted, but at least for rat-derived particles, the relatively large
distance between the top and bottom (generally 100 μm) of the counting chamber led to
64
a great deal of motion of the particles, preventing accurate counting. The use of a
Petroff-Hausser counting chamber (used ordinarily for counting bacteria) would likely
be more appropriate, but even this type of chamber is 10-50 μm deep. Given that the
particles are less than 1 μm in diameter, this might not completely solve the problem of
their movement. Because of this potential problem, other techniques for quantifying
particles published in the literature were pursued.
Counting of bacteria is ordinarily done by measuring the turbidity of cultures
using a spectrophotometer. This technique generally requires calibration using actual
counting for each type of bacteria, but it regardless allows comparisons to make between
different preparations. As suspensions of mast cell-derived particles are visibly turbid,
quantitation using this method was investigated. Purified granules have a characteristic
absorbance spectrum that is dependent on their structural integrity. Granules diluted
into an isoosmolar solution, such as PBS, exhibit an identical spectrum except for
reduction in amplitude due to dilution. If, however, they are diluted into a high salt
solution, such as 5 M NaCl, a treatment that completely solubilizes them, the spectrum is
very different from the undiluted particles (Fig. 8). The absorbance of the particles
either at 280 nm or 600 nm (the traditional wavelength for bacterial turbidity
measurements) is linear with their concentration, implying that this technique can
provide at least a comparative quantitation (Fig. 9).
65
Figure 8: Absorbance spectra of purified mast cell-derived particles
Suspensions of mast cell-derived particles have a distinctive absorbance pattern, that is dependent on its granular structure. The same preparation after dissolution in 5 M NaCl shows greatly reduced absorbance over most of the spectrum.
Figure 9: Absorbance of particle suspensions at 280 and 600 nm are linear with concentration.
In the literature, mast cell-derived particles have generally been quantified using
protein concentration determinations (275, 282), although the quantity has also been
expressed as equivalents of starting mast cell number (281). It is not clear, however, that
the most commonly used techniques for measuring protein concentrations are useful in
66
this application, as they are generally used for measuring the concentrations of soluble
proteins in aqueous solution. As the proteins in mast cell-derived particles are in stable
electrostatic complexes with heparin, it is possible that this will interfere with the
accuracy of the readings. Also, as these suspensions have intrinsic absorbance in the
range measured in such assays, this may also be a complicating factor. Finally, protein
concentration assays are susceptible to interference from certain substances. While
neither heparin nor compound 48/80 is in the list of interfering substances for
commercial assay kits, it was found that heparin interferes with the BCA assay at
concentrations over 100 μg/ml (not shown). Importantly, the concentration of heparin
in particle preparations is probably lower than this.
It was found that the BioRad protein assay (which is a slight modification of the
traditional Bradford assay) provided inconsistent results, and on multiple instances, the
final absorbance reading from the particle sample was lower than the zero standard.
Use of the BCA technique delivered more consistent results, yielding concentrations for
various preparations between 70 and 500 μg/ml. Nevertheless, because of the
considerations above (which vary substantially from experiment to experiment),
diminish confidence in the absolute accuracy of these measurements.
Another potential method for quantifying particles would be to measure the
heparin content of various preparations. The most commonly used assay for this utilizes
the metachromasia that heparin exhibits upon binding certain dyes. Azure A, which is
structurally similar to toluidine blue, is the most commonly used dye in this application,
probably because it exhibits very dramatic metachromasia. The absorbance reading at
the new wavelength is linear with heparin concentration (see Fig. 10). However, it is
linear only over a very narrow range of concentrations (approximately 0.1 - 25 μg/ml),
and again, it may not be as accurate in measuring solid phase heparin. In multiple
67
experiments using this technique, no non-zero measurements were obtained for granule
preparations.
Finally, because mast cell-derived particles are known to contain active
proteases, an attempt was made to quantify preparations by proteolytic activity.
Chymase has a similar substrate spectrum to chymotrypsin, and so the canonical
chymotrypsin substrate BTEE was used. Although chymotrypsin displayed brisk
activity in cleaving this substrate (as measured by changes in UV absorbance over time),
granule preparations exhibited very low or no activity above baseline.
Figure 10: Azura A metachromasia is a quantifiable measure of heparin concentration
In conclusion, many approaches were taken to quantitate granule
preparations. Although protein concentration, heparin concentration, or protease
activity should all be viable techniques in this regard, turbidity/UV absorbance
measurements were found to be far more practical and reproducible. Expressing the
quantity purified as equivalents of the starting number of mast cells would also be an
68
acceptable method, but with the caveat that the same number of mast cells doubtless
yields varying numbers of particles on different days.
2.3.3 Initial Characterization of Mast Cell-Derived Particles
The first of a number of basic studies was to determine particle stability. If they
are to carry inflammatory signals over long distances, they must be able to persist under
conditions similar to those in the extracellular space. As a simple experiment to evaluate
their stability, a preparation of particles suspended in cell culture medium was left at
room temperature and examined microscopically at intervals. As shown in Figure 11,
the particles did not change appreciably over the course of 12 days (even after 11 weeks,
their appearance was the same). This experiment only addresses the intrinsic decay rate
of the particles, and of course, it is not quantitative. However, it does indicate that
under standard conditions, they are very stable.
Figure 11: Mast cell-derived particles are highly stable
(A) DIC micrograph of a suspension of particles at t=0. Micrographs of the same suspensions at t=2 hrs (B), t=23 hrs (C), and t=288 hrs (D).
69
The insolubility of the particles, while making them highly stable potential
vehicles for inflammatory mediators, also makes them quite difficult to study. Methods
were required for the solubilization of granules so that the constituents could be studied
directly. As mentioned above, one way to accomplish this goal is to expose the particles
to very high salt concentrations. Unfortunately, most downstream applications will not
tolerate high salt concentrations, so buffer exchange must be performed. However, it is
reasonable to expect that a return to physiologic osmolarity levels will cause the re-
establishment of the bonds that held the particles together initially. Because the
formation of mast cell granules occurs in a very small space delimited by the granular
membrane, and mediators reach high concentrations, large complexes may be formed.
In solution it is likely that much smaller complexes are formed given the low
concentrations of heparin proteoglycans and basic proteins after granule dissolution.
Although such complexes might be tolerable in downstream applications, it is also
possible that they would interfere. Furthermore, exposure to high ionic strengths
solutions can irreversibly denature proteins, which could compromise results.
Another effective dissolution method is boiling in anionic detergents, such as
SDS. This has permitted analysis of particle-associated proteins by gel electrophoresis,
as detailed below. RapiGest SF (obtained from Waters), a proprietary milder anionic
detergent specifically marketed as improving trypsin digestion as a prelude to mass
spectrometric analysis of proteins, is also capable of dissolving particles. Of course,
although the latter agent is compatible with trypsin proteolysis, proteins are generally
not very good at performing their intended functions after having been boiled in
detergent, so this technique is not optimal for biological experiments requiring activity.
Boiling the particles in the absence of detergent caused them to aggregate, but not to
dissolve. Nonionic detergents, such as Triton X-100, appeared to have no effect on the
70
particles, as would be expected since the forces that hold them together are electrostatic
(see Figure 12).
Figure 12: Purified rat PMCs upon exposure to 0.05% Triton X-100
Alterations in pH change the surface charge characteristics of proteins, so
whether or not particles could be disrupted merely by alterations in pH was
investigated. As mentioned previously, the most abundant proteins in extracellular
granules are highly basic, with an isoelectric point around 9.5. So, it was expected that
exposure of particles to pH 12 would dissociate them. However, exposure to extremes
of pH did not dissolve the particles, although it clearly changed their morphology (see
Figure 13). At pH 2, they probably assume a tightly-packed structure typical of intact
granules (because of the greater number of available charge interactions for cross-
linking). At pH 12, the opposite is likely true, and although it appears not to have been
enough to dissociate them completely, they clearly looked looser and less distinct by
DIC microscopy.
71
Figure 13: Effect of pH on isolated mast cell granules
DIC micrographs of suspensions granules diluted 1:1 in PBS titrated to pH 7.4 (A), 12 (B), and 2 (C). Shown is the edge of a right-most edge of a droplet, so that a range of focal planes can be appreciated. This scenario is supported by research examining the effect of pH on the giant
granules of mast cells obtained from the beige mouse. This mouse has a mutation in the
lyst gene, which encodes the lysosomal trafficking regulator, resulting in an error of
lysosomal storage such that granulated cells have many fewer, but far larger granules
72
(up to 4 μm) (288). There is a rare, homologous condition of immunodeficiency in
humans called Chediak-Higashi syndrome, which has a similar phenotype due to
mutations in the lyst gene (289). Although granules from beige mice are probably
abnormal in ways other than their size, they have proved a useful experimental system
for studying extracellular granules, as the cells will release these normally in response to
activating stimuli, and they exhibit changes similar to those of normal mast cell granules
upon exocytosis (290). It was shown using granules from these mice that the
extracellular granular matrix can undergo condensation, such that it appears as dense as
intracellular granules, under the right conditions. In the presence of 10 mM histamine,
the size of the granules depended on the solution of the pH, correlating strongly with
the transition of histamine with decreasing pH from a monovalent to a divalent cation.
Even in the absence of histamine, the particles shrank in response to lowered pH,
although the effect was not as dramatic. This shrinking effect can also be seen with the
addition of polycations (like polylysine) or even inorganic divalent cations (such as Ca++
or Mg++) (291). Thus, alterations in pH, while apparently able to effect changes in
granule morphology, do not appear to be useful for granule dissolution, at least over the
range tested. This method would also suffer from the drawback that extremes in pH can
also denature and degrade proteins.
A gentler, potentially effective method would be the enzymatic degradation of
heparin by bacterial heparinases. These enzymes, obtained from Flavobacterium
heparinum, have proven useful in the analysis of glycosaminoglycans, as they can
degrade both heparin and its ubiquitous relative, heparan sulfate (292). Unfortunately,
although the activity of the heparinase I used was confirmed with soluble heparin, the
enzyme had no microscopically discernible effects on mast cell-derived particles (even in
extended incubations), although there were unsaturated uronic acid products in the
73
supernatant after this treatment, indicating that some hydrolysis of heparin had
occurred. The likely explanation for this is that the granule appears to exclude larger
proteins from its interior. Interestingly, granule-associated RMCP-I can hydrolyze small
proteins up to about 15 kDa, but it is also active against larger proteins once it has been
purified away from the granule structure and solubilized. Likewise, granular RMCP-1
can be inhibited by small molecule, but not protein protease inhibitors (45). This
suggests that the physiologic targets of chymase may be small proteins. The molecular
weight of heparinase I is 42.8 kDa, suggesting that it cannot access the interior. Thus,
while the enzyme appears able to catalyze some hydrolysis (presumably of exterior
exposed heparin), it cannot dissociate the particle itself.
2.3.4 Analysis of the Mast Cell Secretome
Prior to focusing exclusively on TNF, basic studies on the constituents of mast
cell granules were performed, both with the goal of demonstrating the presence of TNF
and to identify any other inflammatory signals that might be relevant to signaling
between sites of inflammation and draining lymph nodes.
Initially, a cytokine-directed approach was taken, using a multiplex ELISA
system to measure several rat cytokines in purified particles simultaneously. The
analytes were: IL-1 , IL-1 , IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN- , and TNF (Bio-Plex Rat
Cytokine 9-Plex A Panel obtained from Bio-Rad). Although this technique is quite
sensitive, it is intended for use on samples containing soluble cytokines. Unfortunately,
we did not detect cytokines using this method. Because particle-associated proteins are
in insoluble complexes, the lack of cytokine detection in this assay cannot be taken as
proof of their absence.
As an alternative approach, the major proteins of the extracellular granule were
evaluated by SDS-PAGE (Figure 14). Because the supernatant was removed during the
74
purification of these particles, this separation only includes the insoluble fraction of the
secreted products. These findings are consistent with other studies of the protein
constituents of rat mast cell granules (265, 293). Based on the work of others, the major
bands between 25 and 37 kDa could be tentatively identified as the major mast cell
proteases RMCP-1 (~26 kDa), RMCP-5 (~29 kDa), and MC-CPA (multiple bands from 33
- 36 kDa) (266). The identity of the lower molecular weight bands is unknown.
Figure 14: SYPRO Ruby-stained SDS-PAGE separated mast cell granule-associated proteins
Because it appeared that the mixture of granule-associated proteins was of
relatively low complexity, mass spectrometric analysis was performed on in-solution
digests of unseparated particle proteins (Table 2). Although no well-described growth
factors or cytokines were identified in these studies, several patterns emerge. First, the
vast majority of the protein present in extracellular granules is accounted for by the
75
major proteases. Presumably, there is also a fair amount of serglycin, the core protein
for heparin proteoglycans, but this protein is so heavily glycosylated that it is resistant to
trypsin (294) and so would not be detected by this assay.
Table 2: Protein identifications made in six mass spectrometry experiments with five individual granule preparations excluding likely contaminants
Protein # IDs Pi MW (kDa)
6/6 9.96 35
6/6 10.14 25
6/6 9.93 27
3/6 11.66 14.11
3/6 11.05 13.99
4/6 5.46 270.34
3/6 11.86 21.99
3/6 11.91 11.37
3/6 5.15 41.74
3/6 8.19 35.83
2/6 7.42 38.7
2/6 11.96 13.45
2/6 7.84 38.68
1/6 8.54 39.63
1/6 9.39 23.73
Carboxypeptidase B precursor 1/6 6.77 35.24
Protein piccolo 1/6 6.5 555.84
1/6 8.12 36.18
1/6 6.41 532.35
1/6 4.43 84.78
1/6 8.52 50.38
1/6 6.79 171
1/6 8.49 7.78
A number of proteins identified in this study were clearly of intracellular origin,
such as the histones, dynein, and metabolic enzymes. Given what is known about these
proteins, it seems highly unlikely that they are actually loaded into the granule with
other mast cell mediators. It is more probable that these proteins were soluble in the cell
suspension before mast cell activation and that they adhered to the extracellular
granules after their extrusion. Interestingly, most of the proteins identified either have a
76
very basic isoelectric point (histones, eosinophil major basic protein) or have well-
characterized heparin binding activity (amyloid beta A4 precursor (295), fibronectin
(296), and annexins (297)). This evidence provides further support for the idea,
discussed in Chapter 1, that one function of mast cell-derived particles may be to
regulate soluble extracellular heparin-binding proteins.
A quantitative proteomic analysis of granule-associated proteins was also
performed (Table 3). In this experiment, the only proteins identified that were not most
likely serum or keratin contaminants were the mast cell proteases (alcohol
dehydrogenase is added to the mixture as a control for quantitation). The weight ratio
of the proteases (5.41 : 1.49 : 1 for MC-CPA : RMCP-5 : RMCP-1) approximates that
reported previously for rat mast cells. One study found that 50% of the granular protein
was chymase (31), whereas here the value was about 30%. It is possible that the
chymases may have dissociated to some limited extent during particle purification as
well, as they are known to be less tightly bound to the granule than MC-CPA (264). This
could result in an underestimate of the actual levels. Finally, it is worth noting that any
granule-associated protein present must be less than 1% as abundant as MC-CPA on a
molar basis, assuming that the quantity of the least abundant observed protein
represents an approximate lower limit for detection.
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Table 3: Quantitative proteomic analysis of granule-associated proteins
Protein MW (Da) fmol present ng present
Mast cell carboxypeptidase (MC-CPA) 35763 3878.2175 138.7865
Mast cell protease III precursor (RMCP-5) 27551 1384.3846 38.1664
Mast cell protease I precursor (RMCP-1) 28598 896.8084 25.6642
Trypsin precursor EC 3 4 21 4 24393 215.1292 5.2512
Alpha 2 HS glycoprotein precursor Fetuin A Asia 38394 126.081 4.8439
Serum albumin precursor Allergen Bos d 6 69248 122.5014 8.4886
Alcohol dehydrogenase I EC 1 1 1 1 36668 50 1.8346
Alcohol dehydrogenase II EC 1 1 1 1 36577 37.8616 1.3858
Keratin type II cytoskeletal 1 Cytokeratin 1 K 65846 36.6583 2.4153
Hemoglobin beta fetal chain Hemoglobin gamma chai 15849 32.7979 0.5201
As the presence of highly abundant proteins, probably accounting for over 99%
of total granule-associated protein, may have impaired the detection of rarer species, an
attempt was made to exclude these, as their major bands all migrate between 25 and 37
kDa (see Figure 14). Because TNF, the mature, secreted form of which is 17 kDa, was of
particular interest, the analysis was limited to proteins between 14 and 19 kDa by
separating the proteins by SDS-PAGE and then cutting out that region and preparing it
for MS analysis. Again, all three major proteases, but not TNF, were identified (Table 4).
This is not entirely unexpected, as there is already evidence that some smaller molecular
weight bands are probably protease degradation products (293). Human keratins (the
search was limited to rat, but they are highly homologous) are a frequent contaminant in
protein identification experiments. Since histones were identified in multiple separate
experiments (see Table 2), it seems likely that the prominent band around 15 kDa
(Figure 14) is accounted for by these proteins, although protease fragments may also
contribute. The origin of these histones is unclear, but it is probable they are derived
from the small percentage of killed cells and cellular debris in the initial peritoneal
lavage.
78
Table 4: Granule-associated proteins between 14 and 19 kDa identified by MS1
Protein name MW (kDa)
Mast cell protease 1 precursor - Rattus norvegicus (Rat) 25
Mast cell carboxypeptidase A precursor - Rattus norvegicus 35
Chymase precursor - Rattus norvegicus (Rat) 27
Keratin, type I cytoskeletal 10 - Rattus norvegicus (Rat) 56.52
Keratin, type II cytoskeletal 5 - Rattus norvegicus (Rat) 61.84
Histone H2B type 1 - Rattus norvegicus (Rat) 13.99
Keratin, type II cytoskeletal 1 - Rattus norvegicus (Rat) 64.85
Histone H2A type 1-C - Rattus norvegicus (Rat) 14.11
Bone marrow proteoglycan precursor - Rattus norvegicus (Rat) 13.45
Annexin A1 - Rattus norvegicus (Rat) 38.7
Histone H3.1 - Rattus norvegicus (Rat) 15.28
Cofilin-1 - Rattus norvegicus (Rat) 18.53
Hemoglobin subunit beta-1 - Rattus norvegicus (Rat) 15.85
Finally, the soluble proteins released during degranulation were analyzed by
mass spectrometry as well, both to evaluate the presence of TNF and other signals in this
fraction, and also to confirm that effective separation of the two fractions is taking place.
This experiment was controlled by incubating lavage cells in medium and then spinning
them out of this medium, saving it as the control fraction. The cells were then
resuspended in the same medium containing compound 48/80 and incubated under the
same conditions before removing the cells again by centrifugation. Both supernatants
were then centrifuged much faster to sediment extracellular granules in the preparation.
The results of this experiment are expressed as comparisons between identifications
made in the pre- and post-48/80 media.
Several proteins were identified only in the post-compound 48/80 media,
including multiple mast cell-specific proteins, suggesting that the results do reflect
proteins secreted from mast cells (Table 5). Among these were the mast cell proteases
identified in the granule-specific experiments, which is not surprising given their
abundance. The identification of relatively abundant quantities of tryptase, which was
1 Except for the proteases, molecular weights presented here are calculated from sequence.
79
never identified in the particle-associated fraction, confirms that a separate soluble
fraction has been isolated. Unlike human tryptase, which binds strongly to heparin, rat
tryptase does not, and so finding the enzyme among the soluble proteins is not
surprising (298). The storage and activation-related release of dipeptidyl peptidase II by
mast cells has also been previously documented, although its significance remains
unknown (299, 300). The lysosomal hydrolases -hexosaminidase (not shown, but
detected with lower confidence in this experiment and also by using other methods) and
-glucuronidase are known constituents of mast cell granules (301), but the presence of
DNase-2 has not been noted previously. Again, several proteins that are probably
cytoplasmic in origin were identified, suggesting that the treatment (or the extra
centrifugation step) may have caused some cytotoxicity. Neither TNF nor any other
well-described signal was identified in this experiment (aside from a low confidence
identification of interleukin 24, about which essentially nothing is known outside the
context of cancer), but again, such proteins are probably not very abundant compared to
many of the proteins identified here.
80
Table 5: Semiquantitative analysis of soluble proteins released during compound 48/80-induced degranulation
Name MW Control 48/80
Mast cell protease 1 precursor 29 kDa 0 362
Tryptase precursor 30 kDa 0 11
Dipeptidyl-peptidase 2 precursor 55 kDa 0 7
Vimentin 54 kDa 0 6
Mast cell carboxypeptidase A precursor 48 kDa 0 6
Deoxyribonuclease-2-alpha precursor 38 kDa 0 6
Sulfated glycoprotein 1 precursor 61 kDa 0 5
14-3-3 protein theta 28 kDa 0 5
Pyruvate kinase isozymes M1/M2 58 kDa 0 4
78 kDa glucose-regulated protein precursor 72 kDa 0 4
Adenylyl cyclase-associated protein 1 52 kDa 0 3
Glyceraldehyde-3-phosphate dehydrogenase 36 kDa 0 3
Beta-glucuronidase precursor 75 kDa 0 3
Chymase precursor 28 kDa 0 3
Coronin-1A 51 kDa 0 2
Calreticulin precursor 48 kDa 0 2
Protein disulfide-isomerase A3 precursor 57 kDa 0 2
Aldose reductase 36 kDa 0 2
Granulins precursor [Contains: Acrogranin; Granulin-1 63 kDa 0 2
Interestingly, there was a group of proteins whose abundance decreased after
compound 48/80 treatment (Table 6). Included among these were LTA4 hydrolase, an
enzyme required for the synthesis of the neutrophil chemoattractant leukotriene B4, and
leukocyte elastase inhibitor A. Both of these proteins have important potential roles in
inflammation that may be regulated by their adsorption to mast cell-derived particles.
For example, local reductions in leukocyte elastase inhibitor concentrations could
enhance the activity of neutrophil-derived elastase. This phenomenon should be
carefully explored in future studies.
2 Numbers indicate number of identified peptide spectra normalized to percentage of total spectra.
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Table 6: Proteins identified in control supernatants that were reduced after 48/80-induced degranulation
Name MW Control 48/80
L-lactate dehydrogenase A chain 36 kDa 24 8
Leukotriene A-4 hydrolase 69 kDa 12 3
Serum albumin precursor 69 kDa 19 0
Peptidyl-prolyl cis-trans isomerase A 18 kDa 12 0
Creatine kinase B-type 43 kDa 7 0
Malate dehydrogenase, cytoplasmic 36 kDa 7 0
Moesin 68 kDa 5 0
Ubiquitin 9 kDa 5 0
Thymosin beta-10 5 kDa 6 0
Phosphoglycerate kinase 1 45 kDa 6 0
Leukocyte elastase inhibitor A 43 kDa 7 0
Parathymosin 12 kDa 5 0
Cofilin-1 19 kDa 5 0
WD repeat-containing protein 1 66 kDa 3 0
Tropomyosin alpha-3 chain 29 kDa 5 0
Fructose-bisphosphate aldolase A 39 kDa 2 0
TNF and other inflammatory signals were not identified in these studies.
However, this was not entirely surprising, as one study calculated the quantity of
preformed TNF per mast cell at 0.404 fg/rat PMC (254), which is less than 1/1000 of 1%
of the total cellular protein (assuming 100 pg total protein/cell). Still, several interesting
observations were made that have implications for the biological functions of mast cell-
derived particles, which are still essentially unknown.
First, these studies more adequately describe the major protein constituents of
the mast cell secretome than previously available work. In particular, they demonstrate
that rat peritoneal mast cells store large, nearly equivalent quantities of three cell type-
specific proteases, RMCP-1, RMCP-5, and MC-CPA. Although RMCP-1 and RMCP-5
are both chymases, the specificity of RMCP-5 is closer to that of elastase than it is to
chymotrypsin (302). In other words, the extracellular granule matrix contains three
active proteases with different specificities. Although the physiologic substrates for
mast cell proteases remain obscure, perhaps this is partly a function of the fact that they
82
have only been studied in isolation, when, in vivo, they are retained in close proximity
after their secretion. The arrangement suggests cooperative action of the proteases on
substrates, and this has in fact recently been shown for at least two mast cell granule-
associated proteases in the regulation of angiotensin II regulation (169).
Also, these studies provide more evidence for the as yet unproven hypothesis
that these particles regulate levels of important immunomodulatory proteins by
removing them from the soluble aqueous phase of the extracellular environment. This
promising idea should be easy to test, assuming a good candidate molecule could be
designated. From this work, investigating the effect of changes in leukocyte elastase
inhibitor levels would be a reasonable starting point.
2.3.5 Mast Cell-Derived Particles Contain Tumor Necrosis Factor
As a proteomics-based approach to the detection of TNF in mast cell-derived
particles failed, probably due to its low abundance, more specific methods were
pursued. Initially, immunoblotting for TNF on SDS-PAGE separated granule-associated
proteins was utilized. As seen in Figure 15, a polyclonal antibody against TNF detected
a band at ~17 kDa in separated granule proteins, which is the size of mature secreted
TNF. Soluble fractions were also assayed (as before with both a pretreatment control
and a post-compound 48/80 sample). Although a new band appears in the post-
treatment lane at ~55 kDa, this experiment was done in 2% FBS, so the vast majority of
proteins in these lanes is derived from bovine serum. Also, it is associated with the
relative reduction in intensity of a slightly higher molecular weight band at ~60 kDa,
suggesting that the new band may be the result of proteolysis (mediated possibly by
released mast cell proteases).
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Figure 15: Detection of TNF in granule-associated, but not soluble, proteins released by mast cells after activation
Lane 1: control lavage supernatant. Lane 2: lavage supernatant after compound 48/80 treatment. Lane 3: sedimented granule-associated proteins from lavage supernatant after compound 48/80 treatment. Membrane was blotted with a polyclonal anti-human TNF antibody (Cell Signaling Technology #3707). Cross-reactivity with rat TNF was verified using recombinant protein in separate experiments (not shown).
Because this polyclonal anti-TNF antibody was somewhat nonspecific, also
detecting RMCP-1 and MC-CPA to a limited extent among granule-associated proteins
(already a low complexity mixture), this finding was confirmed with a second anti-TNF
antibody (Santa Cruz sc-1350). These results are shown in Figure 16. Soluble TNF was
again assayed, but in this experiment the lavage was split into two parts, one of which
was treated with compound 48/80 in serum-free media in an attempt to limit serum
protein contamination of the supernatant. Although rat PMCs will degranulate in
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response to compound 48/80 in the absence of serum, recovery of particles is difficult
(perhaps because they adhere more readily to cells and surfaces), so the remainder of the
cells (for particle isolation) were treated in the presence of 2% FBS. Soluble proteins
were concentrated over 10-fold before loading on the gel using spin concentrators with a
molecular weight cutoff of 5 kDa. This experiment shows a band in the granule-
associated protein lane 3 that comigrates with recombinant rat TNF (rrTNF). Although
there is also a band in the soluble proteins lane, it appears to be of a slightly greater
molecular weight than rrTNF. However, the release of some soluble TNF by
degranulating mast cells is not incompatible with the hypothesis proposed by this work.
Figure 16: Presence of TNF in granule-associated proteins demonstrated with a second polyclonal anti-human TNF antibody
85
Bizarrely, this totally separate polyclonal anti-TNF antibody obtained from a
different vendor also reacted with RMCP-1. Because there are some relatively abundant
proteins in this size range (see Figure 14) that were mostly accounted for by histones and
other small proteins, an effort was made to detect granule-associated TNF using a
monoclonal antibody, which should theoretically be more specific (clone TN3-19.12,
Sigma T2824). Interestingly, it may have actually been the least specific of the anti-TNF
antibodies used, but it did detect the same band that the others did (Fig. 17). Regardless,
the detection of this 17 kDa species, which is the known size of mature TNF, and which
comigrates with recombinant rat TNF, by three different antibodies is strong evidence
that TNF is in fact released from mast cells in this form.
Figure 17: Detection of TNF in granule-associated proteins using a monoclonal anti-TNF antibody
The detection of TNF was also attempted using the L929 fibroblast cytotoxicity
bioassay and ELISA, but these methods were unsuccessful. This is probably due to the
fact that they are intended to be performed on entirely soluble samples and are not
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likely to detect TNF contained in insoluble complexes. Slight dose-dependent
cytotoxicity was noted in L929 cells treated with purified granules, but it was not
sensitive to TNF neutralization (Fig. 18). It is possible that this effect was caused by
compound 48/80 concentrated within the purified particles, as compound 48/80 is a
polycation and can precipitate heparin in vitro (not shown). This drawback of the
particle purification procedure used in these studies could be addressed in the future by
using a neutral secretagogue, such as calcium ionophores.
Figure 18: Granule-induced cytotoxicity is not neutralized by anti-TNF.
Actinomycin D-treated L929 fibroblasts were treated with nothing (white bars), recombinant TNF, or purified granules (MCGC). Black bars: stimulus plus 1 μg/ml control goat IgG, grey bars: stimulus plus 1 μg/ml neutralizing anti-TNF antibody (R&D Systems AF-410-NA).
Both the cytotoxicity bioassay and ELISA were able to detect TNF, as they easily
quantitated the low levels of de novo synthesized TNF in rat peritoneal lavage cultures
treated with compound 48/80 (Figs. 19 and 20). These experiments indicate that de novo
TNF is not secreted at detectable levels until about an hour after the stimulus,
confirming that the cytokine observed in the other studies at earlier time points is
actually preformed. Also, in the L929 assay a short spike of soluble TNF (as the
insoluble fraction was removed by centrifugation) is observed at 5 minutes that is no
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longer detectable at 15 minutes, consistent with other studies of peritoneal mast cell TNF
release (74). The earliest time point in the ELISA study was 15 minutes, so this spike
may have been missed. Again, the presence of a soluble pool of preformed TNF does
not imply that granule-associated TNF is without importance. The ELISA study, which
goes out to 24 hours, reveals an eventual slow decline in TNF production, but at this
point, there is significant cell death in the lavage culture, which has been maintained in
normal cell culture media supplemented only with FBS. This is not surprising for such a
population of primary cells, so this trend likely does not reflect the pattern that occurs in
vivo.
Figure 19: Release of soluble TNF by rat peritoneal lavage cells treated with compound 48/80 measured by L929 cytotoxicity assay
Expressed as direct reading of cytotoxicity readout -- proportional to TNF release.
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Figure 20: Release of soluble TNF by rat peritoneal lavage cells treated with compound 48/80 measured by ELISA
Finally, in order to produce additional evidence for the storage and release of
TNF by mast cells in association with granules, in vitro mast cell models expressing TNF-
GFP fusion proteins were generated. This was done both in the RBL-2H3 rat basophilic
leukemia cell line, which expressed high levels of the fusion protein, and in BMMCs,
which expressed it less consistently. In both cases, activation was accomplished using
the calcium ionophore ionomycin instead of compound 48/80, as RBL-2H3 cells are not
responsive to 48/80 and BMMCs have reduced responsiveness. BMMCs were first
cocultured with 3T3 fibroblasts to increase their heparin production and enhance
granule maturity. Both cell types released fluorescent particles after activation,
suggesting that the fusion protein remained associated with the granule in the
extracellular environment.
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Figure 21: TNF-GFP fusion protein remains with the granule after exocytosis
Confocal still micrographs from videos of mast cells degranulating in response to ionomycin showing retention of fluorescence in extracellular granules. (A)-(C) TNF-GFP expressing RBL-2H3 cells. (D)-(F) TNF-GFP 3T3 fibroblast-coculture BMMCs.
A few conclusions can be drawn based on the findings of these studies. First, a
significant proportion of the preformed, granular TNF stored by mast cells remains
associated with the granular residue after exocytosis, although there also appears to
exist a soluble pool of TNF. The inability to detect the granule-associated TNF using
several methods implies that it is indeed well-insulated from external interactions while
in this form. It was only observable after the particles had been boiled in Laemmli
buffer for SDS-PAGE. This is consistent with this work's hypothesis, which is that this
insulation is critical to delivering this TNF to its intended target, the draining lymph
node.
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2.3.6 Development of a System for Modeling Mast Cell-Derived Particles
Purifying mast cell-derived particles, even using large numbers of animals, only
yields a limited amount of material. Furthermore, as was suggested in the previous
section, the use of agents like compound 48/80 during the protocol may affect the
activity of the particles. The potential for conducting controlled experiments using
purified particles is relatively poor. What is being investigated in such experiments is
specifically the phenomenon of mast cell mediators being delivered in this insoluble
form, as opposed to as soluble molecules. As no reliable method for dissolving the
particles without using harsh conditions has been developed, this kind of comparison is
difficult to make rigorously.
The granule is held together in the extracellular space by electrostatic interactions
between two polyelectrolytes: heparin and basic proteases. The stability comes from the
large number of such interactions. The fact that no covalent bonds are involved means
that similar complexes can be made in vitro quite easily, simply by mixing solutions of
oppositely charge polyelectrolytes. Upon such mixing, particles form semi-randomly as
the polyelectrolytes precipitate with each other. The process continues until all excess
binding sites are occupied. Thus, there are opportunities for controlling particle size and
composition simply by changing minor variables during fabrication. Also, soluble
molecules included during particle formation will be encapsulated to a lesser or greater
degree (depending primarily on whether or not the cargo molecule binds to one of the
polyelectrolytes initially).
This concept is quite attractive for drug delivery and is in active development. It
is especially favorable for particle formulations of biological therapeutics (especially
proteins) as no harsh conditions are required for the fabrication of the vehicle (303, 304).
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Heparin was chosen as the polyanion component of the synthetic particles, as it
is a major constituent of natural mast cell-derived particles and also because heparin is
widely available. Of course, it must be noted that commercial heparin is produced from
tissue homogenates and is reduced to relatively small pieces during the course of its
processing. As mast cell proteases are not as widely available, alternatives were
considered for the polycation. The first was poly-lysine, which is widely available and
has a very high charge density. Upon adding a 0.1% solution of poly-l-lysine to a 2%
solution of heparin (both in water), turbidity rapidly developed, reflecting the
precipitation of large numbers of particles (Fig. 22).
Figure 22: Particles formed upon addition of 0.1% polylysine to 2% heparin
Scalebar is 15 μm.
This turbidity, measured at 400 nm (a wavelength at which none of the soluble
constituents alone exhibits absorption), correlates linearly with the particle
concentration. As was the case for the natural purified particles themselves, this proved
the most practical method for quantitation (Fig. 23). The synthetic particles were also
similar to mast cell-derived ones in that they were dissolved under the same
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circumstances. A few significant differences were observed. The particles could not be
reliably labeled after fabrication by usual mast cell granule stains, including toluidine
blue and fluorescent avidin. This is probably due to the fact that in mast cell granules,
many anionic sites on heparin are ordinarily bound by histamine and other soluble
molecules, meaning that after the tissue has been fixed and washed, there are ample
binding sites for dyes. In the synthetic particles, on the other hand, the vast majority of
these sites are occupied by polylysine. This fact made the particles difficult to identify in
tissues.
Figure 23: Linear relationship between heparin/polylysine particle concentration and turbidity
To test the effect of delivering TNF in particle vs. soluble form, it was necessary
to develop techniques to encapsulate the cytokine into these particles. TNF
encapsulation was achieved as described in the Methods. This protocol was successful,
as shown in Figure 24. The particles were washed three times in PBS, and little TNF was
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recovered in these washes. Over the course of 24 hours, however, the particles released
a significant quantity of their encapsulation TNF.
Figure 24: Encapsulation of TNF in heparin/polylysine particles
After initial fabrication, the particles were washed three times in PBS -- these washes are shown as indicated. Then they were incubated at room temperature overnight, after which they were centrifuged again and resuspended. The 24 hour supernatant is shown, indicating the amount of TNF released over that period. The boxed lane, containing the microparticles, still contains a substantial amount of TNF after 24 hours.
In addition to not interacting normally with basic dyes, the synthetic particles
showed a much greater tendency to aggregate, probably due to the fact that the content
of polycation is much greater than that found in the natural granules. Polylysine also
has a much higher charge density than mast cell proteases do, meaning that it is even
stickier. Because of this, and for other reasons discussed later, polylysine was eventually
abandoned in favor of chitosan. This polysaccharide, obtained from shellfish, has
unique properties that make it ideal for this application.
Chitosan is also a polycation and fully water-soluble at slightly acidic pH, but at
neutral pH, it is uncharged and insoluble. Due to this property, chitosan forms
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polelectrolyte complexes with heparin and other polyanions when it is charged, but its
insolubility contributes to the structural integrity of the resulting particles once pH
neutrality is restored. Heparin/chitosan particles, then, should not have regions of high
positive charge density as heparin/polylysine particles do. This should make them less
prone to aggregation and also more similar to mast cell-derived particles, the surface of
which is also dominated by negative charge.
2.4 Discussion
Several key findings have been presented in this chapter. First, methods were
established for the purification and study of mast cell-derived particles. Although a
robust protocol for the reliable purification of the particles was developed for rat
peritoneal mast cells, there is still vast room for improvement. Perhaps most
importantly, purification of particles from a source other than peritoneal mast cells
would be good. Some possible alternatives were discussed above, but at present not
enough is truly understood about the processes that govern mast cell differentiation.
The factors that govern the responsiveness of the cells to agonists need to be better
understood, so that a more rational procedure for obtaining and maintaining peritoneal
lavage cells can be used. Finally, there remains a need for a way to completely solubilize
the particles without otherwise irreversibly denaturing their contents. It is possible that
enzymatic digestion with bacterial heparinases or mammalian heparanases, which can
degrade heparin also (305), can be optimized into usefulness.
Limitations of many conventional techniques were recognized that make them
unsuitable (without modification) for the characterization of these particles. Although
turbidity measurements allow comparative quantitation to be calculated, for the
purposes of preparing doses, etc, an absolute method would be preferable, ideally one
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based either on the number of particles or on the total quantity of one of their major
constituents.
These were then characterized biochemically. In agreement with previous work,
the main protein constituents of rat peritoneal mast cell heparin-protein complexes were
identified as RMCP-1, RMCP-5, and MC-CPA. A quantitative analysis of these revealed
their relative quantities and also, importantly, revealed that there are no other detectable
proteins present within 100-fold abundance. The presence of these three proteases with
their different specifities in what appears to be approximate molar equivalence suggests
that future investigations into their physiologic roles should focus on their combined
activity on possible substrates, in the context of the extracellular granule if possible.
Also, the presence in granule preparations of several cationic proteins that,
although it is possible, are probably not actually loaded into mast cell granules, suggests
a scavenger role for mast cell-derived particles. This may be one method for removing
debris, including DNA (which is bound to histones after cell death) from the
extracellular environment. It may also act to bring molecules into close proximity that
have some combined action. Or, it might act simple to regulate the free levels of
heparin-binding proteins in the extracellular milieu. Naturally, this is pure speculation
at this point. It will be exciting to see this story unfold as it is pursued.
The presence of the critical immunomodulatory cytokine TNF was unequivocally
demonstrated in mast cell-derived particles by immunoblotting and by an in vitro
analysis showing the extracellular disposition of a TNF-GFP fusion protein after
experimental degranulation. Some studies also suggested the presence of a soluble pool
of preformed mast cell TNF, which may also be highly relevant for its more local effects.
Finally, a simple protocol for generating synthetic polyelectrolyte complex
particles was developed using anionic heparin and cationic polylysine or chitosan. Such
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synthetic complexes represent an important tool, as they enable control over
experimental conditions that is simply not available using the purified mast cell-derived
particles. Working with such particles is also likely to enable a better appreciation for
the factors that determine the characteristics of natural particles. For example, by
attempting to recreate the granule outside of the cell, insights will certainly be gained as
to the role of pH, the disposition of soluble mediators, etc.
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3. Studies Investigating the Role of Mast Cell-Derived Particle-
Mediated Transport of Inflammatory Mediators in Draining
Lymph Node Responses
3.1 Introduction
3.1.1 Studies on the Fate of Mast Cell-Derived Particles
The first studies on the fate of mast cell-derived particles were performed by
Higginbotham in the 1950s. Using a mouse skin air pouch model of inflammation and
examining connective tissue spreads afterwards, degranulation of mast cells was
observed in response to the injection of a number of substances. Uptake of granules by
fibroblasts (identified by morphology) within 15 - 60 minutes was observed, followed by
their degradation by 3-4 hours. Fibroblasts selectively internalized granules, but not an
innoculum of staphylococci, suggesting a process distinct from classic phagocytosis (50).
A later report by the same group, using antigen as the stimulus, presented evidence that
under conditions of very extensive mast cell degranulation, some material is not rapidly
degraded, but remains extracellular for up to 24 hours or inside fibroblasts for several
days (51).
In 1958, Smith and Lewis made a series of fascinating observations of the fate of
mast cell granules using the rat mesentery. After intraperitoneal injections of either
distilled water or compound 48/80, extensive degranulation of mesenteric mast cells
occurred. In fixed tissue, phagocytosis of granules was observed to begin at 15 - 30
minutes by fibroblasts, macrophages, and incoming leukocytes. By 7 hours, there are
very few extracellular granules, and by 24 hours, there are none. By the latter time point,
granules are beginning to be transformed into amorphous masses of metachromatic
material, and by 4-5 days, no metachromatic material is evident anywhere in the
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mesentery. More interestingly, observations on granule fate were made using the rat
mesentery in vivo. After the injection of distilled water, the mesentery was observed
microscopically with constant superfusion with a buffer solution, and granules were
visualized using toluidine blue as a vital dye. In this system, released granules show
"Brownian motion and freely bounce about." Also, previously immobile tissue
macrophages migrate towards the granules and begin to internalize them. Large
numbers of leukocytes begin to enter the tissue from the vasculature by 2-4 hours.
Fibroblasts do not show any motion, but granules in motion encountering them are
internalized. Again the focus in this study was on phagocytosis. There is no mention of
granules leaving the tissue, but as discussed above, only some areas of the rat mesentery
are drained by lymphatics (306).
Since that time, phagocytosis of granules has also been documented in rat
eosinophils (53), mouse free peritoneal macrophages (52), rat lymph node reticular cells
and macrophages (56), bovine endothelial cells (54), human endothelial cells (307), dove
neurons (308), and rabbit smooth muscle cells (275). In general, these studies thus far
have been phenomenological, and little is known about the mechanisms underlying this
uptake. Aside from phagocytosis, there appears to be no other literature on the fate of
these particles.
3.1.2 The Structure of the Initial Lymphatic Endothelium and Its Permeability to Particulate Material
As discussed briefly in Chapter 1, the endothelium of initial lymphatic vessels is
very highly permeable in comparison to most blood endthelium. The main basis for this
permeability is that, although interendothelial junctions (IEJs) do have many of the same
adhesion molecules found in the blood endothelium, there are significant areas of
discontinuity (309). The structure of junctional complexes between endothelial cells in
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initial lymphatics was recently studied in some detail. Whereas in blood vessels and
downstream large lymphatics, the entire IEJ has junctional molecules, which by
immunofluorescence microscopy appear as dense lines that encircle the entire border of
each individual cell. In contrast, junctional proteins (including VE-cadherin, claudin-5,
zona occludens-1, endothelial cell-selective adhesion molecule (ESAM), and junctional
adhesion molecule- A (JAM-A)) are restricted to "buttons" in initial lymphatic
endothelium, and between these are gaps without junctional proteins of apprxomiately 3
μm. Endothelial cells are still apposed at these gaps, but the cells are not connected to
each other, and these sites correspond to sites of entry for migrating cells (310).
The outside of the initial lymphatic wall is connected to the surrounding
connective tissue by "anchoring filaments" (309), and this connection depends on the
integrity of hyaluronic acid proteoglycans in connective tissue ground substance (311).
When the space between connective tissue elements is increased, such as during edema,
these anchoring filaments exert radial tension on the exterior wall of the vessel, which
dilates the vessel as well as pulling open the noncontinuous sections of IEJs (311).
Scanning electron microscopy of the interior of initial lymphatics during edema reveals
the presence of dramatic openings at junctions (as the underlying connective tissue
fibers can be seen through the opening) well over a micron in width (231).
That the injection of particulate matter can be used to identify lymphatic vessels
has been known for a very long time (312). Many studies of the relationship between
particle size and ability to enter lymphatic vessels have been performed, studied, but it
appears that for particles around 50 nm, there is little or no barrier to lymph node
accumulation (313). Particles above this size can enter lymphatics, but they are more
restricted by extracellular matrix elements (fluid channels in the interstitium are
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ordinarily about 100 nm in diameter (314)), so they take longer to reach the lymphatic
wall. In edema, these "pre-lymphatic" spaces are greatly enlarged.
Given the anatomy of the average connective tissue, it is unsurprising that the
majority of released mast cell-derived particles remain near their cells of origin long
enough to be engulfed by a variety of cells. Still, the finding that after mast cell
degranulation in rat mesentery the granules move enough for it to be visibly perceptible
as Brownian motion suggests that they are not immediately hemmed in after leaving the
mast cell. During a peripheral reaction, it would only take a small percentage of
granules to leave the tissue to carry signals to local lymph nodes.
3.1.3 The Movement of Peripheral Signals to Draining Lymph Nodes: Remote Control
The transport of molecules to draining lymph nodes has received some attention,
and recently a number of exciting reports have emerged showing the control of lymph
node events by signals generated in the periphery. In order to understand how
peripheral signals may control lymph node dynamics, understanding lymph node
structure is crucial. The anatomy of the lymph node can be roughly divided into two
compartments. The first, the sinus system, consists of the subcapsular, paracortical, and
medullary sinuses and is continuous with both the afferent and efferent lymphatics
bringing lymph to the node and taking it away, respectively. It is lined by LYVE-1+ cells
that are functionally analogous to the lymphatic endothelium in larger lymphatic vessels
(and most blood endothelium): it is mostly impermeable to proteins and particles (315).
Though continuous with lymphatic vessels, it is packed with phagocytes and it not a
space through which particles may freely pass. The second lymph node compartment
contains the lymphocytes, which mostly enter the node through specialized high
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endothelial venules (HEVs) and exit by crossing the sinus endothelium and leaving in
efferent lymph.
It has been shown that specialized "conduits" connect the subcapsular sinus to
the high endothelial venules, providing a potential avenue for peripheral-origin signal
molecules, such as chemokines, to control incoming lymphocytes (316). The same report
also presented evidence that molecules above approximately 60-70 kDa cannot enter
these conduits. Given this, mast cell-derived particles are far too large to reach the HEV
from the subcapsular sinus, but they may elute soluble signals near these conduits,
which could in this manner influence HEVs. As shown in Figure 24, synthetic particles
similar to mast cell granules do elute encapsulated TNF over time, so this is plausible.
One of the earliest reports to document the possibility that peripheral signals
may reach lymph nodes and even have functional effects there investigated the role of
luminal presentation of CCL21 on HEVs as a central requirement for naive T cell
recruitment. In this study, cutaneous injection of CCL21 was administered to correct a
defect in T cell recruitment in plt/plt mice, which do not express CCL21. Significantly,
the dose of peripheral CCL21 used in this study was 1 μg (257). In a related study, 2.5
nmol (over 20 μg) of CCL19 or CCL21 was injected to detect its presentation on HEVs
and to induce functional effects (256). These two studies, however, did not assert that
the production of these chemokines in the periphery was the physiologic mechanism
behind the normal expression of the chemokine on HEVs, only that the defect could be
corrected by the peripheral injection of chemokine.
In the first study to describe a role for a peripherally produced inflammatory
mediator in the draining lymph node, and the first to invoke the term "remote control,"
cutaneous inflammation was induced by the injection of complete Freund's
adjuvant/keyhole limpet hemocyanin (CFA/KLH). This was associated with high
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levels of cutaneous expression for the chemokine MCP-1 and significant increases in
protein levels for this chemokine both in the skin and the draining lymph node. No
change in expression levels was observed in the lymph node, and as injected, labeled
chemokine could be detected in the node it was concluded that the rise in LN MCP-1
was a consequence of peripheral production and subsequent drainage. Again, luminal
presentation of the signal on HEVs was a relevant factor, as the lymph node outcome
affected was recruitment of monocytes from the blood into the node (259).
The accumulation in lymph nodes of functional peripheral signals has now also
been documented for TNF (225, 250), VEGF (317), and CCL27 (258), at least. In these
cases, however (aside from TNF), the peripheral signals in question were produced
chronically at high levels during established inflammatory processes. Also, in studies
where chemokines have been injected (as above), truly enormous quantities must be
injected to see functional effects. Preformed TNF, however is present in very small
quantities, so it is unlikely that simple soluble trafficking in lymph can account for its
effects.
3.1.4 Mechanisms Underlying Lymph Node Enlargement During Immune Responses
Changes in lymph node size are primarily referable to accumulation of naive
lymphocytes. Numbers of other cell types change also, but because naive lymphocytes
account for the vast majority of lymph node mass, those changes are dominant. There
are two possibilities for effecting the accumulation of something: one can get more of it
or one can lose less of it. As lymphocytes are continuously shuttling through the node
(as described briefly above), both are options, and in fact it appears that both are
operative.
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A certain proportion of bloodborne lymphocytes will adhere and cross the high
endothelium and enter the lymph node. This process is functionally analogous to the
transmigration of neutrophils and other cells across inflamed blood endothelium, but
with different molecules. Interactions between PNAd and L-selectin mediate rolling,
binding of LFA-1 to ICAM-1 and VLA-4 to VCAM-1 is enhanced by luminal expression
of chemokines, CCL19/21 for T cells and CXCL12 and CXCL13 for B cells (318). The
expression of each of these molecules by HEVs could potentially be modulated in
inflammation to affect changes in the proportion of passing lymphocytes that enter the
node. One study demonstrated, for example, that HEV expression of CCL21 and ICAM-
1 could be upregulated by fever-range hyperthermia (224). The report describing mast
cell-derived TNF-dependent lymph node enlargement observed an associated increase
in LN VCAM-1 levels (225). As TNF upregulates the expression of a variety of adhesion
molecules in the blood endothelium during inflammation, it is conceivable that it has
parallel effects on high endothelial cells.
Inhibition of lymphocyte egress from lymph nodes also appears to contribute to
lymph node enlargement. A study in sheep examining lymphocyte numbers in efferent
lymph observed a significant drop early in responses to various stimuli with near-
simultaneous increases in recruitment (319). The mechanisms behind this remained
obscure until the discovery of a drug, FTY720, a S1P1 sphingosine-1-phosphate receptor
agonist, a drug that causes lymphopenia by preventing lymphocyte egress from
peripheral lymphoid tissues (320). The exact mechanism remains a little unclear, as
agonism at the receptor is required for egress, but excess agonism appears to abrogate it
(321).
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As mast cells do generate extracellular S1P after activation, it is possible that this
is part of how mast cell-dependent lymph node enlargement is effected (322), but more
study will be required to address this hypothesis.
3.2 Materials and Methods
3.2.1 Animals
All experiments involving wild-type mice were done with C57BL/6 mice
(obtained from the NCI animal production facility), usually females 6-8 weeks old. For a
few experiments, KitW-sh/W-sh mast cell-deficient mice (obtained originally from Jackson
Laboratories, then bred at Duke) were used. These mice are on the C57BL/6
background. For experiments involving rats, outbred, male Sprague-Dawley rats
(obtained from Taconic) of various ages were used. All animals were housed under
standard conditions in Duke University facilities.
3.2.1.1 Intraperitoneal Injections
Mice were injected with doses of 5 - 32 μg of compound 48/80 in a volume of 0.1
ml PBS intraperitoneally. After time periods ranging up to 6 hours, they were sacrificed
by CO2 asphyxiation and peritoneal lavage was obtained for downstream analysis.
Rats were injected with a dose of 0.5 mg compound 48/80 in a volume of 10 ml
PBS intraperitoneally. The large volume was required to degranulate resident mast cells
of the thin mesentery. After 30 minutes, the animals were sacrificed by CO2
asphyxiation and peritoneal lavage was collected.
3.2.1.2 Footpad Injections
For all footpad injections, 32 μg compound 48/80, fluorescent microspheres,
purified mast cell-derived particles, or heparin/chitosan microparticles were injected in
a volume of 0.02 ml PBS or normal saline. After the duration of the experiment (up to 24
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hours), the animals were sacrificed as above and popliteal lymph nodes and footpads
collected for analysis. For popliteal lymph node weights, nodes were dissected out and
immediately weighed wet on a highly sensitive scale. Weights were recorded once the
reading had equilibrated and was declining slowly (due to evaporative water loss).
3.2.1.3 Tail Injections
Mice were anesthetized with ketamine/xylazine intraperitoneally and then
injected subcutaneously near the tip of the tail with fluorescent microspheres suspended
in PBS in a volume of 0.01 ml. After 30 minutes the animals were sacrificed and the tails
examined on an epifluorescent and confocal microscope in whole mount.
3.2.1.4 Epicutaneous PMA application
For in vivo tracking studies, 10 μg PMA (Sigma) in acetone (1 μg/μl) or acetone
alone were applied in two sequential treatments to the footpads of mice anesthetized
with pentobarbital. Footpads were allowed to dry completely between applications.
DLNs and footpads were harvested after 2 hours.
3.2.1.5 Mast Cell Reconstitution
For experiments requiring the reconstitution of KitW-sh/W-sh mice with TNF-GFP or
mMCP5-GFP expressing BMMCs, the cells were produced as discussed in Chapter 2.
106 cells were injected into mouse footpads in a volume of 0.02 ml PBS. 5 weeks later,
animals were considered reconstituted and were injected with compound 48/80 or PBS
in the footpad and then sacrificed for tissue harvest.
3.2.2 Flow Cytometry
For immunophenotyping of mast cell-derived particle-internalizing cells,
peritoneal cells were collected by lavage in cold RPMI 1640 cell culture media
supplemented with 10% FBS. These were blocked in this solution for one hour on ice.
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Then an anti-CD11b mAb (clone M1/70, BD 550993) conjugated to PerCP-Cy5.5 was
added at a dilution of 1:200, and the cells were incubated on ice for one hour. Cells were
washed in cold PBS and centrifuged at 500 x g for 10 minutes at 4° C. They were then
resuspended in 0.5 ml of 4% paraformaldehyde and incubated for one hour on ice. Then
cells were washed in PBS and recentrifuged. They were resuspended then in the
blocking buffer as above containing 0.05% saponin for permeabilization. After 30
minutes of permeabilization, AlexaFluor488-conjugated avidin (Invitrogen A-21370) was
added at a dilution of 1:2000. Labeling was carried out for one hour, and then cells were
recentrifuged and finally reuspsended in PBS with 1% BSA before flow cytometry was
performed.
3.2.3 Microscopy
Mouse mesentery whole mounts were prepared as for rats described in Chapter
2, except that for staining they were fixed for a few seconds in methanol, then rapidly
stained with methylene blue/azure A and eosin.
LYVE-1 immunohistochemistry was performed as followed. Paraffin embedded
sections were deparaffinized and brought through graded alcohols to distilled water.
Then they were washed two times 5 minutes in TBS + 0.025% Triton X-100. Blocking
was done in TBS + 10% FBS + 1% BSA for 2 hours at room temperature. The primary
antibody was an anti-LYVE-1 rabbit pAb (Abcam ab14917) and was used at a dilution of
1:100 in the blocking solution. Slides were washed twice for 5 minutes each with TBS +
0.025% Triton X-100 before placing them in 0.3% H2O2 for 15 minutes at room
temperature to quench endogenous peroxidases. Then a secondary goat anti-rabbit
conjugated to HRP (Bio-Rad 170-6515) was added at a dilution of 1:1000 in TBS + 1%
BSA for one hour at room temperature. After three washes of 5 minutes each in TBS,
DAB substrate was added for 10 minutes at room temperature, after which slides were
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washed in tap water for 5 minutes. Then slides were counterstained with 0.1% toluidine
blue pH 2 for 2 minutes before dehydration and mounting in Permount.
3D deconvolution of confocal volumes was achieved using AutoQuant X
(AutoQuant Imaging, Inc.). 3D rendering of confocal volumes was done in AutoQuant
X or Volocity (improvision). The enhanced depth of field brightfield micrograph was
constructed from an image stack in ImageJ (NIH) using the Extended Depth of Field
plugin (Alan Hadley). Enhanced contrast toluidine blue image created by selecting
metachromatic (purple) areas from the image in GIMP (GIMP Development Team)
using the select-by-color tool and subtracting all other areas.
3.2.4 Encapsulation of TNF in Heparin/Chitosan Microparticles
In order to load particles with TNF, 5 ng recombinant murine TNF was vortexed
for 10 minutes in 1.25 ml 1% heparin before the stepwise addition of 1% chitosan
solution as described in Section 2.2.8.
3.3 Results
3.3.1 Local Fates of Mast Cell-Derived Particles
As it has been previously reported that mast cell-derived particles are
phagocytosed by nearby cells after their release, this was investigated as an alternate fate
to entering lymphatics and trafficking to local secondary lymphoid tissues. First, the
fate of granules released by free peritoneal mast cells was evaluated. It was found in the
mouse that after injection of a mast cell activator such as compound 48/80, granules
were rapidly taken up by other free peritoneal cells, apparently mostly macrophages.
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Figure 25: Toluidine blue-stained mouse peritoneal lavage 1 hour after IP injection of compound 48/80
A flow cytometric analysis of peritoneal lavage cells after IP compound 48/80
treatment revealed that it is in fact CD11bhi macrophages that are responsible for most of
the phagocytosis of extracellular granules (Fig. 26).
Figure 26: CD11bhi peritoneal macrophages take up released mast cell granules
Red solid curve represents intracellular avidin-FITC staining of peritoneal macrophages (gated on CD11bhi cells) from a PBS-injected mouse. Black and yellow lines show avidin-FITC staining of peritoneal macrophages from two mice each injected with compound 48/80.
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A time course of granule fate was performed using compound 48/80-treated rat
peritoneal lavage cells ex vivo (Fig. 27). A large number of particles was released within
seconds of treatment, and these are much larger and less distinct that native, intact
granules. They gradually adhered to other lavage cells (probably also macrophages)
and were beginning to be internalized by 10 minutes. Once internalized, the granules
become much smaller and more darkly staining, appearing much like native granules in
mast cells. By 24 hours, viability in the culture diminishes (only 80% by trypan blue
exclusion). But even after 24 hours, granules can clearly be discerned in the cytoplasm
of presumed macrophages, although they are now much smaller, suggesting that some
degradation has occurred.
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Figure 27: Kinetics of mast cell granule uptake and degradation by peritoneal macrophages
Duration of treatment: (A) 1 minute, (B) 5 minutes, (C) 10 minutes, (D) 45 minutes, (E) 12 hours, (F) 24 hours Serosal cavities are probably not representative of most connective tissues, which
ordinarily present much more formidable barriers to the movement of particles, due to
the high concentration of extracellular matrix molecules. Therefore, mast cell-derived
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particle phagocytosis was also examined in more typical tissues, including mouse
mesentery and footpad (Fig. 28). As early as 2.5 hours after activation in thin mouse
mesentery, no extracellular granules are evident in the tissue, those that remain in the
tissue having been internalized by fibroblasts. Six hours after activation in mouse
footpad, the majority of extracellular granules have been gathered into groups, also
suggesting internalization (compare the picture at 30 minutes, Fig. 5).
Figure 28: Phagocytosis of mast cell-derived particles in tissues
(A) Whole mounted mouse mesentery 2.5 hrs after IP injection of compound 48/80, stained with methylene blue/azure A and eosin. (B) Low magnification confocal micrograph of mouse footpad 6 hrs after compound 48/80 injection. Green: heparin labeled with avidin, showing the locations of extracellular and intracellular granules. Red: c-kit labeling, showing the position of mast cell membranes.
These data confirm the findings of others that released mast cell granules can be
removed from the tissue by nearby cells, including macrophages and fibroblasts. The
significance of this is unknown, although as discussed previously it might have
modulatory effects on the cells taking up the particles, or it may serve as a mechanism
for regulating the levels of heparin-binding molecules in tissues. Finally, it may be
advantageous to limit the duration of local effects for granule-associated mediators.
Although many particles are clearly removed by phagocytosis, this finding does not
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imply that this is the fate of all. In fact, it does not account for the possibility that some
particles may be leaving the tissue entirely, as will be shown below.
3.3.2 Mast Cell-Derived Particles Can Enter Initial Lymphatics
Given the anatomy of connective tissues, there are three potential fates for
particles the size of mast cell granules. First, they could remain in the extracellular
space, but this appears not to be the case outside of the first hour or so. Second, they
may be removed from the extracellular space by phagocytes, and this clearly does occur.
Finally, they may enter initial lymphatic vessels, which as discussed above, are highly
permeable. Especially in inflammatory situations where edema is present,
discontinuities between adjacent endothelial cells should easily admit particles the size
of released granules. This possibility has not been investigated previously.
Initially, the fate of exogenous trackable particles was investigated. Using the
mouse tail, which has a superficial hexagonal lymphatic network that is relatively easy
to image in whole mount (323), 1 μm, 0.5 μm, and 0.1 μm diameter fluorescent
polystyrene microspheres were injected subcutaneously. 30 minutes later, all three
populations of microspheres could be visualized within the lymphatic network (Fig 29).
Figure 29: Fluorescent microspheres inside superficial lymphatic vessels of mouse tail
(A) Green fluorescent 1 μm diameter microspheres. (B) Red fluorescent 0.5 μm diameter microspheres. (C) Blue fluorescent 0.1 μm diameter microspheres (difficult to see on background of tissue autofluorescence).
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Experiments directly examining interactions between mast cells and initial
lymphatics were initially carried out in the rat mesentery, a tissue containing a very high
density of mast cells. The tissue can also be examined in whole mount, enabling the
entire connective tissue drainage to be visualized. Finally, although not all mesenteric
"windows" (the translucent, extremely thin areas between the gut and vascular bundles)
contain lymphatic vessels, some possess extensive networks (see Figure 30).
Figure 30: Network of initial lymphatic vessels in rat mesentery windows
Green: lymphatic vessels (LYVE-1), Blue: mast cells (heparin) -- inset: higher magnification view showing proximity of mast cells to lymphatic vessels.
Although mesentery-resident mast cells are separated from the peritoneal space
by a layer of mesothelium, they can be activated by an intraperitoneal injection of
compound 48/80. In fact, very dramatic degranulation is evident 30 minutes after such
a treatment. Widespread colocalization between mast cell-derived particles and initial
lymphatics occurred (Fig. 31).
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Figure 31: Colocalization between mast cell-derived particles and initial lymphatics after intraperitoneal compound 48/80 instillation
(A) Confocal micrograph of LYVE-1 staining, showing the position of lymphatic vessels. (B) Avidin staining, showing mast cells and extracellular particles. (C) Merge. (D) Image showing only areas of signal colocalization.
In several areas, granules were clearly inside vessels, although this could not be
proven definitively in these preparations as the vessels are collapsed during processing.
Such an area is shown in Figure 32.
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Figure 32: Mast cell-derived particles inside an initial lymphatic vessel in rat mesentery
(A) Confocal micrograph of LYVE-1 staining for lymphatics (B) Heparin staining (avidin) showing mast cell particles. (C) Merge.
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Because it was difficult to determine the topographical relationship between
particles and lymphatic vessels in mesentery whole mounts, cross sections were
examined. This proved quite difficult, as the mesentery collapses on itself when
removed from its attaching structures. Also, only a few areas contain lymphatic vessels,
and identifying these before tissue processing for sectioning is difficult. To identify
these areas, mesentery windows were cut in half and one half was processed for LYVE-1
and avidin staining in whole mount, and the other half for sectioning. Tissue blocks
matching a half found to have lymphatics in whole mount were then prepared for
sectioning. Immunohistochemistry for LYVE-1 combined with toluidine blue
counterstaining revealed both mast cells and lymphatics in sections (Fig. 33). Very few
patent vessels were found, but the experiment did suggest that lymphatic vessels fill the
vast majority of the space between mesothelial layers, suggesting that particles
colocalizing with vessels are indeed inside those vessels.
Figure 33: Cross section of rat mesenteric lymphatic vessel demonstrated by LYVE-1 IHC counterstained with toluidine blue
Brown areas indicate deposition of peroxidase reaction product. Contrast has been enhanced so that the unstained borders of the upper loop of mesentery are visible. The mast cell in this image (below the right hand end of the open lymphatic) is in another section of mesentery that is closely apposed to the one with the lymphatic.
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Peripheral initial lymphatics in the mouse footpad after compound 48/80
injection were examined to identify patent vessels near mast cells. In the footpad,
edema develops rapidly after compound 48/80 instillation, while in the mesentery this
does not really occur, as the tissue is not very expandable and most areas are not served
by nearby blood vessels. Dilated lymphatics were observed in this tissue, and they were
frequently near to degranulating mast cells. A micrograph of one such vessel containing
a clearly intraluminal mast cell-derived particle is shown (Fig. 34).
Figure 34: A mast cell-derived particle in the lumen of an initial lymphatic vessel
(A) Confocal micrograph of a lymphatic vessel cross-section (green: LYVE-1) with an adjacent degranulating mast cell (blue: heparin). (B) 3D isosurface reconstruction of confocal volume shown in (A) demonstrating luminal particle. (C) and (D) Another angle of view with (C) and without (D) LYVE-1 signal showing no significant overlap and showing that intraluminal particle is in fact an isolated particle. Scale: 8 μm.
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These studies clearly demonstrate that mast cell-derived particles can enter
peripheral lymphatic vessels. Although it would appear that this is the fate of a
minority of particles (the majority of which are phagocytosed in the local tissue), the
transport of small quantities of potent signals to draining lymphoid tissue can have very
significant effects.
3.3.3 Mast Cell-Derived Particles Can Traffic to Local Lymph Nodes
Experiments on the ability of peripheral particles to reach draining lymph nodes
were performed using exogenous fluorescent microspheres. These were injected into
the mouse footpad and after 30 minutes, the popliteal lymph node was evaluated for the
presence of microspheres. It was reasoned that 30 minutes was not enough time for
microspheres to be actively carried to the lymph node by phagocytes, and so their
presence would be proof that they had entered the vessels without assistance and had
drained there as independent objects. Migration of cells into lymphatics is an active
process that requires complicated maneuvers, including directed migration and crossing
the lymphatic endothelium. This entails changes in transcription. Time is also required
for phagocytosis. The microspheres used are so bright that they can easily be
appreciated with the lymph node in whole mount on a slide. Although, clearly, this
permits only a limited focal plane, it gives some sense for the distribution of particles in
the lymph node subcapsular sinus (where afferent lymph enters the node). These data
are shown in Figure 35. As can easily be appreciated from these images, particles
entering the lymph node are widely distributed in the subcapsular sinus, suggesting that
even after they have entered they are able to cover significant distances. Lymph nodes
draining the site of a PBS injection did not display fluorescence (not shown).
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Figure 35: Peripherally injected fluorescent microspheres rapidly appear in the draining lymph node
(A) 1 μm diameter fluorescent microspheres. (B) 0.5 μm diameter fluorescent microspheres. (C) 0.1 μm diameter fluorescent microspheres.
As mast cell-derived particles are readily identified in tissues, popliteal lymph
nodes were also examined after the footpad injection of compound 48/80. Indeed, many
free particles were observed in the subcapsular sinus of lymph nodes after this treatment
(Fig. 36). Unfortunately, the origin of the particles identified in these studies could not
be ascertained with certainty, because no dose of compound 48/80 could be found that
activated footpad mast cells without also activating subcapsular sinus-resident mast
cells.
This problem was later addressed by the use of epicutaneous application of
PMA, a treatment known to cause dermal mast cell degranulation a (64). As PMA is a
lipid soluble substance, it was reasoned that it should not reach lymph nodes in
appreciable quantities. Indeed, when popliteal lymph nodes were examined after this
treatment, no detectable mast cell degranulation had occurred (Fig. 37), whereas it was
dramatic after compound 48/80 injection. After this treatment, too, mast cell-derived
particles could be observed in the draining lymph node.
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Figure 36: Mast cell-derived particles in the subcapsular sinus of the murine popliteal lymph node after footpad injection of compound 48/80
(A) Popliteal lymph node section stained with acidic toluidine blue. Background staining of nuclei and structural elements is blue, while staining of mast cell-derived particles is purple due to heparin metachromasia. This image was made using a stack of images at slightly different focal planes to extend the depth of field. (B) Same image, but only metachromatic areas are shown in black. Scale; 30 μm.
121
Figure 37: Mast cell-derived particles in popliteal lymph node after epicutaneous footpad PMA application
Toludine blue-stained sections of DLN tissue 2 hours after (A) injection with 32 μg compound 48/80 (scale: 50 μm for A-D), (B) topical application of vehicle alone, or (C) topical application of 10 μg PMA. (D) and inset (E) Free metachromatic granules could be visualized within the DLNs of PMA-treated footpads; scale: 20 μm. In another attempt to circumvent the problem of lymph node-resident mast cells,
local footpad mast cell reconstitution of mast cell deficient KitW-sh/W-sh mice was explored.
BMMCs expressing the TNF-GFP or mMCP5-GFP fusion protein were used for the
reconstitution, so that in addition to demonstrating the movement of the particles, the
movement of the fusion protein could also be assessed. mMCP5 is a mast cell chymase
that is an abundant, granule-associated protease (homologous to RMCP-5) that was
intended as a positive control for granule association. These BMMCs were injected into
the footpads of KitW-sh/W-sh mice, and five weeks later they were injected with compound
48/80 or PBS, after which the popliteal lymph nodes were assessed for the presence of
particles and the fusion protein. Unfortunately, the reconstitution was not very
effective, except in the lymph node itself, which had an apparently normal complement
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of resident mast cells. Detection of the fusion protein could also not be detected. Only
one LN draining a PBS injection was sectioned (the other nodes were homogenized for
fusion protein detection). Interestingly, there were enormous numbers of apparently
extracellular granules in these lymph nodes, despite the fact that no activating stimulus
was given (Fig. 38). The significance of this is unknown, although, speculatively,
perhaps the initial reconstitution bolus of cells sensitized the animals to GFP and this is
the outcome of the ongoing response against the grafting cells.
Figure 38: Popliteal lymph node of a mast cell deficient mouse footpad-reconstituted with mMCP5-GFP expressing BMMCs and injected with PBS
This section was darkly stained with toluidine blue. The reddish-brown coloring is probably hemosiderin deposition from bleeding that occurred either during the initial reconstitution or in the PBS injection.
Whether or not footpad-injected mast cell-derived particles could be detected in
popliteal lymph nodes was also examined. To avoid the resident mast cell problem,
mast cell-deficient mice were used again. Although this experiment was attempted
several times using particles purified from rat peritoneal mast cells, particles could not
be identified in the draining lymph node. This may be due to the use of compound
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48/80 in the purification of rat mast cell-derived particles. When particles of mouse
origin were purified using ionomycin as the secretagogue, however, they were
detectable in the popliteal lymph node after footpad injection (Fig. 39).
Figure 39: Presence of purified mouse mast cell-derived particles in popliteal lymph node after footpad injection
(A) Toluidine blue staining of DLN tissue sections from a sash mouse injected with isolated MC-derived particles; scale: 50 μm. (B) Same area as in A) showing only metachromatic staining. The location of the edge of the DLN is depicted by a dashed line. (C) Inset from A; scale: 10 μm.
Because heparin/chitosan microparticles were used in later experiments on the
functional aspects of mast cell-derived particles, it was necessary to demonstrate that
these also could reach the draining lymph node after peripheral application.
Microparticles were labeled with FITC-conjugated polylysine by adding a small quantity
of FITC-PLL to the heparin solution before combining it with chitosan. After injection,
they could easily be detected in the popliteal lymph node (Fig. 40).
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Figure 40: Heparin/chitosan microparticles in the popliteal lymph node 30 minutes after footpad injection
Green: FITC-polylysine labeled heparin/chitosan microparticles. Blue: LYVE-1 staining showing the lymph node sinus system.
3.3.4 Mast Cell-Derived Particles and Their Associated TNF Mediate Lymph Node Enlargement
At this point, it has been demonstrated both that extracellular mast cell granules
retain preformed TNF and that these particles can reach the draining lymph node from
peripheral sites of inflammation. As it is known that mast cell-derived TNF is required
for mast cell-induced hypertrophy (225), it remains only to show causation between
these correlated processes.
Although it has already been shown that peripheral mast cell degranulation
leads to lymph node enlargement (225), it is unknown whether or not mast cell-derived
particles by themselves, in the absence of soluble mediators can mediate this effect. To
investigate this, particles were purified from rat peritoneal mast cells and injected into
murine footpad. Twenty-four hours later, significant popliteal lymph node enlargement
was observed (Fig. 41).
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Figure 41: Popliteal lymph node enlargement twenty-four hours after footpad injection of purified rat mast cell-derived particles
n=7, p=0.03 by two-tailed, paired t test.
To demonstrate that TNF is the critical principle in mast cell-derived particles
determining lymph enlargement, particles were purified from wild-type and Tnf-/- mice.
These were then injected into the footpads of mice and their effects on lymph node size
evaluated at twenty-four hours. As shown in Figure 42, only particles derived from
wild-type mice could elicit lymph node enlargement, strongly suggesting that TNF is the
required factor for this activity.
In order to exclude the possibility that there are meaningful differences between
wild-type and TNF knockout mast cell-derived particles other than lack of TNF that
might account for the differences observed in the previous experiment, lymph node
enlargement experiments were also carried out using TNF-loaded heparin-based
microparticles. Initial experiments using heparin/polylysine microparticles were
abandoned after it was discovered that these particles could cause lymph node
enlargement without TNF. This is perhaps unsurprising, as polylysine (like many
126
polycations) is known to degranulate mast cells and probably elicits inflammation on its
own (24).
Figure 42: Mast cell-derived particles purified from wild-type mice elicit popliteal lymph node enlargement after footpad injection while those from TNF
knockout mice do not
n=3 for each group, p=0.004 by two-tailed, unpaired t test.
Chitosan, on the other hand, is a favored material for drug delivery and other in
vivo applications, as it is very biocompatible and is not inflammatory. So, TNF-loaded
heparin/chitosan microparticles were used. As the actual amount of TNF encapsulated
in the particles could not be ascertained for technical reasons (although this should be
possible), the dose was determined assuming 100% encapsulation. Other studies using
model cargo molecules and rTNF suggest that encapsulation is almost never more than
30% using the procedures described here (not shown). As shown in Figure 43,
heparin/chitosan microparticles carrying no more than 1 ng TNF elicited much greater
levels of lymph node enlargement than 10 ng of soluble TNF. Furthermore, empty
particles did not cause hypertrophy, and mixing soluble TNF with soluble heparin and
chitosan (at neutral pH no particles form) did not enhance its potency. This implies that
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its delivery in particle form in particular (and not simply a combinatorial effect with
heparin or chitosan) that mediates the greater than ten-fold increase in potency
achieved.
Figure 43: TNF delivered inside heparin/chitosan microparticles elicits more lymph node hypertrophy than soluble TNF
Less than 1 ng rTNF encapsulated in particles similar to MC granule particles elicits more LN enlargement than 10 ng of soluble protein when injected into the footpads of mice (black bars: particles with and without encapsulated TNF, open bars: soluble TNF, hatched bar: soluble TNF mixed with soluble chitosan and heparin, n=3 for each group, * indicates p<0.05 for comparison vs. 10 ng soluble TNF, ** indicates p<0.01 for comparisons vs. particles without TNF, 1 ng soluble TNF, and 1 ng soluble TNF mixed with soluble chitosan and heparin, data analyzed by one-way analysis of variance followed by Tukey's Multiple Comparisons test).
3.4 Discussion
In this chapter, evidence has been presented discussing the fate of mast cell-
derived particles after degranulation and their role in mediating lymph node
enlargement during inflammation. The release of insoluble particles from the granules
of mast cells is both morphologically striking and perplexing. Heparin was the
component by which mast cells were discovered, due to its unusual interactions with
128
basic dyes (1). The basis of this interaction is also the property that enables the
structural integrity of mast cell-derived particles: its extremely high negative charge
density. Although specific structural motifs govern heparin's interaction with certain
proteins (such as antithrombin III), its charge also contributes to many of its protein
interactions. Interestingly, many proteins heparin interacts with are also important
extracellular signaling molecules, such as cytokines and growth factors. The physiologic
significance of this fact is unknown. The reason for this is that, in general, heparin has
only been used as a convenient tool to investigate the interactions of proteins with
heparan sulfate. These are also certainly important, as heparan sulfate is ubiquitous in
mammalian biology. But heparin proteoglycans are found throughout the phylum
Chordata, even if they are stored and produced only by one cell type, mast cells. It is
rapidly becoming clear, however, that this cell type is physiologically important in host
responses to infection and injury. Given heparin's interaction with so many signaling
molecules, and the importance of said molecules in inflammatory responses, it is
surprising that this area has not received more attention.
The work described herein contributes to the existing body of work concerning
the local fates of heparin-based mast cell granules, in that they are avidly internalized by
a variety of cell types. This is very likely to have physiologic significance both in the
removal of injurious substances from tissues (many of which are cationic and
presumably would bind to heparin) and in the regulation of host-derived heparin-
binding molecules.
In addition to this fate, which does appear to be the destiny of the great majority
of mast cell-derived particles, the evidence presented in this chapter demonstrates that
such particles can also enter initial lymphatic vessels and are then carried to and
deposited in the local draining lymph node. Together with the work presented in
129
Chapter 2 showing that these particles contain TNF, these experiments suggest that mast
cell-derived particles do, in fact, carry the cytokine from the site of their release in the
periphery to the draining lymph node.
The biological relevance of this phenomenon was shown by experiments
indicating that this mechanism for transporting TNF to the draining lymph node does
enhance lymph node enlargement. Granules obtained from TNF knockout mice did not
elicit lymph node changes, while these changes were impressive after administration of
wild-type granules. The specific importance of "particulate-ness" was demonstrated by
directly comparing the efficacy of soluble TNF to particle-based TNF, which was shown
to be over ten times more potent for eliciting LN hypertrophy.
The mechanistic details of what exactly happens when mast cell-derived particles
arrive in the node remain to be explored. Does TNF act directly on the high endothelial
venule to enhance lymphocyte recruitment? This would seem the simplest explanation,
and previous experiments have shown that TNF enhances LN VCAM-1 expression,
which contributes to the recruitment of lymphocytes (225). While synthetic particles
certainly elute TNF, it is not known whether natural particles behave similarly. Also, it
is possible that the action of extracellular heparanases may enhance this process.
Whether or not egress is affected by mast cells is also unknown. It is known that
mast cells express sphingosine kinases and that extracellular sphingosine-1-phosphate is
generated after activation (322). It is tempting to speculate that granule-associated
sphingosine kinase might bring this process directly to the lymph node. As the granule
is deposited in the subcapsular sinus initially, such S1P would be in a good position to
shut down lymphocyte exit routes.
These questions await experimental treatment in the future. This work
establishes that this highly novel form of communication between peripheral sites of
130
inflammation and local secondary lymphoid tissue occurs and that it is of biological
relevance.
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4. Conclusions and Discussion
In conclusion, the existence of a novel form of extracellular communication over
long distances has been demonstrated. The use of the circulatory system to deliver
extracellular signals is well known, as endocrine signaling involves the transport of
hormones from their secreting cells to distant targets. However, this has mainly been
studied in the context of signals that reach easily measurable concentrations. Such
hormones, for example insulin, are secreted in great quantities by large populations of
cells devoted to that purpose. This kind of signaling is relatively untargeted, insofar as
sufficient concentrations are reached in the circulation that the soluble mediator can be
detected easily by most cells in the body, or at least by a receptor-bearing subset.
Endocrine signaling is also important during immunologic responses, take for example
the induction of the systemic acute phase response following initiation of inflammation
(324).
This work suggests that there may also exist a much less obvious, but equally
important, parallel secondary system of long distance inflammatory communication
earlier in responses, involving much lower concentration of signals. Because most
inflammatory signals are not very long-lived, it is reasonable to expect that adaptations
must have developed in order to a) prolong the survival of extracellular signals, and b)
ensure their delivery to specific targets. This kind of adaptation is already well known
for many blood-borne endocrine signals whose solubility and targeting are regulated by
serum carrier proteins (325, 326). The delivery of TNF to draining lymph nodes during
inflammation by mast cell-derived particles appears to provide an example of such an
adaptation. As opposed to the kind of untargeted signaling discussed above, mast cell-
derived particles seem to be specialized for the delivery of very small quantities of cargo
from one specific site (the site of inflammation) to another specific site (targets in the
132
draining lymph node that can mediate changes favoring the development of adaptive
immunity). Heparin, the predominant component of mast cell particles, has the highest
negative charge density of any molecule known in mammalian biology, which likely
minimizes interaction with adjacent cells and other structures in the extracellular
environment, this charge favoring their movement into the lymphatic drainage (327).
On the other hand, the size of extracellular granules (while likely retarding their
movement through peripheral tissues) would also prevent them from moving through
the lymph node to the efferent lymph, resulting in their sequestration in this target
organ (328). This scenario is favored by our finding that packaging TNF in similar
heparin-based synthetic particles greatly enhances its potency relative to soluble TNF.
This work also makes important contributions to the basic fund of knowledge on
mast cells. It is the opinion of this author that science as an enterprise has far too much
disdain for descriptive science, which is the foundation of all science. This is not
specifically a defense of this work, although clearly it has some descriptive aspects. The
assumption, made by many, that the time for mere observation is past, is incorrect as
new techniques are constantly being developed that permit more exact depictions of
biological structures and processes than were possible previously. The human genome
project is a good example, and there are many more techniques that need to be applied
to description in a comprehensive manner in order to more firmly establish the anatomy
of biology. Although this work only inches towards this goal, high quality proteomic
descriptions of every cell type should be available. A little more information on what
mast cells are made of, nevertheless, is a step in the right direction.
Synchronization and collaboration between the periphery and the draining
secondary lymphoid tissue is crucial for a rapid and effective host response to infection.
DLN enlargement alone can shorten the time between pathogen entry and antigen
133
presentation by increasing the chances that the extremely rare circulating lymphocytes
carrying receptors for antigens specific to the infection are present. The delivery of
inflammatory signals by this unusual mechanism may help to explain how this MC-
dependent process occurs so rapidly given the small amount of prestored cytokine
available. It is also possible that particle-associated molecules other than TNF might be
important in aspects of lymph node remodeling; MC proteases are clearly present in
large quantities, and other low abundance signaling molecules may also be present. For
example, there is some evidence that MCs also prestore fibroblast growth factor-2 (42)
and vascular endothelial growth factor (329), two well known heparin-binding growth
factors, which could be involved in the regulation of lymph node vascular remodeling
during immune responses.
MCs reside at the crossroads between the external environment, the blood
vasculature, and the lymphatic system. They are ideally situated to orchestrate events
during the earliest stages of immune responses, and it seems likely that this is their
physiologic role in mammalian biology. In fact, their proximity to these critical
structures suggests that they influence them directly in many ways in addition to those
that have already been described. This work indicates one way in which these
important cells may also influence events in distant lymphoid tissues.
Finally, many other immune cells including neutrophils (330), eosinophils (331),
basophils (332), and cytotoxic T lymphocytes (333) store biologically active compounds
in granules. The granular matrix in these cell types is comprised of serglycin
proteoglycans which, in addition to their role in storage, can contribute to the
extracellular function of the bound mediators (334). To date, only the local effects of
such complexes have been investigated. Our studies suggest that they may also be
specialized to deliver their cargo of signaling proteins to far more distant targets.
134
References
1. Ehrlich, P. 1877. Beitraege zur Kenntniss der Anilinfaerbungen und ihrer Verwendung in der mikroskopischen Technik. Arch. mikr. anat. u. entwcklngsmech. 13:263.
2. de Barros, C.M., Andrade, L.R., Allodi, S., Viskov, C., Mourier, P.A., Cavalcante,
M.C., Straus, A.H., Takahashi, H.K., Pomin, V.H., Carvalho, V.F., et al. 2007. The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma. J Biol Chem 282:1615-1626.
3. Matsuda, H., Watanabe, N., Kiso, Y., Hirota, S., Ushio, H., Kannan, Y., Azuma,
M., Koyama, H., and Kitamura, Y. 1990. Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice. J Immunol 144:259-262.
4. Lantz, C.S., Boesiger, J., Song, C.H., Mach, N., Kobayashi, T., Mulligan, R.C.,
Nawa, Y., Dranoff, G., and Galli, S.J. 1998. Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 392:90-93.
5. R.C. Wagner, F.E.H. Transmission electron micrograph of a mast cell adjacent to
a venule and arteriole. 6. Katz, H.R., Raizman, M.B., Gartner, C.S., Scott, H.C., Benson, A.C., and Austen,
K.F. 1992. Secretory granule mediator release and generation of oxidative metabolites of arachidonic acid via Fc-IgG receptor bridging in mouse mast cells. J Immunol 148:868-871.
7. Latour, S., Bonnerot, C., Fridman, W.H., and Daeron, M. 1992. Induction of
tumor necrosis factor-alpha production by mast cells via Fc gamma R. Role of the Fc gamma RIII gamma subunit. J Immunol 149:2155-2162.
8. Silva, M.R.E., Bier, O., and Aronson, M. 1951. Histamine Release by
Anaphylatoxin. Nature 168:465-466. 9. Dias Da Silva, W., and Lepow, I.H. 1967. Complement as a mediator of
inflammation. II. Biological properties of anaphylatoxin prepared with purified components of human complement. J Exp Med 125:921-946.
10. Gorski, J.P., Hugli, T.E., and Muller-Eberhard, H.J. 1979. C4a: the third
anaphylatoxin of the human complement system. Proc Natl Acad Sci U S A 76:5299-5302.
11. Alam, R., Welter, J.B., Forsythe, P.A., Lett-Brown, M.A., and Grant, J.A. 1989.
Comparative effect of recombinant IL-1, -2, -3, -4, and -6, IFN-gamma, granulocyte-macrophage-colony-stimulating factor, tumor necrosis factor-alpha, and histamine-releasing factors on the secretion of histamine from basophils. J Immunol 142:3431-3435.
135
12. Taylor, A.M., Galli, S.J., and Coleman, J.W. 1995. Stem-cell factor, the kit ligand, induces direct degranulation of rat peritoneal mast cells in vitro and in vivo: dependence of the in vitro effect on period of culture and comparisons of stem-cell factor with other mast cell-activating agents. Immunology 86:427-433.
13. Subramanian, N., and Bray, M.A. 1987. Interleukin 1 releases histamine from
human basophils and mast cells in vitro. J Immunol 138:271-275. 14. Jasani, B., Kreil, G., Mackler, B.F., and Stanworth, D.R. 1979. Further studies on
the structural requirements for polypeptide-mediated histamine release from rat mast cells. Biochem J 181:623-632.
15. Hagermark, O., Hokfelt, T., and Pernow, B. 1978. Flare and itch induced by
substance P in human skin. J Invest Dermatol 71:233-235. 16. Theoharides, T.C., Betchaku, T., and Douglas, W.W. 1981. Somatostatin-induced
histamine secretion in mast cells. Characterization of the effect. Eur J Pharmacol 69:127-137.
17. Piotrowski, W., and Foreman, J.C. 1986. Some effects of calcitonin gene-related
peptide in human skin and on histamine release. Br J Dermatol 114:37-46. 18. Fjellner, B., and Hagermark, O. 1981. Studies on pruritogenic and histamine-
releasing effects of some putative peptide neurotransmitters. Acta Derm Venereol 61:245-250.
19. Kurose, M., and Saeki, K. 1981. Histamine release induced by neurotensin from
rat peritoneal mast cells. Eur J Pharmacol 76:129-136. 20. Hirai, Y., Yasuhara, T., Yoshida, H., Nakajima, T., Fujino, M., and Kitada, C. 1979.
A new mast cell degranulating peptide "mastoparan" in the venom of Vespula lewisii. Chem Pharm Bull (Tokyo) 27:1942-1944.
21. Wakamatsu, K., Okada, A., Miyazawa, T., Ohya, M., and Higashijima, T. 1992.
Membrane-bound conformation of mastoparan-X, a G-protein-activating peptide. Biochemistry 31:5654-5660.
22. Higashijima, T., Uzu, S., Nakajima, T., and Ross, E.M. 1988. Mastoparan, a
peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). J Biol Chem 263:6491-6494.
23. Ortner, M.J., and Chignell, C.F. 1981. Spectroscopic studies of rat mast cells,
mouse mastocytoma cells, and compound 48/80--III. Evidence for a protein binding site for compound 48/80. Biochem Pharmacol 30:1587-1594.
24. Padawer, J. 1970. Reaction of Rat Mast Cells to Polylysine. Journal of Cell Biology
47:352-&.
136
25. Supajatura, V., Ushio, H., Nakao, A., Akira, S., Okumura, K., Ra, C., and Ogawa, H. 2002. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest 109:1351-1359.
26. Matsushima, H., Yamada, N., Matsue, H., and Shimada, S. 2004. TLR3-, TLR7-,
and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J Immunol 173:531-541.
27. Malaviya, R., Gao, Z., Thankavel, K., van der Merwe, P.A., and Abraham, S.N.
1999. The mast cell tumor necrosis factor alpha response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48. Proc Natl Acad Sci U S A 96:8110-8115.
28. Kramer, S., Sellge, G., Lorentz, A., Krueger, D., Schemann, M., Feilhauer, K.,
Gunzer, F., and Bischoff, S.C. 2008. Selective activation of human intestinal mast cells by Escherichia coli hemolysin. J Immunol 181:1438-1445.
29. Lagunoff, D., and Rickard, A. 1983. Evidence for Control of Mast-Cell Granule
Protease Insitu by Low Ph. Experimental Cell Research 144:353-360. 30. Uvnaes, B. 1964. Release Processes in Mast Cells and Their Activation by Injury.
Ann N Y Acad Sci 116:880-890. 31. Schwartz, L.B., Riedel, C., Caulfield, J.P., Wasserman, S.I., and Austen, K.F. 1981.
Cell association of complexes of chymase, heparin proteoglycan, and protein after degranulation by rat mast cells. J Immunol 126:2071-2078.
32. Uvnas, B., Aborg, C.H., and Bergendorff, A. 1970. Storage of histamine in mast
cells. Evidence for an ionic binding of histamine to protein carboxyls in the granule heparin-protein complex. Acta Physiol Scand Suppl 336:1-26.
33. Forsberg, E., Pejler, G., Ringvall, M., Lunderius, C., Tomasini-Johansson, B.,
Kusche-Gullberg, M., Eriksson, I., Ledin, J., Hellman, L., and Kjellen, L. 1999. Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme. Nature 400:773-776.
34. Humphries, D.E., Wong, G.W., Friend, D.S., Gurish, M.F., Qiu, W.T., Huang, C.,
Sharpe, A.H., and Stevens, R.L. 1999. Heparin is essential for the storage of specific granule proteases in mast cells. Nature 400:769-772.
35. Fillion, G.M., Slorach, S.A., and Uvnas, B. 1970. The release of histamine, heparin
and granule protein from rat mast cells treated with compound 48-80 in vitro. Acta Physiol Scand 78:547-560.
36. Rohlich, P., Anderson, P., and Uvnas, B. 1971. Electron microscope observations
on compounds 48-80-induced degranulation in rat mast cells. Evidence for sequential exocytosis of storage granules. J Cell Biol 51:465-483.
137
37. McCaffrey, T.A., Falcone, D.J., and Du, B. 1992. Transforming growth factor-beta 1 is a heparin-binding protein: identification of putative heparin-binding regions and isolation of heparins with varying affinity for TGF-beta 1. J Cell Physiol 152:430-440.
38. Gospodarowicz, D., Ferrara, N., Schweigerer, L., and Neufeld, G. 1987. Structural
characterization and biological functions of fibroblast growth factor. Endocr Rev 8:95-114.
39. Senger, D.R., Perruzzi, C.A., Feder, J., and Dvorak, H.F. 1986. A highly conserved
vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res 46:5629-5632.
40. Handel, T.M., Johnson, Z., Crown, S.E., Lau, E.K., and Proudfoot, A.E. 2005.
Regulation of protein function by glycosaminoglycans--as exemplified by chemokines. Annu Rev Biochem 74:385-410.
41. Boesiger, J., Tsai, M., Maurer, M., Yamaguchi, M., Brown, L.F., Claffey, K.P.,
Dvorak, H.F., and Galli, S.J. 1998. Mast cells can secrete vascular permeability factor/ vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E-dependent upregulation of fc epsilon receptor I expression. The Journal of experimental medicine 188:1135-1145.
42. Qu, Z., Kayton, R.J., Ahmadi, P., Liebler, J.M., Powers, M.R., Planck, S.R., and
Rosenbaum, J.T. 1998. Ultrastructural immunolocalization of basic fibroblast growth factor in mast cell secretory granules. Morphological evidence for bfgf release through degranulation. J Histochem Cytochem 46:1119-1128.
43. Lantz, M., Thysell, H., Nilsson, E., and Olsson, I. 1991. On the binding of tumor
necrosis factor (TNF) to heparin and the release in vivo of the TNF-binding protein I by heparin. J Clin Invest 88:2026-2031.
44. Lantz, M., Thysell, H., Nilsson, E., and Olsson, I. 1991. On the binding of tumor
necrosis factor (TNF) to heparin and the release in vivo of the TNF-binding protein I by heparin. J Clin Invest 88: 2026-2031.
45. Le Trong, H., Neurath, H., and Woodbury, R.G. 1987. Substrate specificity of the
chymotrypsin-like protease in secretory granules isolated from rat mast cells. Proc Natl Acad Sci U S A 84:364-367.
46. Saksela, O., Moscatelli, D., Sommer, A., and Rifkin, D.B. 1988. Endothelial cell-
derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol 107:743-751.
47. Pillarisetti, S., Paka, L., Sasaki, A., Vanni-Reyes, T., Yin, B., Parthasarathy, N.,
Wagner, W.D., and Goldberg, I.J. 1997. Endothelial cell heparanase modulation of lipoprotein lipase activity. Evidence that heparan sulfate oligosaccharide is an extracellular chaperone. J Biol Chem 272:15753-15759.
138
48. Bengtsson-Olivecrona, G., and Olivecrona, T. 1985. Binding of active and inactive forms of lipoprotein lipase to heparin. Effects of pH. Biochem J 226:409-413.
49. Ellyard, J.I., Simson, L., Bezos, A., Johnston, K., Freeman, C., and Parish, C.R.
2007. Eotaxin selectively binds heparin. An interaction that protects eotaxin from proteolysis and potentiates chemotactic activity in vivo. J Biol Chem 282:15238-15247.
50. Higginbotham, R.D., Dougherty, T.F., and Jee, W.S. 1956. Fate of shed mast cell
granules. Proc Soc Exp Biol Med 92:256-261. 51. Carter, P.B., Higginbotham, R.D., and Dougherty, T.F. 1957. The local response of
tissue mast cells to antigen in sensitized mice. J Immunol 79:259-264. 52. Lindahl, U., Pertoft, H., and Seljelid, R. 1979. Uptake and degradation of mast-
cell granules by mouse peritoneal macrophages. Biochem J 182:189-193. 53. Welsh, R.A., and Geer, J.C. 1959. Phagocytosis of mast cell granule by the
eosinophilic leukocyte in the rat. Am J Pathol 35:103-111. 54. Atkins, F.M., Friedman, M.M., and Metcalfe, D.D. 1985. Biochemical and
microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells. Lab Invest 52:278-286.
55. Baggiolini, M., Horisberger, U., and Martin, U. 1982. Phagocytosis of mast cell
granules by mononuclear phagocytes, neutrophils and eosinophils during anaphylaxis. Int Arch Allergy Appl Immunol 67:219-226.
56. Miyata, K., and Takaya, K. 1985. Acid-phosphatase activity of reticular cells and
macrophages in the lymph node of the rat after ingestion of mast-cell granules. Histochemistry 83:201-205.
57. Miyata, K., and Takaya, K. 1985. Uptake of released mast cell granules by
reticular cells of the rat lymph node. Cell Tissue Res 240:49-55. 58. Kitamura, Y., Go, S., and Hatanaka, K. 1978. Decrease of mast cells in W/Wv
mice and their increase by bone marrow transplantation. Blood 52:447-452. 59. Qureshi, R., and Jakschik, B.A. 1988. The role of mast cells in thioglycollate-
induced inflammation. J Immunol 141:2090-2096. 60. Harris, R.R., Wilcox, D., Bell, R.L., and Carter, G.W. 1998. The role of tissue mast
cells in polyacrylamide gel-induced inflammation in mice. Inflamm Res 47:104-108.
61. Ikai, K., Danno, K., Horio, T., and Narumiya, S. 1985. Effect of ultraviolet
irradiation on mast cell-deficient W/Wv mice. The Journal of investigative dermatology 85:82-84.
139
62. Walsh, L.J. 1995. Ultraviolet B irradiation of skin induces mast cell degranulation and release of tumour necrosis factor-alpha. Immunol Cell Biol 73:226-233.
63. Frangogiannis, N.G., Lindsey, M.L., Michael, L.H., Youker, K.A., Bressler, R.B.,
Mendoza, L.H., Spengler, R.N., Smith, C.W., and Entman, M.L. 1998. Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 98:699-710.
64. Wershil, B.K., Murakami, T., and Galli, S.J. 1988. Mast cell-dependent
amplification of an immunologically nonspecific inflammatory response. Mast cells are required for the full expression of cutaneous acute inflammation induced by phorbol 12-myristate 13-acetate. J Immunol 140:2356-2360.
65. Rao, T.S., Shaffer, A.F., Currie, J.L., and Isakson, P.C. 1994. Role of mast cells in
calcium ionophore (A23187)-induced peritoneal inflammation in mice. Inflammation 18:187-192.
66. Suzuki, N., Horiuchi, T., Ohta, K., Yamaguchi, M., Ueda, T., Takizawa, H., Hirai,
K., Shiga, J., Ito, K., and Miyamoto, T. 1993. Mast cells are essential for the full development of silica-induced pulmonary inflammation: a study with mast cell-deficient mice. American journal of respiratory cell and molecular biology 9:475-483.
67. Matsuda, H., Kawakita, K., Kiso, Y., Nakano, T., and Kitamura, Y. 1989.
Substance P induces granulocyte infiltration through degranulation of mast cells. The journal of immunology 142:927-931.
68. Yano, H., Wershil, B.K., Arizono, N., and Galli, S.J. 1989. Substance P-induced
augmentation of cutaneous vascular permeability and granulocyte infiltration in mice is mast cell dependent. The journal of clinical investigation 84:1276-1286.
69. Harris, R.R., Komater, V.A., Marett, R.A., Wilcox, D.M., and Bell, R.L. 1997. Effect
of mast cell deficiency and leukotriene inhibition on the influx of eosinophils induced by eotaxin. Journal of leukocyte biology 62:688-691.
70. Ribeiro, R.A., Souza-Filho, M.V., Souza, M.H., Oliveira, S.H., Costa, C.H., Cunha,
F.Q., and Ferreira, H.S. 1997. Role of resident mast cells and macrophages in the neutrophil migration induced by LTB4, fMLP and C5a des arg. International Archives of Allergy and Immunology 112:27-35.
71. Nishida, M., Uchikawa, R., Tegoshi, T., Yamada, M., Matsuda, S., Hyoh, Y., and
Arizono, N. 1999. Migration of neutrophils is dependent on mast cells in nonspecific pleurisy in rats. APMIS 107:929-936.
72. Ramos, B.F., Qureshi, R., Olsen, K.M., and Jakschik, B.A. 1990. The importance of
mast cells for the neutrophil influx in immune complex-induced peritonitis in mice. J Immunol 145:1868-1873.
140
73. Ramos, B.F., Zhang, Y., Qureshi, R., and Jakschik, B.A. 1991. Mast cells are critical for the production of leukotrienes responsible for neutrophil recruitment in immune complex-induced peritonitis in mice. J Immunol 147:1636-1641.
74. Zhang, Y., Ramos, B.F., and Jakschik, B.A. 1992. Neutrophil recruitment by
tumor necrosis factor from mast cells in immune complex peritonitis. Science 258:1957-1959.
75. Baumann, U., Chouchakova, N., Gewecke, B., Köhl, J., Carroll, M.C., Schmidt,
R.E., and Gessner, J.E. 2001. Distinct tissue site-specific requirements of mast cells and complement components C3/C5a receptor in IgG immune complex-induced injury of skin and lung. The journal of immunology 167:1022-1027.
76. Gershon, R.K., Askenase, P.W., and Gershon, M.D. 1975. Requirement for
vasoactive amines for production of delayed-type hypersensitvity skin reactions. The Journal of experimental medicine 142:732-747.
77. Askenase, P.W., Van Loveren, H., Kraeuter-Kops, S., Ron, Y., Meade, R.,
Theoharides, T.C., Nordlund, J.J., Scovern, H., Gerhson, M.D., and Ptak, W. 1983. Defective elicitation of delayed-type hypersensitivity in W/Wv and SI/SId mast cell-deficient mice. The journal of immunology 131:2687-2694.
78. Takizawa, H., Ohta, K., Hirai, K., Misaki, Y., Horiuchi, T., Kobayashi, N., Shiga,
J., and Miyamoto, T. 1989. Mast cells are important in the development of hypersensitivity pneumonitis. A study with mast-cell-deficient mice. The journal of immunology 143:1982-1988.
79. Kolaczkowska, E., Seljelid, R., and Plytycz, B. 2001. Role of mast cells in
zymosan-induced peritoneal inflammation in Balb/c and mast cell-deficient WBB6F1 mice. J Leukoc Biol 69:33-42.
80. Mullaly, S.C., and Kubes, P. 2006. The role of TLR2 in vivo following challenge
with Staphylococcus aureus and prototypic ligands. J Immunol 177:8154-8163. 81. Iuvone, T., Den Bossche, R.V., D'Acquisto, F., Carnuccio, R., and Herman, A.G.
1999. Evidence that mast cell degranulation, histamine and tumour necrosis factor alpha release occur in LPS-induced plasma leakage in rat skin. British journal of pharmacology 128:700-704.
82. Leal-Berumen, I., Conlon, P., and Marshall, J.S. 1994. IL-6 production by rat
peritoneal mast cells is not necessarily preceded by histamine release and can be induced by bacterial lipopolysaccharide. The journal of immunology 152:5468-5476.
83. Malaviya, R., Ikeda, T., Ross, E., and Abraham, S.N. 1996. Mast cell modulation
of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 381:77-80.
84. Echtenacher, B., Mannel, D.N., and Hultner, L. 1996. Critical protective role of
mast cells in a model of acute septic peritonitis. Nature 381:75-77.
141
85. Malaviya, R., and Abraham, S.N. 2000. Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis. J Leukoc Biol 67:841-846.
86. Mercer-Jones, M.A., Shrotri, M.S., Heinzelmann, M., Peyton, J.C., and Cheadle,
W.G. 1999. Regulation of early peritoneal neutrophil migration by macrophage inflammatory protein-2 and mast cells in experimental peritonitis. Journal of leukocyte biology 65:249-255.
87. Siebenhaar, F., Syska, W., Weller, K., Magerl, M., Zuberbier, T., Metz, M., and
Maurer, M. 2007. Control of Pseudomonas aeruginosa skin infections in mice is mast cell-dependent. Am J Pathol 170:1910-1916.
88. Thakurdas, S.M., Melicoff, E., Sansores-Garcia, L., Moreira, D.C., Petrova, Y.,
Stevens, R.L., and Adachi, R. 2007. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. J Biol Chem 282:20809-20815.
89. Mokhtarian, F., and Griffin, D.E. 1984. The role of mast cells in virus-induced
inflammation in the murine central nervous system. Cellular immunology 86:491-500.
90. Galli, S.J., Wershil, B.K., and Mekori, Y.A. 1987. Analysis of mast cell function in
biological responses not involving IgE. International Archives of Allergy and Applied Immunology 82:269-271.
91. Siflinger-Birnboim, A., Del Vecchio, P.J., Cooper, J.A., Blumenstock, F.A.,
Shepard, J.M., and Malik, A.B. 1987. Molecular sieving characteristics of the cultured endothelial monolayer. J Cell Physiol 132:111-117.
92. Riley, J.F. 1953. Histamine in tissue mast cells. Science 118:332. 93. Snyder, S.H., and Axelrod, J. 1965. Tissue Metabolism of Histamine -C14 in Vivo.
Fed Proc 24:774-776. 94. Kapeller-Adler, R. 1965. Histamine Catabolism in Vitro and in Vivo. Fed Proc
24:757-765. 95. Ash, A.S., and Schild, H.O. 1966. Receptors mediating some actions of histamine.
Br J Pharmacol Chemother 27:427-439. 96. Heltianu, C., Simionescu, M., and Simionescu, N. 1982. Histamine receptors of
the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules. The journal of cell biology 93:357-364.
97. Majno, G., Palade, G.E., and Schoefl, G.I. 1961. Studies on inflammation. II. The
site of action of histamine and serotonin along the vascular tree: a topographic study. J Biophys Biochem Cytol 11:607-626.
142
98. Bakker, R.A., Timmerman, H., and Leurs, R. 2002. Histamine receptors: specific ligands, receptor biochemistry, and signal transduction. Clin Allergy Immunol 17:27-64.
99. Kelm, M., Feelisch, M., Krebber, T., Motz, W., and Strauer, B.E. 1993.
Mechanisms of histamine-induced coronary vasodilatation: H1-receptor-mediated release of endothelium-derived nitric oxide. J Vasc Res 30:132-138.
100. Sheldon, R., Moy, A., Lindsley, K., Shasby, S., and Shasby, D.M. 1993. Role of
myosin light-chain phosphorylation in endothelial cell retraction. Am J Physiol 265:L606-612.
101. Moy, A.B., Shasby, S.S., Scott, B.D., and Shasby, D.M. 1993. The effect of
histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells. J Clin Invest 92:1198-1206.
102. Hong, S.L., and Deykin, D. 1982. Activation of phospholipases A2 and C in pig
aortic endothelial cells synthesizing prostacyclin. J Biol Chem 257:7151-7154. 103. Hanafy, K.A., Krumenacker, J.S., and Murad, F. 2001. NO, nitrotyrosine, and
cyclic GMP in signal transduction. Med Sci Monit 7:801-819. 104. Teixeira, M.M., Williams, T.J., and Hellewell, P.G. 1993. Role of prostaglandins
and nitric oxide in acute inflammatory reactions in guinea-pig skin. Br J Pharmacol 110:1515-1521.
105. Schroder, H., Strobach, H., and Schror, K. 1992. Nitric oxide but not prostacyclin
is an autocrine endothelial mediator. Biochem Pharmacol 43:533-537. 106. Draijer, R., Atsma, D.E., van der Laarse, A., and van Hinsbergh, V.W. 1995.
cGMP and nitric oxide modulate thrombin-induced endothelial permeability. Regulation via different pathways in human aortic and umbilical vein endothelial cells. Circ Res 76:199-208.
107. Kuhlencordt, P.J., Rosel, E., Gerszten, R.E., Morales-Ruiz, M., Dombkowski, D.,
Atkinson, W.J., Han, F., Preffer, F., Rosenzweig, A., Sessa, W.C., et al. 2004. Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells. Am J Physiol Cell Physiol 286:C1195-1202.
108. Garcia, J.G., Davis, H.W., and Patterson, C.E. 1995. Regulation of endothelial cell
gap formation and barrier dysfunction: role of myosin light chain phosphorylation. J Cell Physiol 163:510-522.
109. Goeckeler, Z.M., and Wysolmerski, R.B. 1995. Myosin light chain kinase-
regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation. J Cell Biol 130:613-627.
143
110. Baenziger, N.L., Fogerty, F.J., Mertz, L.F., and Chernuta, L.F. 1981. Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells. Cell 24:915-923.
111. McIntyre, T.M., Zimmerman, G.A., Satoh, K., and Prescott, S.M. 1985. Cultured
endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate. J Clin Invest 76:271-280.
112. Morley, J., Page, C.P., and Paul, W. 1983. Inflammatory actions of platelet
activating factor (Pafacether) in guinea-pig skin. Br J Pharmacol 80:503-509. 113. Lorant, D.E., Patel, K.D., McIntyre, T.M., McEver, R.P., Prescott, S.M., and
Zimmerman, G.A. 1991. Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils. J Cell Biol 115:223-234.
114. Weibel, E.R., and Palade, G.E. 1964. New Cytoplasmic Components in Arterial
Endothelia. J Cell Biol 23:101-112. 115. Hattori, R., Hamilton, K.K., Fugate, R.D., McEver, R.P., and Sims, P.J. 1989.
Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140. J Biol Chem 264:7768-7771.
116. Wagner, D.D., Olmsted, J.B., and Marder, V.J. 1982. Immunolocalization of von
Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol 95:355-360.
117. Bonfanti, R., Furie, B.C., Furie, B., and Wagner, D.D. 1989. PADGEM (GMP140) is
a component of Weibel-Palade bodies of human endothelial cells. Blood 73:1109-1112.
118. Utgaard, J.O., Jahnsen, F.L., Bakka, A., Brandtzaeg, P., and Haraldsen, G. 1998.
Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells. J Exp Med 188:1751-1756.
119. Wolff, B., Burns, A.R., Middleton, J., and Rot, A. 1998. Endothelial cell "memory"
of inflammatory stimulation: human venular endothelial cells store interleukin 8 in Weibel-Palade bodies. J Exp Med 188:1757-1762.
120. Subramaniam, M., Koedam, J.A., and Wagner, D.D. 1993. Divergent fates of P-
and E-selectins after their expression on the plasma membrane. Mol Biol Cell 4:791-801.
121. Mayadas, T.N., Johnson, R.C., Rayburn, H., Hynes, R.O., and Wagner, D.D. 1993.
Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell 74:541-554.
144
122. Denis, C.V., Andre, P., Saffaripour, S., and Wagner, D.D. 2001. Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice. Proc Natl Acad Sci U S A 98:4072-4077.
123. Jones, D.A., Abbassi, O., McIntire, L.V., McEver, R.P., and Smith, C.W. 1993. P-
selectin mediates neutrophil rolling on histamine-stimulated endothelial cells. Biophys J 65:1560-1569.
124. Asako, H., Kurose, I., Wolf, R., DeFrees, S., Zheng, Z.L., Phillips, M.L., Paulson,
J.C., and Granger, D.N. 1994. Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules. J Clin Invest 93:1508-1515.
125. Talreja, J., Kabir, M.H., M, B.F., Stechschulte, D.J., and Dileepan, K.N. 2004.
Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components. Immunology 113:224-233.
126. Delneste, Y., Lassalle, P., Jeannin, P., Joseph, M., Tonnel, A.B., and Gosset, P.
1994. Histamine induces IL-6 production by human endothelial cells. Clin Exp Immunol 98:344-349.
127. Jeannin, P., Delneste, Y., Gosset, P., Molet, S., Lassalle, P., Hamid, Q.,
Tsicopoulos, A., and Tonnel, A.B. 1994. Histamine induces interleukin-8 secretion by endothelial cells. Blood 84:2229-2233.
128. Miki, I., Kusano, A., Ohta, S., Hanai, N., Otoshi, M., Masaki, S., Sato, S., and
Ohmori, K. 1996. Histamine enhanced the TNF-alpha-induced expression of E-selectin and ICAM-1 on vascular endothelial cells. Cellular immunology 171:285-288.
129. Li, Y., Chi, L., Stechschulte, D.J., and Dileepan, K.N. 2001. Histamine-induced
production of interleukin-6 and interleukin-8 by human coronary artery endothelial cells is enhanced by endotoxin and tumor necrosis factor-alpha. Microvascular Research 61:253-262.
130. Sato, N., Goto, T., Haranaka, K., Satomi, N., Nariuchi, H., Mano-Hirano, Y., and
Sawasaki, Y. 1986. Actions of tumor necrosis factor on cultured vascular endothelial cells: morphologic modulation, growth inhibition, and cytotoxicity. J Natl Cancer Inst 76:1113-1121.
131. Brett, J., Gerlach, H., Nawroth, P., Steinberg, S., Godman, G., and Stern, D. 1989.
Tumor necrosis factor/cachectin increases permeability of endothelial cell monolayers by a mechanism involving regulatory G proteins. J Exp Med 169:1977-1991.
132. Schutze, S., Berkovic, D., Tomsing, O., Unger, C., and Kronke, M. 1991. Tumor
necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase C. J Exp Med 174:975-988.
145
133. Ferro, T., Neumann, P., Gertzberg, N., Clements, R., and Johnson, A. 2000. Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha. Am J Physiol Lung Cell Mol Physiol 278:L1107-1117.
134. Wojciak-Stothard, B., Entwistle, A., Garg, R., and Ridley, A.J. 1998. Regulation of
TNF-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells. J Cell Physiol 176:150-165.
135. Beynon, H.L., Haskard, D.O., Davies, K.A., Haroutunian, R., and Walport, M.J.
1993. Combinations of low concentrations of cytokines and acute agonists synergize in increasing the permeability of endothelial monolayers. Clin Exp Immunol 91:314-319.
136. Bevilacqua, M.P., Pober, J.S., Mendrick, D.L., Cotran, R.S., and Gimbrone, M.A.,
Jr. 1987. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci U S A 84:9238-9242.
137. Pober, J.S., Lapierre, L.A., Stolpen, A.H., Brock, T.A., Springer, T.A., Fiers, W.,
Bevilacqua, M.P., Mendrick, D.L., and Gimbrone, M.A., Jr. 1987. Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin 1 species. J Immunol 138:3319-3324.
138. Osborn, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., Chi-Rosso, G.,
and Lobb, R. 1989. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59:1203-1211.
139. Strieter, R.M., Kunkel, S.L., Showell, H.J., Remick, D.G., Phan, S.H., Ward, P.A.,
and Marks, R.M. 1989. Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta. Science 243:1467-1469.
140. Sica, A., Wang, J.M., Colotta, F., Dejana, E., Mantovani, A., Oppenheim, J.J.,
Larsen, C.G., Zachariae, C.O., and Matsushima, K. 1990. Monocyte chemotactic and activating factor gene expression induced in endothelial cells by IL-1 and tumor necrosis factor. J Immunol 144:3034-3038.
141. Wen, D.Z., Rowland, A., and Derynck, R. 1989. Expression and secretion of
gro/MGSA by stimulated human endothelial cells. EMBO J 8:1761-1766. 142. Haskill, S., Peace, A., Morris, J., Sporn, S.A., Anisowicz, A., Lee, S.W., Smith, T.,
Martin, G., Ralph, P., and Sager, R. 1990. Identification of three related human GRO genes encoding cytokine functions. Proc Natl Acad Sci U S A 87:7732-7736.
143. Viemann, D., Goebeler, M., Schmid, S., Klimmek, K., Sorg, C., Ludwig, S., and
Roth, J. 2004. Transcriptional profiling of IKK2/NF-kappa B- and p38 MAP kinase-dependent gene expression in TNF-alpha-stimulated primary human endothelial cells. Blood 103:3365-3373.
146
144. Magder, S., Neculcea, J., Neculcea, V., and Sladek, R. 2006. Lipopolysaccharide and TNF-alpha produce very similar changes in gene expression in human endothelial cells. J Vasc Res 43:447-461.
145. Feyerabend, T.B., Hausser, H., Tietz, A., Blum, C., Hellman, L., Straus, A.H.,
Takahashi, H.K., Morgan, E.S., Dvorak, A.M., Fehling, H.J., et al. 2005. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol 25:6199-6210.
146. Huang, C., Friend, D.S., Qiu, W.T., Wong, G.W., Morales, G., Hunt, J., and
Stevens, R.L. 1998. Induction of a selective and persistent extravasation of neutrophils into the peritoneal cavity by tryptase mouse mast cell protease 6. J Immunol 160:1910-1919.
147. Compton, S.J., Cairns, J.A., Holgate, S.T., and Walls, A.F. 1998. The role of mast
cell tryptase in regulating endothelial cell proliferation, cytokine release, and adhesion molecule expression: tryptase induces expression of mRNA for IL-1 beta and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells. J Immunol 161:1939-1946.
148. Vu, T.K., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. 1991. Molecular cloning
of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057-1068.
149. Steinhoff, M., Corvera, C.U., Thoma, M.S., Kong, W., McAlpine, B.E., Caughey,
G.H., Ansel, J.C., and Bunnett, N.W. 1999. Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase. Exp Dermatol 8:282-294.
150. Shpacovitch, V.M., Brzoska, T., Buddenkotte, J., Stroh, C., Sommerhoff, C.P.,
Ansel, J.C., Schulze-Osthoff, K., Bunnett, N.W., Luger, T.A., and Steinhoff, M. 2002. Agonists of proteinase-activated receptor 2 induce cytokine release and activation of nuclear transcription factor kappaB in human dermal microvascular endothelial cells. J Invest Dermatol 118:380-385.
151. Vergnolle, N. 1999. Proteinase-activated receptor-2-activating peptides induce
leukocyte rolling, adhesion, and extravasation in vivo. J Immunol 163:5064-5069. 152. Lindner, J.R., Kahn, M.L., Coughlin, S.R., Sambrano, G.R., Schauble, E.,
Bernstein, D., Foy, D., Hafezi-Moghadam, A., and Ley, K. 2000. Delayed onset of inflammation in protease-activated receptor-2-deficient mice. J Immunol 165:6504-6510.
153. Klarenbach, S.W., Chipiuk, A., Nelson, R.C., Hollenberg, M.D., and Murray, A.G.
2003. Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases. Circ Res 92:272-278.
147
154. Suzuki, H., Motley, E.D., Eguchi, K., Hinoki, A., Shirai, H., Watts, V., Stemmle, L.N., Fields, T.A., and Eguchi, S. 2009. Distinct roles of protease-activated receptors in signal transduction regulation of endothelial nitric oxide synthase. Hypertension 53:182-188.
155. Meyer, M.C., Creer, M.H., and McHowat, J. 2005. Potential role for mast cell
tryptase in recruitment of inflammatory cells to endothelium. Am J Physiol Cell Physiol 289:C1485-1491.
156. Sendo, T., Sumimura, T., Itoh, Y., Goromaru, T., Aki, K., Yano, T., Oike, M., Ito,
Y., Mori, S., Nishibori, M., et al. 2003. Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells. Cell Signal 15:773-781.
157. Houliston, R.A., Keogh, R.J., Sugden, D., Dudhia, J., Carter, T.D., and Wheeler-
Jones, C.P. 2002. Protease-activated receptors upregulate cyclooxygenase-2 expression in human endothelial cells. Thromb Haemost 88:321-328.
158. Chi, L., Li, Y., Stehno-Bittel, L., Gao, J., Morrison, D.C., Stechschulte, D.J., and
Dileepan, K.N. 2001. Interleukin-6 production by endothelial cells via stimulation of protease-activated receptors is amplified by endotoxin and tumor necrosis factor-alpha. J Interferon Cytokine Res 21:231-240.
159. Proud, D., Siekierski, E.S., and Bailey, G.S. 1988. Identification of human lung
mast cell kininogenase as tryptase and relevance of tryptase kininogenase activity. Biochem Pharmacol 37:1473-1480.
160. Imamura, T., Dubin, A., Moore, W., Tanaka, R., and Travis, J. 1996. Induction of
vascular permeability enhancement by human tryptase: dependence on activation of prekallikrein and direct release of bradykinin from kininogens. Lab Invest 74:861-870.
161. Kozik, A., Moore, R.B., Potempa, J., Imamura, T., Rapala-Kozik, M., and Travis, J.
1998. A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens. J Biol Chem 273:33224-33229.
162. Rubinstein, I., Nadel, J.A., Graf, P.D., and Caughey, G.H. 1990. Mast cell chymase
potentiates histamine-induced wheal formation in the skin of ragweed-allergic dogs. J Clin Invest 86:555-559.
163. Reilly, C.F., Tewksbury, D.A., Schechter, N.M., and Travis, J. 1982. Rapid
conversion of angiotensin I to angiotensin II by neutrophil and mast cell proteinases. J Biol Chem 257:8619-8622.
164. Suzuki, H., Caughey, G.H., Gao, X.P., and Rubinstein, I. 1998. Mast cell chymase-
like protease(s) modulates Escherichia coli lipopolysaccharide-induced vasomotor dysfunction in skeletal muscle in vivo. J Pharmacol Exp Ther 284:1156-1164.
148
165. Schechter, N.M., Irani, A.M., Sprows, J.L., Abernethy, J., Wintroub, B., and Schwartz, L.B. 1990. Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell. J Immunol 145:2652-2661.
166. Peterson, M.W., Gruenhaupt, D., and Shasby, D.M. 1989. Neutrophil cathepsin G
increases calcium flux and inositol polyphosphate production in cultured endothelial cells. J Immunol 143:609-616.
167. Peterson, M.W. 1989. Neutrophil cathepsin G increases transendothelial albumin
flux. J Lab Clin Med 113:297-308. 168. Camussi, G., Tetta, C., Bussolino, F., and Baglioni, C. 1988. Synthesis and release
of platelet-activating factor is inhibited by plasma alpha 1-proteinase inhibitor or alpha 1-antichymotrypsin and is stimulated by proteinases. J Exp Med 168:1293-1306.
169. Lundequist, A., Tchougounova, E., Abrink, M., and Pejler, G. 2004. Cooperation
between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II. J Biol Chem 279:32339-32344.
170. Azizkhan, R.G., Azizkhan, J.C., Zetter, B.R., and Folkman, J. 1980. Mast cell
heparin stimulates migration of capillary endothelial cells in vitro. J Exp Med 152:931-944.
171. Lagunoff, D., and Rickard, A. 1999. Mast cell granule heparin proteoglycan
induces lacunae in confluent endothelial cell monolayers. Am J Pathol 154:1591-1600.
172. Kanwar, Y.S., and Farquhar, M.G. 1979. Presence of heparan sulfate in the
glomerular basement membrane. Proc Natl Acad Sci U S A 76:1303-1307. 173. Levi-Schaffer, F., Dayton, E.T., Austen, K.F., Hein, A., Caulfield, J.P., Gravallese,
P.M., Liu, F.T., and Stevens, R.L. 1987. Mouse bone marrow-derived mast cells cocultured with fibroblasts. Morphology and stimulation-induced release of histamine, leukotriene B4, leukotriene C4, and prostaglandin D2. J Immunol 139:3431-3441.
174. Benyon, R.C., Robinson, C., and Church, M.K. 1989. Differential release of
histamine and eicosanoids from human skin mast cells activated by IgE-dependent and non-immunological stimuli. Br J Pharmacol 97:898-904.
175. Miller, D.K., Gillard, J.W., Vickers, P.J., Sadowski, S., Leveille, C., Mancini, J.A.,
Charleson, P., Dixon, R.A., Ford-Hutchinson, A.W., Fortin, R., et al. 1990. Identification and isolation of a membrane protein necessary for leukotriene production. Nature 343:278-281.
176. Bach, M.K., Brashler, J.R., and Morton, D.R., Jr. 1984. Solubilization and
characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: a novel, particulate glutathione S-transferase. Arch Biochem Biophys 230:455-465.
149
177. Raulf, M., Stuning, M., and Konig, W. 1985. Metabolism of leukotrienes by L-gamma-glutamyl-transpeptidase and dipeptidase from human polymorphonuclear granulocytes. Immunology 55:135-147.
178. Soter, N.A., Lewis, R.A., Corey, E.J., and Austen, K.F. 1983. Local effects of
synthetic leukotrienes (LTC4, LTD4, LTE4, and LTB4) in human skin. J Invest Dermatol 80:115-119.
179. Dahlen, S.E., Bjork, J., Hedqvist, P., Arfors, K.E., Hammarstrom, S., Lindgren,
J.A., and Samuelsson, B. 1981. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory response. Proc Natl Acad Sci U S A 78:3887-3891.
180. Sjostrom, M., Jakobsson, P.J., Heimburger, M., Palmblad, J., and Haeggstrom, J.Z.
2001. Human umbilical vein endothelial cells generate leukotriene C4 via microsomal glutathione S-transferase type 2 and express the CysLT(1) receptor. Eur J Biochem 268:2578-2586.
181. Peres, C.M., Aronoff, D.M., Serezani, C.H., Flamand, N., Faccioli, L.H., and
Peters-Golden, M. 2007. Specific leukotriene receptors couple to distinct G proteins to effect stimulation of alveolar macrophage host defense functions. J Immunol 179:5454-5461.
182. Datta, Y.H., Romano, M., Jacobson, B.C., Golan, D.E., Serhan, C.N., and
Ewenstein, B.M. 1995. Peptido-leukotrienes are potent agonists of von Willebrand factor secretion and P-selectin surface expression in human umbilical vein endothelial cells. Circulation 92:3304-3311.
183. McIntyre, T.M., Zimmerman, G.A., and Prescott, S.M. 1986. Leukotrienes C4 and
D4 stimulate human endothelial cells to synthesize platelet-activating factor and bind neutrophils. Proc Natl Acad Sci U S A 83:2204-2208.
184. Kanaoka, Y., Maekawa, A., Penrose, J.F., Austen, K.F., and Lam, B.K. 2001.
Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase. J Biol Chem 276:22608-22613.
185. Maekawa, A., Austen, K.F., and Kanaoka, Y. 2002. Targeted gene disruption
reveals the role of cysteinyl leukotriene 1 receptor in the enhanced vascular permeability of mice undergoing acute inflammatory responses. J Biol Chem 277:20820-20824.
186. Mencia-Huerta, J.M., Razin, E., Ringel, E.W., Corey, E.J., Hoover, D., Austen,
K.F., and Lewis, R.A. 1983. Immunologic and ionophore-induced generation of leukotriene B4 from mouse bone marrow-derived mast cells. J Immunol 130:1885-1890.
150
187. Malmsten, C.L., Palmblad, J., Uden, A.M., Radmark, O., Engstedt, L., and Samuelsson, B. 1980. Leukotriene B4: a highly potent and stereospecific factor stimulating migration of polymorphonuclear leukocytes. Acta Physiol Scand 110:449-451.
188. Ford-Hutchinson, A.W., Bray, M.A., Doig, M.V., Shipley, M.E., and Smith, M.J.
1980. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286:264-265.
189. Byrum, R.S., Goulet, J.L., Snouwaert, J.N., Griffiths, R.J., and Koller, B.H. 1999.
Determination of the contribution of cysteinyl leukotrienes and leukotriene B4 in acute inflammatory responses using 5-lipoxygenase- and leukotriene A4 hydrolase-deficient mice. J Immunol 163:6810-6819.
190. Roberts, L.J., 2nd, Lewis, R.A., Oates, J.A., and Austen, K.F. 1979. Prostaglandin
thromboxane, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid production by ionophore-stimulated rat serosal mast cells. Biochim Biophys Acta 575:185-192.
191. Roberts, L.J., 2nd, Sweetman, B.J., Lewis, R.A., Austen, K.F., and Oates, J.A. 1980.
Increased production of prostaglandin D2 in patients with systemic mastocytosis. N Engl J Med 303:1400-1404.
192. Flower, R.J., Harvey, E.A., and Kingston, W.P. 1976. Inflammatory effects of
prostaglandin D2 in rat and human skin. Br J Pharmacol 56:229-233. 193. Mills, D.C., and Macfarlane, D.E. 1974. Stimulation of human platelet adenylate
cyclase by prostaglandin D2. Thromb Res 5:401-412. 194. Rahman, A., Inoue, T., Ago, J., Ishikawa, T., and Kamei, C. 2007. Interactive effect
of histamine and prostaglandin D2 on nasal allergic symptoms in rats. Eur J Pharmacol 554:229-234.
195. Mencia-Huerta, J.M., Lewis, R.A., Razin, E., and Austen, K.F. 1983. Antigen-
initiated release of platelet-activating factor (PAF-acether) from mouse bone marrow-derived mast cells sensitized with monoclonal IgE. J Immunol 131:2958-2964.
196. Schleimer, R.P., MacGlashan, D.W., Jr., Peters, S.P., Pinckard, R.N., Adkinson,
N.F., Jr., and Lichtenstein, L.M. 1986. Characterization of inflammatory mediator release from purified human lung mast cells. Am Rev Respir Dis 133:614-617.
197. Vercellotti, G.M., Moldow, C.F., Wickham, N.W., and Jacob, H.S. 1990.
Endothelial cell platelet-activating factor primes neutrophil responses: amplification of endothelial activation by neutrophil products. J Lipid Mediat 2 Suppl:S23-30.
198. Camussi, G., Bussolino, F., Salvidio, G., and Baglioni, C. 1987. Tumor necrosis
factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor. J Exp Med 166:1390-1404.
151
199. Bussolino, F., Camussi, G., Aglietta, M., Braquet, P., Bosia, A., Pescarmona, G., Sanavio, F., D'Urso, N., and Marchisio, P.C. 1987. Human endothelial cells are target for platelet-activating factor. I. Platelet-activating factor induces changes in cytoskeleton structures. J Immunol 139:2439-2446.
200. Barzaghi, G., Sarau, H.M., and Mong, S. 1989. Platelet-activating factor-induced
phosphoinositide metabolism in differentiated U-937 cells in culture. J Pharmacol Exp Ther 248:559-566.
201. Bussolino, F., Silvagno, F., Garbarino, G., Costamagna, C., Sanavio, F., Arese, M.,
Soldi, R., Aglietta, M., Pescarmona, G., Camussi, G., et al. 1994. Human endothelial cells are targets for platelet-activating factor (PAF). Activation of alpha and beta protein kinase C isozymes in endothelial cells stimulated by PAF. J Biol Chem 269:2877-2886.
202. Pirotzky, E., Page, C.P., Roubin, R., Pfister, A., Paul, W., Bonnet, J., and
Benveniste, J. 1984. PAF-acether-induced plasma exudation in rat skin is independent of platelets and neutrophils. Microcirc Endothelium Lymphatics 1:107-122.
203. Humphrey, D.M., McManus, L.M., Hanahan, D.J., and Pinckard, R.N. 1984.
Morphologic basis of increased vascular permeability induced by acetyl glyceryl ether phosphorylcholine. Lab Invest 50:16-25.
204. Jiang, Y., Wen, K., Zhou, X., Schwegler-Berry, D., Castranova, V., and He, P.
2008. Three-dimensional localization and quantification of PAF-induced gap formation in intact venular microvessels. Am J Physiol Heart Circ Physiol 295:H898-906.
205. Ishii, S., Kuwaki, T., Nagase, T., Maki, K., Tashiro, F., Sunaga, S., Cao, W.H.,
Kume, K., Fukuchi, Y., Ikuta, K., et al. 1998. Impaired anaphylactic responses with intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor. J Exp Med 187:1779-1788.
206. Myers, A., Ramey, E., and Ramwell, P. 1983. Glucocorticoid protection against
PAF-acether toxicity in mice. Br J Pharmacol 79:595-598. 207. Shibamoto, T., Liu, W., Cui, S., Zhang, W., Takano, H., and Kurata, Y. 2008. PAF,
rather than histamine, participates in mouse anaphylactic hypotension. Pharmacology 82:114-120.
208. Gordon, J.R., and Galli, S.J. 1990. Mast cells as a source of both preformed and
immunologically inducible TNF-alpha/cachectin. Nature 346:274-276. 209. Burd, P.R., Rogers, H.W., Gordon, J.R., Martin, C.A., Jayaraman, S., Wilson, S.D.,
Dvorak, A.M., Galli, S.J., and Dorf, M.E. 1989. Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J Exp Med 170:245-257.
152
210. Lin, T.J., Garduno, R., Boudreau, R.T., and Issekutz, A.C. 2002. Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta. J Immunol 169:4522-4530.
211. Plaut, M., Pierce, J.H., Watson, C.J., Hanley-Hyde, J., Nordan, R.P., and Paul,
W.E. 1989. Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophores. Nature 339:64-67.
212. Hultner, L., Kolsch, S., Stassen, M., Kaspers, U., Kremer, J.P., Mailhammer, R.,
Moeller, J., Broszeit, H., and Schmitt, E. 2000. In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9. J Immunol 164:5556-5563.
213. Tachimoto, H., Ebisawa, M., Hasegawa, T., Kashiwabara, T., Ra, C., Bochner,
B.S., Miura, K., and Saito, H. 2000. Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-gamma. J Allergy Clin Immunol 106:141-149.
214. Wakahara, S., Fujii, Y., Nakao, T., Tsuritani, K., Hara, T., Saito, H., and Ra, C.
2001. Gene expression profiles for Fc epsilon RI, cytokines and chemokines upon Fc epsilon RI activation in human cultured mast cells derived from peripheral blood. Cytokine 16:143-152.
215. Varadaradjalou, S., Feger, F., Thieblemont, N., Hamouda, N.B., Pleau, J.M., Dy,
M., and Arock, M. 2003. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells. Eur J Immunol 33:899-906.
216. Kendall, J.C., Li, X.H., Galli, S.J., and Gordon, J.R. 1997. Promotion of mouse
fibroblast proliferation by IgE-dependent activation of mouse mast cells: role for mast cell tumor necrosis factor-alpha and transforming growth factor-beta 1. J Allergy Clin Immunol 99:113-123.
217. Nakajima, T., Inagaki, N., Tanaka, H., Tanaka, A., Yoshikawa, M., Tamari, M.,
Hasegawa, K., Matsumoto, K., Tachimoto, H., Ebisawa, M., et al. 2002. Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcepsilon receptor I cross-linking: an interspecies comparison. Blood 100:3861-3868.
218. Watson, M.L., Lewis, G.P., and Westwick, J. 1989. Increased vascular
permeability and polymorphonuclear leucocyte accumulation in vivo in response to recombinant cytokines and supernatant from cultures of human synovial cells treated with interleukin 1. Br J Exp Pathol 70:93-101.
219. Williams, M.R., Kataoka, N., Sakurai, Y., Powers, C.M., Eskin, S.G., and McIntire,
L.V. 2008. Gene expression of endothelial cells due to interleukin-1 beta stimulation and neutrophil transmigration. Endothelium 15:73-165.
153
220. Abe, Y., Sekiya, S., Yamasita, T., and Sendo, F. 1990. Vascular hyperpermeability induced by tumor necrosis factor and its augmentation by IL-1 and IFN-gamma is inhibited by selective depletion of neutrophils with a monoclonal antibody. J Immunol 145:2902-2907.
221. Maruo, N., Morita, I., Shirao, M., and Murota, S. 1992. IL-6 increases endothelial
permeability in vitro. Endocrinology 131:710-714. 222. Desai, T.R., Leeper, N.J., Hynes, K.L., and Gewertz, B.L. 2002. Interleukin-6
causes endothelial barrier dysfunction via the protein kinase C pathway. J Surg Res 104:118-123.
223. Watson, C., Whittaker, S., Smith, N., Vora, A.J., Dumonde, D.C., and Brown, K.A.
1996. IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes. Clin Exp Immunol 105:112-119.
224. Chen, Q., Fisher, D.T., Clancy, K.A., Gauguet, J.M., Wang, W.C., Unger, E., Rose-
John, S., von Andrian, U.H., Baumann, H., and Evans, S.S. 2006. Fever-range thermal stress promotes lymphocyte trafficking across high endothelial venules via an interleukin 6 trans-signaling mechanism. Nat Immunol 7:1299-1308.
225. McLachlan, J.B., Hart, J.P., Pizzo, S.V., Shelburne, C.P., Staats, H.F., Gunn, M.D.,
and Abraham, S.N. 2003. Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat Immunol 4:1199-1205.
226. Metz, M., Piliponsky, A.M., Chen, C.C., Lammel, V., Abrink, M., Pejler, G., Tsai,
M., and Galli, S.J. 2006. Mast cells can enhance resistance to snake and honeybee venoms. Science 313:526-530.
227. Ohhashi, T., Kawai, Y., and Azuma, T. 1978. The response of lymphatic smooth
muscles to vasoactive substances. Pflugers Arch 375:183-188. 228. Casley-Smith, J.R., and Florey, H.W. 1961. The structure of normal small
lymphatics. Q J Exp Physiol Cogn Med Sci 46:101-106. 229. Wenzel-Hora, B.I., Berens von Rautenfeld, D., Majewski, A., Lubach, D., and
Partsch, H. 1987. Scanning electron microscopy of the initial lymphatics of the skin after use of the indirect application technique with glutaraldehyde and MERCOX as compared to clinical findings. Lymphology 20:126-144.
230. Casley-Smith, J.R., and Bolton, T. 1973. Electron microscopy of the effects of
histamine and thermal injury on the blood and lymphatic endothelium, and the mesothelium of the mouse's diaphragm, together with the influence of coumarin and rutin. Experientia 29:1386-1388.
231. Castenholz, A. 1987. Structural and functional properties of initial lymphatics in
the rat tongue: scanning electron microscopic findings. Lymphology 20:112-125. 232. Price, G.M., Chrobak, K.M., and Tien, J. 2008. Effect of cyclic AMP on barrier
function of human lymphatic microvascular tubes. Microvasc Res 76:46-51.
154
233. Breslin, J.W., Yuan, S.Y., and Wu, M.H. 2007. VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism. Lymphat Res Biol 5:105-113.
234. Bianchi, L., Lorenzoni, P., Bini, L., Weber, E., Tani, C., Rossi, A., Agliano, M.,
Pallini, V., and Sacchi, G. 2007. Protein expression profiles of Bos taurus blood and lymphatic vessel endothelial cells. Proteomics 7:1600-1614.
235. Banerji, S., Ni, J., Wang, S.X., Clasper, S., Su, J., Tammi, R., Jones, M., and
Jackson, D.G. 1999. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 144:789-801.
236. Breiteneder-Geleff, S., Soleiman, A., Kowalski, H., Horvat, R., Amann, G.,
Kriehuber, E., Diem, K., Weninger, W., Tschachler, E., Alitalo, K., et al. 1999. Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol 154:385-394.
237. Srinivasan, R.S., Dillard, M.E., Lagutin, O.V., Lin, F.J., Tsai, S., Tsai, M.J.,
Samokhvalov, I.M., and Oliver, G. 2007. Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Genes Dev 21:2422-2432.
238. Harrison, R.L., Ewert, A., and Folse, D.S. 1986. Presence of Weibel-Palade bodies
in lymphatic endothelium of the cat. Lymphology 19:170-171. 239. Leak, L.V., Cadet, J.L., Griffin, C.P., and Richardson, K. 1995. Nitric oxide
production by lymphatic endothelial cells in vitro. Biochem Biophys Res Commun 217:96-105.
240. Lahdenranta, J., Hagendoorn, J., Padera, T.P., Hoshida, T., Nelson, G.,
Kashiwagi, S., Jain, R.K., and Fukumura, D. 2009. Endothelial nitric oxide synthase mediates lymphangiogenesis and lymphatic metastasis. Cancer Res 69:2801-2808.
241. Hammer, T., Tritsaris, K., Hubschmann, M.V., Gibson, J., Nisato, R.E., Pepper,
M.S., and Dissing, S. 2009. IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation. Microvasc Res.
242. Leak, L.V., Saunders, M., Day, A.A., and Jones, M. 2000. Stimulation of
plasminogen activator and inhibitor in the lymphatic endothelium. Microvasc Res 60:201-211.
243. Johnson, L.A., Clasper, S., Holt, A.P., Lalor, P.F., Baban, D., and Jackson, D.G.
2006. An inflammation-induced mechanism for leukocyte transmigration across lymphatic vessel endothelium. J Exp Med 203:2763-2777.
244. Pegu, A., Qin, S., Fallert Junecko, B.A., Nisato, R.E., Pepper, M.S., and Reinhart,
T.A. 2008. Human lymphatic endothelial cells express multiple functional TLRs. J Immunol 180:3399-3405.
155
245. Korhonen, H., Fisslthaler, B., Moers, A., Wirth, A., Habermehl, D., Wieland, T., Schutz, G., Wettschureck, N., Fleming, I., and Offermanns, S. 2009. Anaphylactic shock depends on endothelial Gq/G11. J Exp Med 206:411-420.
246. Bhoola, K.D., Figueroa, C.D., and Worthy, K. 1992. Bioregulation of kinins:
kallikreins, kininogens, and kininases. Pharmacol Rev 44:1-80. 247. Williams, T.J. 1983. Vascular permeability changes induced by complement-
derived peptides. Agents Actions 13:451-455. 248. Jawdat, D.M., Rowden, G., and Marshall, J.S. 2006. Mast cells have a pivotal role
in TNF-independent lymph node hypertrophy and the mobilization of Langerhans cells in response to bacterial peptidoglycan. J Immunol 177:1755-1762.
249. Suto, H., Nakae, S., Kakurai, M., Sedgwick, J.D., Tsai, M., and Galli, S.J. 2006.
Mast cell-associated TNF promotes dendritic cell migration. J Immunol 176:4102-4112.
250. Demeure, C.E., Brahimi, K., Hacini, F., Marchand, F., Peronet, R., Huerre, M., St-
Mezard, P., Nicolas, J.F., Brey, P., Delespesse, G., et al. 2005. Anopheles mosquito bites activate cutaneous mast cells leading to a local inflammatory response and lymph node hyperplasia. J Immunol 174:3932-3940.
251. Gordon, J.R., and Galli, S.J. 1991. Release of both preformed and newly
synthesized tumor necrosis factor alpha (TNF-alpha)/cachectin by mouse mast cells stimulated via the Fc epsilon RI. A mechanism for the sustained action of mast cell-derived TNF-alpha during IgE-dependent biological responses. J Exp Med 174:103-107.
252. Gamble, J.R., Harlan, J.M., Klebanoff, S.J., and Vadas, M.A. 1985. Stimulation of
the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc Natl Acad Sci U S A 82:8667-8671.
253. Sallusto, F., Cella, M., Danieli, C., and Lanzavecchia, A. 1995. Dendritic cells use
macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med 182:389-400.
254. Bissonnette, E.Y., Chin, B., and Befus, A.D. 1995. Interferons differentially
regulate histamine and TNF-alpha in rat intestinal mucosal mast cells. Immunology 86:12-17.
255. Rampart, M., De Smet, W., Fiers, W., and Herman, A.G. 1989. Inflammatory
properties of recombinant tumor necrosis factor in rabbit skin in vivo. J Exp Med 169:2227-2232.
256. Baekkevold, E.S., Yamanaka, T., Palframan, R.T., Carlsen, H.S., Reinholt, F.P.,
von Andrian, U.H., Brandtzaeg, P., and Haraldsen, G. 2001. The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment. J Exp Med 193:1105-1112.
156
257. Stein, J.V., Rot, A., Luo, Y., Narasimhaswamy, M., Nakano, H., Gunn, M.D., Matsuzawa, A., Quackenbush, E.J., Dorf, M.E., and von Andrian, U.H. 2000. The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J Exp Med 191:61-76.
258. Huang, V., Lonsdorf, A.S., Fang, L., Kakinuma, T., Lee, V.C., Cha, E., Zhang, H.,
Nagao, K., Zaleska, M., Olszewski, W.L., et al. 2008. Cutting edge: rapid accumulation of epidermal CCL27 in skin-draining lymph nodes following topical application of a contact sensitizer recruits CCR10-expressing T cells. J Immunol 180:6462-6466.
259. Palframan, R.T., Jung, S., Cheng, G., Weninger, W., Luo, Y., Dorf, M., Littman,
D.R., Rollins, B.J., Zweerink, H., Rot, A., et al. 2001. Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues. J Exp Med 194:1361-1373.
260. Beutler, B., Mahoney, J., Le Trang, N., Pekala, P., and Cerami, A. 1985.
Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells. J Exp Med 161:984-995.
261. Ferraiolo, B.L., Moore, J.A., Crase, D., Gribling, P., Wilking, H., and Baughman,
R.A. 1988. Pharmacokinetics and tissue distribution of recombinant human tumor necrosis factor-alpha in mice. Drug Metab Dispos 16:270-275.
262. Lagunoff, D., and Benditt, E.P. 1963. Proteolytic enzymes of mast cells. Ann N Y
Acad Sci 103:185-198. 263. Lagunoff, D., Phillips, M.T., Iseri, O.A., and Benditt, E.P. 1964. Isolation and
Preliminary Characterization of Rat Mast Cell Granules. Lab Invest 13:1331-1344. 264. Schwartz, L.B., Riedel, C., Schratz, J.J., and Austen, K.F. 1982. Localization of
carboxypeptidase A to the macromolecular heparin proteoglycan-protein complex in secretory granules of rat serosal mast cells. J Immunol 128:1128-1133.
265. Abe, T., Swieter, M., Imai, T., Hollander, N.D., and Befus, A.D. 1990. Mast cell
heterogeneity: two-dimensional gel electrophoretic analyses of rat peritoneal and intestinal mucosal mast cells. Eur J Immunol 20:1941-1947.
266. Befus, A.D., Chin, B., Pick, J., Evans, S., Osborn, S., and Forstrom, J. 1995.
Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A. J Immunol 155:4406-4411.
267. Benyon, R.C., Enciso, J.A., and Befus, A.D. 1993. Analysis of human skin mast
cell proteins by two-dimensional gel electrophoresis. Identification of tryptase as a sialylated glycoprotein. J Immunol 151:2699-2706.
157
268. Schwartz, L.B., Irani, A.M., Roller, K., Castells, M.C., and Schechter, N.M. 1987. Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells. J Immunol 138:2611-2615.
269. Irani, A.M., Goldstein, S.M., Wintroub, B.U., Bradford, T., and Schwartz, L.B.
1991. Human mast cell carboxypeptidase. Selective localization to MCTC cells. J Immunol 147:247-253.
270. Tannenbaum, S., Oertel, H., Henderson, W., and Kaliner, M. 1980. The biologic
activity of mast cell granules. I. Elicitation of inflammatory responses in rat skin. J Immunol 125:325-335.
271. Higginbotham, R.D. 1959. On the participation of micellophagosis in the
resistance of tissue to injurious agents. Int Arch Allergy Appl Immunol 15:195-222. 272. Higginbotham, R.D., and Karnella, S. 1971. The significance of the mast cell
response to bee venom. J Immunol 106:233-240. 273. Higginbotham, R.D. 1965. Mast cells and local resistance to Russell's viper
venom. J Immunol 95:867-875. 274. Lomonte, B., Moreno, E., Tarkowski, A., Hanson, L.A., and Maccarana, M. 1994.
Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling. J Biol Chem 269:29867-29873.
275. Decorti, G., Klugmann, F.B., Crivellato, E., Malusa, N., Furlan, G., Candussio, L.,
and Giraldi, T. 2000. Biochemical and microscopic evidence for the internalization of drug-containing mast cell granules by macrophages and smooth muscle cells. Toxicol Appl Pharmacol 169:269-275.
276. Henderson, W.R., Chi, E.Y., and Klebanoff, S.J. 1980. Eosinophil peroxidase-
induced mast cell secretion. J Exp Med 152:265-279. 277. Subba Rao, P.V., Friedman, M.M., Atkins, F.M., and Metcalfe, D.D. 1983.
Phagocytosis of mast cell granules by cultured fibroblasts. J Immunol 130:341-349. 278. Hartmann, T., Ruoss, S.J., Raymond, W.W., Seuwen, K., and Caughey, G.H. 1992.
Human tryptase as a potent, cell-specific mitogen: role of signaling pathways in synergistic responses. Am J Physiol 262:L528-534.
279. Cairns, J.A., and Walls, A.F. 1997. Mast cell tryptase stimulates the synthesis of
type I collagen in human lung fibroblasts. J Clin Invest 99:1313-1321. 280. Shah, B.A., Li, Y., Stechschulte, D.J., and Dileepan, K.N. 1995. Phagocytosis of
mast cell granules results in decreased macrophage superoxide production. Mediators Inflamm 4:406-412.
158
281. Ito, N., Li, Y., Suzuki, T., Stechschulte, D.J., and Dileepan, K.N. 1998. Transient degradation of NF-kappaB proteins in macrophages after interaction with mast cell granules. Mediators Inflamm 7:397-407.
282. Kokkonen, J.O., and Kovanen, P.T. 1985. Low density lipoprotein degradation by
rat mast cells. Demonstration of extracellular proteolysis caused by mast cell granules. J Biol Chem 260:14756-14763.
283. Kokkonen, J.O., and Kovanen, P.T. 1989. Proteolytic enzymes of mast cell
granules degrade low density lipoproteins and promote their granule-mediated uptake by macrophages in vitro. J Biol Chem 264:10749-10755.
284. Ma, H., and Kovanen, P.T. 1995. IgE-dependent generation of foam cells: an
immune mechanism involving degranulation of sensitized mast cells with resultant uptake of LDL by macrophages. Arterioscler Thromb Vasc Biol 15:811-819.
285. Lindstedt, K.A., Kokkonen, J.O., and Kovanen, P.T. 1992. Soluble heparin
proteoglycans released from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor-mediated phagocytosis. J Lipid Res 33:65-75.
286. Blenkinsopp, W.K. 1967. Mast cell proliferation in adult mice. Nature 214:930-931. 287. Montgomery, R.I., Lidholt, K., Flay, N.W., Liang, J., Vertel, B., Lindahl, U., and
Esko, J.D. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci U S A 89:11327-11331.
288. Chi, E.Y., and Lagunoff, D. 1975. Abnormal mast cell granules in the beige
(Chediak-Higashi syndrome) mouse. J Histochem Cytochem 23:117-122. 289. Nagle, D.L., Karim, M.A., Woolf, E.A., Holmgren, L., Bork, P., Misumi, D.J.,
McGrail, S.H., Dussault, B.J., Jr., Perou, C.M., Boissy, R.E., et al. 1996. Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome. Nat Genet 14:307-311.
290. Poon, K.C., Liu, P.I., and Spicer, S.S. 1981. Mast cell degranulation in beige mice
with the Chediak-Higashi defect. Am J Pathol 104:142-149. 291. Curran, M.J., and Brodwick, M.S. 1991. Ionic control of the size of the vesicle
matrix of beige mouse mast cells. J Gen Physiol 98:771-790. 292. Yang, V.C., Linhardt, R.J., Bernstein, H., Cooney, C.L., and Langer, R. 1985.
Purification and characterization of heparinase from Flavobacterium heparinum. J Biol Chem 260:1849-1857.
293. Lagunoff, D., and Pritzl, P. 1976. Characterization of rat mast cell granule
proteins. Arch Biochem Biophys 173:554-563.
159
294. Yurt, R.W., Leid, R.W., Jr., and Austen, K.F. 1977. Native heparin from rat peritoneal mast cells. J Biol Chem 252:518-521.
295. Schubert, D., LaCorbiere, M., Saitoh, T., and Cole, G. 1989. Characterization of an
amyloid beta precursor protein that binds heparin and contains tyrosine sulfate. Proc Natl Acad Sci U S A 86:2066-2069.
296. Stathakis, N.E., and Mosesson, M.W. 1977. Interactions among heparin, cold-
insoluble globulin, and fibrinogen in formation of the heparin-precipitable fraction of plasma. J Clin Invest 60:855-865.
297. Kassam, G., Manro, A., Braat, C.E., Louie, P., Fitzpatrick, S.L., and Waisman,
D.M. 1997. Characterization of the heparin binding properties of annexin II tetramer. J Biol Chem 272:15093-15100.
298. Braganza, V.J., and Simmons, W.H. 1991. Tryptase from rat skin: purification and
properties. Biochemistry 30:4997-5007. 299. Sannes, P.L., McDonald, J.K., Allen, R.C., and Spicer, S.S. 1979. Cytochemical
localization and biochemical characterization of dipeptidyl aminopeptidase II in macrophages and mast cells. J Histochem Cytochem 27:1496-1498.
300. Struckhoff, G., and Heymann, E. 1986. Rat peritoneal mast cells release
dipeptidyl peptidase II. Biochem J 236:215-219. 301. Chiu, H., and Lagunoff, D. 1972. Histochemical comparison of vertebrate mast
cells. Histochem J 4:135-144. 302. Karlson, U., Pejler, G., Tomasini-Johansson, B., and Hellman, L. 2003. Extended
substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity. J Biol Chem 278:39625-39631.
303. Peng, B.G., Liu, S.Q., Kuang, M., He, Q., Totsuka, S., Huang, L., Huang, J., Lu,
M.D., Liang, L.J., Leong, K.W., et al. 2002. Autologous fixed tumor vaccine: a formulation with cytokine-microparticles for protective immunity against recurrence of human hepatocellular carcinoma. Jpn J Cancer Res 93:363-368.
304. Tiyaboonchai, W., Woiszwillo, J., Sims, R.C., and Middaugh, C.R. 2003. Insulin
containing polyethylenimine-dextran sulfate nanoparticles. Int J Pharm 255:139-151.
305. Freeman, C., and Parish, C.R. 1998. Human platelet heparanase: purification,
characterization and catalytic activity. Biochem J 330 ( Pt 3):1341-1350. 306. Smith, D.E., and Lewis, Y.S. 1958. Phagocytosis of granules from disrupted mast
cells. Anat Rec 132:93-111. 307. DeSchryver-Kecskemeti, K., Williamson, J.R., Jakschik, B.A., Clouse, R.E., and
Alpers, D.H. 1992. Mast cell granules within endothelial cells: a possible signal in the inflammatory process? Mod Pathol 5:343-347.
160
308. Greenberg, G., and Burnstock, G. 1983. A novel cell-to-cell interaction between mast cells and other cell types. Exp Cell Res 147:1-13.
309. Leak, L.V., and Burke, J.F. 1966. Fine structure of the lymphatic capillary and the
adjoining connective tissue area. Am J Anat 118:785-809. 310. Baluk, P., Fuxe, J., Hashizume, H., Romano, T., Lashnits, E., Butz, S., Vestweber,
D., Corada, M., Molendini, C., Dejana, E., et al. 2007. Functionally specialized junctions between endothelial cells of lymphatic vessels. J Exp Med 204:2349-2362.
311. CasleySmith, J.R. 1967. Electron microscopical observations on the dilated
lymphatics in oedmatous regions and their collapse following hyaluronidase administration. Br J Exp Pathol 48:680-686.
312. Pullinger, B.D., and Florey, H.W. 1935. Some observations on the structure and
functions of lymphatics - Their behaviour in local oedema. British Journal of Experimental Pathology 16:49-61.
313. Nakajima, M., Takeda, M., Kobayashi, M., Suzuki, S., and Ohuchi, N. 2005.
Nano-sized fluorescent particles as new tracers for sentinel node detection: experimental model for decision of appropriate size and wavelength. Cancer Sci 96:353-356.
314. Casley-Smith, J.R. 1980. The fine structure and functioning of tissue channels and
lymphatics. Lymphology 13:177-183. 315. Wrobel, T., Dziegiel, P., Mazur, G., Zabel, M., Kuliczkowski, K., and Szuba, A.
2005. LYVE-1 expression on high endothelial venules (HEVs) of lymph nodes. Lymphology 38:107-110.
316. Gretz, J.E., Norbury, C.C., Anderson, A.O., Proudfoot, A.E., and Shaw, S. 2000.
Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J Exp Med 192:1425-1440.
317. Halin, C., Tobler, N.E., Vigl, B., Brown, L.F., and Detmar, M. 2007. VEGF-A
produced by chronically inflamed tissue induces lymphangiogenesis in draining lymph nodes. Blood 110:3158-3167.
318. Miyasaka, M., and Tanaka, T. 2004. Lymphocyte trafficking across high
endothelial venules: dogmas and enigmas. Nat Rev Immunol 4:360-370. 319. Cahill, R.N., Frost, H., and Trnka, Z. 1976. The effects of antigen on the migration
of recirculating lymphocytes through single lymph nodes. J Exp Med 143:870-888. 320. Suzuki, S., Enosawa, S., Kakefuda, T., Shinomiya, T., Amari, M., Naoe, S.,
Hoshino, Y., and Chiba, K. 1996. A novel immunosuppressant, FTY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation. Transplantation 61:200-205.
161
321. Pham, T.H., Okada, T., Matloubian, M., Lo, C.G., and Cyster, J.G. 2008. S1P1 receptor signaling overrides retention mediated by G alpha i-coupled receptors to promote T cell egress. Immunity 28:122-133.
322. Prieschl, E.E., Csonga, R., Novotny, V., Kikuchi, G.E., and Baumruker, T. 1999.
The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering. J Exp Med 190:1-8.
323. Hagendoorn, J., Padera, T.P., Kashiwagi, S., Isaka, N., Noda, F., Lin, M.I., Huang,
P.L., Sessa, W.C., Fukumura, D., and Jain, R.K. 2004. Endothelial nitric oxide synthase regulates microlymphatic flow via collecting lymphatics. Circ Res 95:204-209.
324. Suffredini, A.F., Fantuzzi, G., Badolato, R., Oppenheim, J.J., and O'Grady, N.P.
1999. New insights into the biology of the acute phase response. J Clin Immunol 19:203-214.
325. Bischoff, F., and Pilhorn, H.R. 1948. The state and distribution of steroid
hormones in biologic systems; solubilities of testosterone, progesterone and alpha-estradiol in aqueous salt and protein solution and in serum. J Biol Chem 174:663-682.
326. Gordon, A.H., Gross, J., O'Connor, D., and Pitt-Rivers, R. 1952. Nature of the
circulating thyroid hormone-plasma protein complex. Nature 169:19-20. 327. Patel, H.M., Boodle, K.M., and Vaughan-Jones, R. 1984. Assessment of the
potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery. Failure of 99m-technetium marker to represent intact liposomes in lymph nodes. Biochim Biophys Acta 801:76-86.
328. Hirano, K., and Hunt, C.A. 1985. Lymphatic transport of liposome-encapsulated
agents: effects of liposome size following intraperitoneal administration. J Pharm Sci 74:915-921.
329. Grutzkau, A., Kruger-Krasagakes, S., Baumeister, H., Schwarz, C., Kogel, H.,
Welker, P., Lippert, U., Henz, B.M., and Moller, A. 1998. Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206. Mol Biol Cell 9:875-884.
330. Niemann, C.U., Abrink, M., Pejler, G., Fischer, R.L., Christensen, E.I., Knight,
S.D., and Borregaard, N. 2007. Neutrophil elastase depends on serglycin proteoglycan for localization in granules. Blood 109:4478-4486.
331. Erjefalt, J.S., Greiff, L., Andersson, M., Matsson, E., Petersen, H., Linden, M.,
Ansari, T., Jeffery, P.K., and Persson, C.G. 1999. Allergen-induced eosinophil cytolysis is a primary mechanism for granule protein release in human upper airways. Am J Respir Crit Care Med 160:304-312.
162
332. Metcalfe, D.D., Bland, C.E., and Wasserman, S.I. 1984. Biochemical and functional characterization of proteoglycans isolated from basophils of patients with chronic myelogenous leukemia. J Immunol 132:1943-1950.
333. Masson, D., Peters, P.J., Geuze, H.J., Borst, J., and Tschopp, J. 1990. Interaction of
chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent on pH. Biochemistry 29:11229-11235.
334. Veugelers, K., Motyka, B., Frantz, C., Shostak, I., Sawchuk, T., and Bleackley, R.C.
2004. The granzyme B-serglycin complex from cytotoxic granules requires dynamin for endocytosis. Blood 103:3845-3853.
163
Biography
Christian Anton Kunder was born July 20, 1979, in New Jersey, USA to Rudolf
and Patricia Kunder. He attended Providence Day School in Charlotte, NC for high
school, graduating in 1997. Then, he matriculated at the University of Pennsylvania,
from which he graduated summa cum laude in the spring of 2001 with a B.A. in Biology
and Biological Basis of Behavior. In the fall of 2001, he entered the Duke University
School of Medicine, and is currently pursuing dual M.D. and Ph.D. degrees in the
Medical Scientist Training Program. His doctoral thesis is entitled "Interactions of Mast
Cells With the Lymphatic System: Delivery of Peripheral Signals to Lymph Nodes by
Mast Cell-Derived Particles." Upon completion of his doctorate, he intends to complete
medical school and then pursue residency training in pathology.