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PCRJFICAï'ION AND PAR= CHARACTERIZAITON OF A NOVEL
MYELOSUPPRESSIVE FACTOR FRûM BONE MARROW
Rodney Peter DeKoter
Department of Microbiology and Immunology
Submitted in partial fuMlment
of the requirements for the degree of
Doctor of Philosophy
Faculty of Graduate Studies
The University of Western Ontario
London, Ontario
Apnl, 1996
O Rodney P. DeKoter 1996
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ABSTRACT
Bone marrow (BM) is the major site of hernatopoiesis in adult mammals; it also
contains several types of immunoregulatory activity, including veto activity and natural
suppressor activity. Nanual suppressor (NS) ceils inhibit cellular proliferative responses
in a dose-dependent, antigen-nonspecific manner. The phenotype of NS cells suggests
these cells are myeloid lineage hematopoietic progenitor cells. NS activity is mediated
by a soluble factor.
Previous results from this laboratory showed that supematants of mouse BM NS
cells were inhibitory for various in vitro proliferative responses. Work was undertaken
to p a the factor responsible for this activity based on inhibition of proliferation of the
murÏne myelornonocytic ce11 line WEHE-3. h order to scale up production, BM-derived
inhibitory activity was prepared from supematants of rat bone marrow cells (BMC).
The rat BMC which produced the activity were shown to be phenotypically similar to
mouse BM NS cells.
Characterization of the BM-derived inhibitory activity showed it to be a low mo-
lecular weight (MW) substance, which was soluble in polar solvents, resistant to
proteolytic enzymes, and negatively charged. Based on these characteristics the BM-
derived inhibitory factor was purified by solid phase extraction, gel filtration chroma-
tography, anion exchange chromatography, normal phase high performance liquid chro-
matography (HEC), and reversed-phase HPLC. Analysis of the purified preparation by
electrospray mass spectrometry showed that it contained a limited number of ions. One
of these ions, m/z 373, was repeatably found to be unique to the biologically active
fraction, which suggested it was the inhibitory molecule. Structural analysis of m/z 373
indicated it was not a simple peptide or nucleic acid structure but may be a unique struc-
ture. Lnhibitory factors produced by NS cells may have important hemoregulatory and
immunoregulatory activities.
DEDICATION
To my parents, Lamy and Wilhelrnina
Although only my n m e appears as author on this thesis, the work described here
could not have been completed without the support of many people. I would fmt of all
like to thank my supervisor, Dr. Kim Singhal, for his patience and guidance in alI aspects
of graduate school me, from the lab bench to the seminar room. The experiments pre-
sented here could not have been perfomed without the collaboration (and fiiendship!) of
Dr. Chao Lin and Mr. XinFeng Jiang. Several advisors were integral in the development
of this project: Dr. Kang Howson-Jan, Dr. Wahid Khalil, Dr. Alan Weedon and Dr. Bhagi
Singh.
1 thank my colleagues in the Singhal lab with whom 1 had the privilege of working
over the years: Mike Parsons, David Ford, Wai Gin Fong, Sean Frost, and Masud
Khandaker. Special thanks go to Colin Anderson and al1 membea of the Carroll lab, past
and present, for their friendship and in making the fifth floor a fun place to work.
1 thank my family for their unfailing love and support.
Finally (and above all!) 1 thank Marietta Drost. It's been a wonderful year. The next will be even better.
. . C E R ' C A T E OF E-ATION .............................................................................. u
... ABSTRACT .................................................................................................................... rn DEDICATION ................................................................................................................. iv
ACKNOWLEDGEMENTS ......................... ......,.. ................................................... v
TABLE OF CONTENTS ................................................................................................. vi
LIST OF FIGURES ............................................................ x
LIST OF TABLES ............................. ........., ................................. xi . .
ABBREVDWONS .................... ,., ................... .,, ...................................................... m
CHAPTER 1 . INTRODUCTION AND GENEIiAL REVIEW ...................................... 1
1.1 The Immune System ..................................... ... .......................................................... 2 - 1.2 Bone Marrow ............................................................................................................. 3
1.3 Bone Marrow and Hematopoiesis ............................................................................. 4
1.3.1 Stem Cells and Clonal Analysis ........................................................................ 5
1.3.2 Hernatopoietic Growth Factors .......................................................................... 7
1 .3.3 Hematopoietic Negative Regulators .......................................................... 10
1.3.3.1 Evidence for Hematopoietic Negative Regulators .................................... 10
1 .3.3 -2 Isolation of Hematopoietic Negative Regulators ...................................... 12
1.3.3.3 Summary .................................................................................................... 14
1.4 Bone Marrow and Immune Regulation ................................................................... 14
1.4.1 Veto Activity .......... .. ......................................................................................... 15
1.4.2 Natural Suppressor Activity ............................... .. ....................................... 20
1.4.2.1 Discovery of Murine BM Suppressor Activity ......................................... 20
1 A2.2 Murine Neonatal Spleen Suppressor Activity .......................................... 21
1.4.2.3 Murine TLI-Induced Spleen Suppressor Activity ..................................... 22
1.4.2.4 Murine 89Sr-Induced Spleen Suppressor Activity ..................................... 23
1.4.2.5 Murine Cyclophosphamide-Induced Spleen Suppressor Activity ............ 24
1.4.2.6 Murine Adjuvant-Induced Spleen Suppressor Activity ............................ 24
1 .4.2.7 Murine Pregnancy-Induced Spleen Suppressor Activity .......................... 25
1.4.2.8 Murine GVHD-Induced Spleen Suppressor Activity ................................ 25
1.4.2.9 Summary .................................................................................................... 26
1.4.3 Characterïzation of Murine BM NS Cells ............................................. 2 7
1.4.4 Adult BM NS cells in Other Species .............................................................. 29
1.4.5 In I/i»o Biological Activities of Murine BM NS Celis .................................... 30 1.4.6 Evidence That Murine BM NS Cells Act Through a Soluble Mediator .......... 31
3.1.12 Solvent Extractions ....................................................................................... 5 3
3.1.13 Enzymatic Digestion ...................................................................................... 54
3.1.14 Ion Exchange Chromatography ................................................................... 54
3.1.15 Gel Filtration ........................... .,.. ............................................................ 55 3.1.16 Anion Exchange FPLC .................................................................................. 55
3.1.17 High Performance Liquid Chromatography (IIPLC) ..................................... 56
3.1.18 Amino (NY)-HPLC ..................................................................................... 5 6
3.1.19 Reverse-Phase HPLC .................................................................................... 5 6
3.1.20 Liquid Chromatography-Mass Spectrornetry (LC-MS) ...................... .... ..... 57 3.1.21 Unit of Activity ............................................................................................... 57
3.1.22 Statistics .......................................................................................................... 58 3.2 Results ...................... ..., ........................................................................................ 58
3.2.1 Effect of Murine BMC Supernatants on Cell Lines ......................................... 58 3.2.2 C 1 8 Cartridge Extraction of Rat Tissue Supematants ...................................... 58 3.2.3 Effect of Rat BM C 18 Extracts on WEHI-3 Viability ...................................... 59
............ 3.2.4 Effect of Elutriated Rat BMC Supematants on WEHI-3 Proliferation 64
.. 3.2.5 PreLiminary Charactenzation of the Inhibitory Factor .............................. ... 64
3 . 2.5. 1 Didysis ........... ,. ......................................................................................... 64
3.2.5.2 SoIubility ................................................................................................... 72 3.2.5.3 Susceptibility to Proteolytic Enzymes ................................................... 75
3.2.5.4 Ion Exchange Chromatography ................................................................. 80 3.2.5.5 Gel Filtration Chromatography ............................................................... 80
3.2.6 Establishment of a Standard Curve for rat BM-Denved Inhibitory Activity ......... 84 3.2.7 Effect of BM-Denved Inhibitory Activity on Proliferation of
Myeloid Cell Lines ................................................................................... 89
........................... 3.2.8 Scale-Up of Production of BM-Denved Inhibitory Activity 89 3.2.9 Purification of BM-Derived uihibitory Activity ........................................... 92
3.2.9.1 Gel Filtration ...................................................................................... 92
3 .2.9.2 Anion Exchange FPLC ......................................................................... 9 8 3.2.9.3 Amino w) HPLC ................................................................................. 9 8 3.2.9.4 Reversed-f hase HPLC ........................................................................... 101
3 -2.10 Characterization of BM-Derived Inhibitory Activity .......................................................................... Using Mass Spectrometry 106
3.2.10.1 Electrospray MS Analysis of F'urifîed BM-Derived Inhibitory Activity .... 106 3.2.10.2 Identification of an Ion Associated With BM-Derived
Inhibitory Activity Using LC-MS ............................................................ 109
3 .2.10.3 Daughter Ion Analysis of mlz 373 ......................................................... 109
3.2.11 Summary of Results ...................... .... .................................................... 112
CHAPTER 4 . DISCUSSION AND FUTURE WORK ............................................ 116
4.1 Discussion ......................................,........................ 117 ....................... ........................................................... 4.1.1 Reliminary Work .... 117
......................................................................................... 4.1.2 Biological Activity 119 4.1.3 Purification ............................... ...,. ................................................................. 120
.............................................................................................. 4.1 -4 Characterization 122 .................................................................................................... 4.1.5 Simcance 123
4.2 Future Work ........................................................................................................... 124
4.2.1 Scale-Up of BM-Derived Inbibitory Factor Production ................................. 124 4.2.2 Final Structural EIucidation ............................................................................ 124
4.2.2.1 Meîhylation analysis ............................ .... ................................................ 125 ............................. 4.2.2.2 Fast Atom Bombardment (FAB) M a s Spectrometry 125
.......................................... 4.2.2.3 Nuclear Magnetic Resonance Spectroscopy 125 ................................................................... 4.2.2.4 Ion Trap M a s Spectrometry 126
................................................................................. 4.2.2.5 Chernical Synthesis 126 4.2.3 BioIogical Studies ..... ...................................................................................... 126
4.2.3.1 Cellular Source and Target ...................................................................... 126 4.2.3.2 In Vitro Myelopoiesis ......................................................................... 127
................................................................................ 4.2.3.3 Stem Ce11 Protection 127 ................... ........................................... 4.2.3 -4 Cancer Therapy ...............,... 127
4.2.3.5 Autoimmunity and Inflammation ............................................................ 128 4.3 Conclusions ....................................................................................................... 128
REFERENCES ............................................................................................................. 129
LIST OF FLGURES Figure Description Page
....................................................................................... 1.1 Veto Activiv ........... ...... 18
1.2 A Model of Naturd Suppression ............................................................................ 37
3.1 Cl8 Extracts of rat BM Supernatants are More Suppressive .......................... for WEHI-3 Proliferation than Extracts kom Other Tissues 61
3.2 Cl8 Extracts of BM-Derived Inhibitory Activity are not Cytotoxic for WEHI-3 Cells ............................... .. ......................................................... 63
3.3 Countedow Cenaifugai Elutriation of Rat BMC ...................... .... ........... 66. 67
3.4 Supernatants of Elutriated Rat BMC are Directly Suppressive for WEHI-3 Cell Proliferation ........................................................................ 69
3.5 C 18 Extracts of Elutriated rat BMC Supernatants are Suppressive ................................................. for WEHI-3 Cell Proliferation ................. ..., 71
3.6 Inhibitory Activity in BM and Spleen Cl8 Extracts is Dialyzable ......................... 74
3.7 Gel Filtration Chromatography of BM Cl8 Extracts .............................................. 83
3.8 Inhibition of WEHI-3 CeU Proliferation by BM-Derived Inhibitory Activity Prepared by Cl 8 Extraction and Gel Filtration Chromatography .................. 86
3.9 Standard Curve of BM-Derived Inhibitory Activity .......................................... 88
3.10 BM-Derived Inhibitory Activity Suppresses ProMeration of Myeloid Cell Lines ..... 91
3.11 Rat BMC Decrease in Viability and in Inhibitory Activity Production Over Four Days of Culture ............................................................................. 94
3.12 Gel Filtration Chromatography ......................................................................... 97
........................................................................................ 3.13 Anion Exchange FPLC 100
............................................................................................ 3.14 Amino (NtZ) HPLC 103
.......................................................................................... 3 . 15 Reverse-Phase HPLC 105
........ 3.16 Ionspray Mass Spectrometry of Med BM-Derived Inhibitory Activity 108
3.17 Correlation of m/z 373 With Biological Activity by LC-MS .............................. 111
3.18 Daughter Ion Analysis of the Ion 373+ ............................................................... 114
LIST OF TABLES
Table Description Page 3.1 HexaneWater Partition of Cl8 Extracts. pH 3.0 .................... ... .................... 76 3.2 Hexanemater Partition of Cl8 Extracts, pH 7.0 .............................................. 7 7
3.3 Methylene ChlondelWater Partition of C 18 Extracts, pH 3.0 ................................. 78 ................................................................ 3.4 Enzymatic Digestion of BM Cl8 Extract 79
......................... 3.5 Fractionation of BM Cl8 Extract by Anion and Cation Exchange 81
3 -6 Purification Table ................................................................................................... 9 5
Ab - ALS- BCG - BEV-E - BM - BMC - BSF - CFA - cm - CFU-G - CFU-GM - CFU-M - CFU-S - CID - CM - ConA - CTL - DE& - ECM - EI - ES - FAB - FACS - FCS - GC- G-CSF - GM-CSF - GSL - GVHD - HBSS - HLA - HPLC - HPP-CFU -
IL - LIF - LPS - LTC-IC - M-CSF - MHC - M P - l a - MLC - MLR - MS - M W - NK - NMR - NP-HF'LC - NS - NZB - PFC -
ABBREVLATIONS Antibody Anti-Lymphocyte S e m Bacillus Calmette-Guérin Blast Forming Unit-Erythrocyte Bone Marrow Bone Marrow Cell Bone Marrow Derived Suppressor Factor Complete Freund's Adjuvant Colony Foming Unit Colony Forming Unit-Granulocyte Colony Fonning Unit-Granulocyte-Macrophage Colony Forming Unit-Macrophage Colony Forming Unit-Spleen Collision-Induced Dissociation Carboxymethy 1 Concanavalin A Cytotoxic T Lymphocyte Diethyl Aminoethyl ExtraceUular Matrix Elec trou impact Embryonic Stem Fast Atom Bombardment Fluorescence-Activated Ce11 Sorting Fetal Calf Serum Gas Chromatography Granulocyte-Colony Stimulating Factor Granulocyte-Macrophage-Colony Stimulating Factor Gl ycosphingolipid Graft Versus Host Disease Hank's Balanced Saline Solution Human Leukocyte Antigen Hïgh Performance Liquid Chromatography High Proliferative Potential-Colony Fonning Unit Immunoglobulin Interferon Interleukin Leukemia Inhibi tory Factor Lipopolysaccharide Long Term Culture-Initiating Cell Macrophage-Colony Stimulating Factor Major Histocompatibility CornpIex Macrophage Inflammatory Protein- l -Alpha Mixed Leukocyte Culture Mixed Lymphocyte Reac tion Mass Spectrometer Molecular Weight Natural Killer Nuclear Magnetic Resonance Normal Phase High Performance Liquid Chromatography Naturd Suppressor New Zealand Black Plaque Forrning Cell
RF - RP-HPLC - SCF - SC1 - SPANS - 89Sr - SRBC - TCR -
Radio Frequency Reversed-Phase High Performance Liquid Chromatography Stem CeU Factor Stem CeU Inhibitor Splenic Pregnancy-Associated N a m Suppressor Strontium-89 Sheep Red Blood CeU T Cell Receptor Transfonning Growth Factor-Beta Total Lymphoid Irradiation Turnour Necrosis Factor
CHAPTER 1
INTRODUCTION AND GENERAL REVIEW
1.1 The Immune Svstem
The immune system consists of a dif ise network of cells primarily of the lym-
phoid and myeloid lineages. The immune system protects theebody against invasive and
infectious organisms, which may otherwise cause disease and kill the host (Janeway Jr.
and Travers, 1994). Therefore one of the most important properties of the immune sys-
tem is that it discriminates foreign organisms from "self", a property which is calied self
tolerance (Janeway Jr. and Travers, 1994). Tolerance to self and reactivity to foreign
organisms occur in the body at biochemical and cellular levels. The alternative pathway
of the complement cascade and natural antibody are biochernical defenses against for-
eign organisms; while at the cellular Ievel lymphocytes, macrophages, and a series of
related cells including splenic dendritic cells and epidermal Langerhans cells defend the
host (Janeway Jr. andTravers, 1994; Janeway Jr., 1992)- Cell-rnediated irnmunity can be
further divided into innate immunity, which is present at ali tirnes and depends on con-
served receptors; and adaptive irnmunity, which is mediated by clonal selection of
lymphocytes and depends on remangement of receptor genes in B cells (Tonegawa,
1983) and T cells (Hedrick et al. 1984; Yanagi el al. 1984).
The cel1s of the marnmalian immune system are generated in and mature in pn-
mary lymphoid organs, including bone mmow and thymus. Immune cells mature fur-
ther in secondary Lymphoid organs, including lymph nodes, spleen, and gut-associated
lymphoid tissues. Lyrnphoid and myeloid immune cells develop from hematopoietic
stem cells in the bone marrow (Janeway Jr. and Travers, 1994).
Several methods of studying immune responses in vitro helped to advance under-
standing of immune regulation. One technique pivotal to the sîudy of the immune sys-
tem was the histolytic plaque-fomiing cell (PFC) assay, in which B lymphocytes previ-
ously sensitized to erythrocyte antigens are immobilized in a field of erythrocytes. The
B lymphocytes produce anti-sheep red blood cell (SRBC) antibody which in the pres-
ence of complement lyses the SRBC, producing clear plaques (Jerne et al. 1974). This
technique allowed the quantification of antigen-specinc B lymphocytes derived from lym-
phoid organs. An important addition to this technique was the Mishell and Dutton in vitro
culture system, in which the effect of adding or deleting cells or soluble factors on the
developrnent of an immune response could be studied (Mishell and Dutton, 1967). A
second type of important technique was the mixed lymphocyte reaction (MLR), which
allowed quantification and measurement of specificity of cellular responses for the first
tirne (Bach and Hirschom, 1964). PFC and MLR assays allowed extensive investigation
of the nature of immune regulation.
Three major types of cellular imrnunoregulation have been described. First, clonal
deletion or inactivation is the removal of unwanted clones of immune ceUs by anergy
(Jenkins. 1992) or apoptosis (Surh and Sprent, 1994) of the reactive ceus. Second, sup-
pression is the active inhibition of an immune ce11 by a suppressor ce11 or factor (Gershon,
1975; Bloom et al. 1992). Finally, idiotypic regulation is the regulation of immune cells
by a network of complementary receptor idiotopes and idiotypes (Urbain, 1986). Theo-
retically, immune regulation exists to ensure that one clone of cells does not completely
dominate the immune system; the immune systern depends on maintenance of diversity
(Janeway Jr. and Travers, 1994).
1.2 Bone Marrow
Bune marrow in humans consists of 30-50 mgkg of body weight, making it one of
the largest organs of the body (Wickramasinge, 1975). It is dso one of the rnost complex.
Bone marrow is radially organized, with blood flow from the capillary bed dong the
inside surface of endosteal bone flowing towards the central vein through the venous
sinuses m e r and Williams, 1995). The cell walls of the venous sinuses consist of
endothelid cells called "barrier cells", which have a remarkable ability to proliferate and
increase in surface area in response to demands for more hematopoietic cells (Yoder and
1995). Hematopoiesis occurs only in extravascular space, and is guided by a
complex extracellular ma&, or ECM (Yoder and Williams, 1995). The cells of the
ECM include fibroblasts, macrophages, and adipocytes, in addition to developing stem
and progenitor cells (Mayani et al. 1993). Important molecules of the ECM include the
colIagens, glycoproteins (fibronectin, laminin, thrombospondin, hernonectin, tenascin),
and g~ycosaminoglycaus (hyaluronic acid, chondroitin, dermatan, and heparan sulphate)
voder and Williams, 1995)- The most primitive hematopoietic cells are located dong
the bone endosteum; these move towards the central vein dong hematopoietic "cords" as
they proliferate and differentiate (Yoder and Williams, 1995). The ECM plays a role of
physical support and dynamic regulation in hematopoiesis (Mayani et al. 1993).
1.3 Bone Marrow and Hernatopoiesis
Hematopoiesis, the process of blood ce11 formation, occurs in several sites during
mammalian development. In the rnouse, the earliest detectable colony-forming uni&
( C m appear in the yolk sac at day 8 of embryogenesis. CFU migrate from the yolk sac
and initiate hematopoiesis in the iiver by day 10- 11 of embryogenesis and in the bone
marrow and spleen by day 15 (Metcalf and Moore, 1971). By two weeks of age the bone
marrow is the primary hematopoietic organ in the mouse (Till and McCulloch, 1961;
Lord and Dexter, 1995).
Tt has been hypothesized that the pluripotent stem ce11 differentiates into lym-
phoid and myeloid stem cells, which in turn differentiate into Iymphoid and myeloid
progenitor cells. The lymphoid lineage includes B andT lymphocytes, which arise from
lymphocytic progenitors in bone marrow. B lymphocytes arise from an early progenitor
common to myeloid cells (Hirayama et al. 1992; Cumano and Paige, 1992) and differen-
tiate in bone marrow according to a well-characterized series of steps for which discrete
markers are known (Jacobsen and Osmond, 1990). Progenitors of T lymphocytes are
continually released from bone marrow throughout aduIt Me (Donskoy and Goldschneider,
1992) but very Little about their early differentiation is known (Golunski and Palacios,
1994). Differentiation of T cells once they enter the thymus is well charactenzed, and
like B ceus, progresses through a series of steps for which discrete markers are h o w n
(Groettrup and von Boehmer, 1993). Progenitor cells of the myeloid lineage have the
potentid to differentiate into granulocytes (including neutrophils, basophils and
eosinophils), monocytes/macrophages, dendritic cells, and rnegakaryocytes (Inaba et al.
1993).
1.3.1 Stem Cells and Clonal Analvsis
Stem cells have been defined in an important review by Potten and Loeffler (1990)
as follows: "undifferentiated cells capable of (a) proliferation, (b) self maintenance, (c)
the production of a large number of differentiated, functional progeny, (d) regenerating
the tissue after injury, and (e) a flexibility in the use of these options". Hematopoietic
stem cells with these properties have been isolated by ff ow cytometry with the cell sur-
face marker phenotype Thyl. 1" Lin- Sca-1' in the mouse wchida and Weissman, 1992)
and CD34+ HLA-DR+ CD38- in the human (Leon and Terstappen, 1992). These cellular
populations are still somewhat heterogeneous in morphology and long-term reconstitut-
ing activity, but can be further subdivided on the basis of supravital staining and other
cell surface markers.
Stem cells represent less than 0.1% of adult BMC and therefore theoretically
have tremendous potential to give nse to daughter cells. There are at least two possible
explanations for how this potential is regulated. There may be 1) enough stem cells so
that single cells c m in tum differentiate and repopulate the tissues for the lifetirne of the
animal (also known as the "clonal succession7' theory), or 2) A small nurnber of original
stem cells are capable of a high degree of proliferation and self-renewal (Lord and Dex-
ter, 1995). Experiments with send transfer of stem cells show that stem cells can self-
renew, but that some capacity to differentiate is lost at each ce11 division (Brecher et al.
1993).
The detection of single precursor cells by observing the colonies to which they
give rise is known as clonal analysis. The colony-fomiing unit spleen (CFU-S) tech-
nique, published by T i and McCulloch in 196 1, was the Fust description of clonal analysis
for mouse hematopoietic stem cells. They found that when hematopoietic cells were
injected into irradiated mice these cells would migrate to the spleen and form visible
colonies of differentiating ceiIs after 8-12 days. This was the E s t assay which could be
used to quanti@ the number of stem cells in a cell preparaiion and therefore study fac-
tors controlling hernatopoiesis. The CFU-S provided the first biological evidence for
the existence of a single type of pluripotent stem cell which gives rise to al1 blood line-
ages. 1t is now known that the day 12 CFU-S is not the earliest pluripotent stem celI but
a closely related transition cell (Lord and Dexter, 1995).
The technique of using semisolid media for in vitm clonal analysis was discov-
ered independently by Donald Metcalf in Melbourne, Australia and Leo Sachs at the
Weizmann Institute in Israel (Metcalf, 1994). Clonal assay systems that use soft agar or
methylcellulose as semisolid media now include the BFU-E, CFU-G, CFü-M, CFU-
GM, and CFU-Mix (Moore and Belmont, 1993)- These culture systems have been used
to map differentiation pathways of hematopoietic progenitors, in particular of the my-
eloid lineage (Metcalf, 1994).
ln vitro analysis of the hematopoietic stem ce11 has been advaiiced by several
long-term culture techniques. Among the earliest in vitro hematopoietic culture systems
was the Dexter system which generated primariiy macrophages and granulocytes (Dex-
ter and Lajtha, 1974), and the Whitlock-Witte system which generated primarily B cells
(Whitlock and Witte, 1982). More recent techniques for in vitro stem ce11 analysis in-
clude the long term culture-initiating ceU @.TC-IC) assay (Sutherland et al. 1990) and
the culture of embryonic stem (ES) ce11 lines (Wurst and Joyner, 1993). Other types of
culture system include the CFU-Blast (CFU-BI), and the high proliferative potential
colony forrning ceU assay (HPP-CFU) (Moore and Belmont, 1993).
1.3.2 Hematopoietic Growth Factors
The f k t hematopoietic growth factor described was erythropoietin, which in 1906
was hypothesized to be the factor active in promothg growth of chicken red blood cells
(reviewed in Metcalf and Nicola, 1995). Therefore the concept that growth of cellular
Lineages depends on specific growth factors has been in existence for many years. How-
ever, it is only in the last twenty years that many more growth factors have been punfied,
characterized and rnolecuIarly cloned (Callard, 1990a). A growth factor is theoreticdy a
molecule which promotes growth and differentiation of a specific ceLIu1a.r Lineage, al-
though most cloned growth factors have highly pleiotropic effects. Growth factors may
act by suppressing apoptosis in target cells, dowing these ceils to proliferate and differ-
entiate according to their genetic program (Metcalf, 1994).
One of the central questions in the study of hematopoiesis is whether growth fac-
tors are sufficient to maintain stable numbers of blood cells, or whether excess numbers
of blood cells are produced and must be down-regulated by a negative feedback system
(Metcalf and Nicola. 1995). In erythropoiesis there does seem to be negative feedback of
erythrocyte production by high oxygen levels (MetcaIf and Nicola, 1995)- Donald Metcalf
concluded, afier a lifetime of studying the myeloid colony-stimulating factors, that "at
present, outside the erythroid system there is no substantive evidence for the existence of
regulatory mechanisms to monitor blood ce11 levels and to initiate corrections in
hernopoiesis where necessary" (Metcalf and Nicola, 1995). There is, however, indirect
evidence for the existence of such regulatory mechanisms. The followlng sections will
briefly review the properties of some growth factors, with emphasis on the myeloid line-
age, and will discuss the evidence for the existence of negative regulatory factors.
There are a large number of well-characterized growth factors for the lymphoid
heage, a detailed review of which is beyond the scope of this thesis. These factors
include IL-2, IL4, IL-7, EN-aIPIy, and T W a (Callard er al. 1990b). The growth fac-
tors which support proliferation and differentiation of the myeloid lineage are among the
best characterized of all soluble factors (Clark and Karnen, 1987). Growth factors with
effects on myeloid progenitor cells include stem cell factor, leukernia inhibitory factor,
IL-1, IL-6, erythropoietin, M-CSF, G-CSF, GM-CSF, IL-3, IL-5, and thrombopoietin.
Stem Cell Factor: Stem ce11 factor (SCF) (also called Steel factor or c-kit ligand)
was originaliy characterized by a mutation in mice at the steel locus 1990). Mice
with the defective Steel gene have severe hematopoietic defects, including the complete
absence of several hematopoietic lineages. The receptor for SCF, the c-kit tyrosine ki-
nase, is coded for at the white spotting locus in rnice. SCF? which was purified and hlly
characterized in 1990, may be a "survival factor" for stem cells (Zsebo et al. 1990;
Godin et al. 1991). SCF can stimulate proliferation of progenitor ceUs when acting
together with other CSFs (Moheux et al. 1991).
Leukentia Zn hibitory Factor: Leukernia inhibitory factor (LIF) was originally
purified on the basis of its being able to induce differentiation in the Ml myeloid leukernia
ce11 line (Hilton et al. 1988). Upon sequencing it was quickly discovered that it was
identical to embryonic stem (ES) cell differentiation inhibitory activity (DIA), a soluble
factor capable of maintaining ES cell lines in their pluripotent state (Hilton, 1992). Pu-
rified recombinant LIF c m maintain ES cells for an indefinite number of passages and
therefore has made a huge contribution to the technology for producing transgenic rnice
(Williams el al. 1988; Wurst and Joyner, 1993). In vivo, LIF production appears local-
ized to the metrial gland on day 4 of munne embryogenesis (Hilton, 1992). The produc-
tion of LIF knockout mice has c o n h e d an important role for LIF in vivo, since LIF -/
- female mice are sterile and have greatly reduced levels of progenitor cells including the
CN-S, B N - E , and CFU-GM (Escary et al. 1993).
Inrerleukin-1: Interleukin-1 (IL-1) is a 12- 15 kDa glycoprotein produced by and
acting on many cells in the body. IL- 1 does not by itself stimulate hematopoiesis in vitro
but can act s ynergisticdy with other cytokines including GM-CSF, G-CSF, IL-3 and M-
CSF (Moore et al. 1990; di Giovine and Duff, 1990).
Interleukin-6: Interleukin-6 (IL-6) is a 22-29 kDa glycoprotein which, Like IL-1,
is produced by many cells in the body and is highly pleiotropic (Van Snick, 1990). Like
IL-1, IL-6 does not act in vitro by itself but can act synergisticdy with GM-CSF, M-CSF
and IL-3 in inducing proliferation of granulocytic and mast ce11 precursors (Tsujino et al.
1993).
Er-ythroooietin: Erythropoietin is a polypeptide of 18,236 Da (30.4 kDa
glycosylated) produced by kidney, liver and bone marrow cells. Erythropoietin s h u -
lates proliferation and daerentiation of erythroid progenitors in bone marrow. Erythro-
poietin production is regulated by relative oxygen levels in red blood cells (Garnick,
1990; Metcdf and Nicola, 1995).
Macro~haae Colonv-Stimulatina Factor: Macrophage colony-stimulating factor
(M-CSF) is a glycoprotein of 18-26 kDa produced by monocytes, fibroblasts, and en-
dothelial cells. M-CSF was the £ k t colony-stimulating factor to be purified in 1975.
The major function of M-CSF is to stimulate dinerentiation of macrophages fiom mono-
cytic precursors (Clark and Kamen, 1987; Metcalf and Nicola, 1995).
Grunuloqvte Coloq-Stimulatinp Factor: Granulocyte colony-stimulating factor
(G-CSF) is a polypeptide of 19,06 1 Da (22-25 kDa glycosylated) produced by monocytes
and fibroblasts. G-CSF stimulates primarily neutrophil colony growth from granulocytic
progenitors (Clark and Kamen, 1987; Metcûlf and Nicola, 1995).
Granulocvte-Macrophage Colonv-Stimulatinp Focroc Granulocyte-macrophage
colony stimulating factor (GM-CSF) is a polypeptide of 14,700 Da (24-33 kDa
glycosylated) produced by T cells, endothelid cells, and fibroblasts. In addition to pro-
moting differentiation into granulocytes/macrophages GM-CSF stimulates growth of the
myeloid ce11 common progenitor (Clark and Kamen, 1987; Metcalf and Nicola, 1995).
Interleukin-3: Interleukin-3 &3), also calied multi-CSF, is a polypeptide of
16,200 Da (22-34 kDa glycosylated) produced by T cells and some myelomonocytic
leukemias. IL3 stimulates growth of the myeloid common progenitor in a fashion simi-
lar to GM-CSF (Clark and Kamen, 1987; Metcalf and Nicola, 1995).
Interleukin-5: Interleukin-5 (IL-5) is also known as "eosinophil difîerentiation
factor". IL-5 is a glycoprotein of 30 kDa produced by T cells. The major fûnction of IL-
5 seems to be the stimulation of differentiation of eosinophils from eosinophilic precur-
sors in vitro. IL5 is aIso a maturation factor for B ceus, up-regulating IgM and IgA
production (Sanderson et al. 1988). The genes for IL-3, IL-4, IL-5 and GM-CSF have a
high degree of homology and are structurally related (Sanderson et ai. 1988). GM-CSF,
IL-3 and IL-5 receptors share a common subunit, the beta chah; therefore there may be
"cross-talk" between signals from these three cytokines (Sanderson et al. 1988; Lopez et
al. 1992).
Thrornbopoietin: A growth factor for megakaryocytes cailed thrornbopoietin or
c-Mpl ligand has recentiy been described (de Sauvage et al. 1994). Thrombopoietin is a
38 kDa protein which acts as a growth and differentiation factor for megakaryocyte pro-
geni tors.
1.3.3 Hematopoietic Negative Remrlators
As reviewed above, it is accepted that hematopoiesis is regulated by growth fac-
tors. However, it is not clear whether hematopoiesis regulated by growth factors alone or
by a balance of positive and negative growth regulatory factors. There is some evidence
that the effects of growth factors can be reversed by inhibitory factors, indicating the
existence of circuits of hematopoietic regdation.
1.3.3.1 Circumstantial Evidence for Hematopoietic Negative Regulators
In the absence of direct evidence for regulatory circuits, only two situations are
acceptable as evidence for the existence of negative regulators of hematopoiesis in vivo:
1) situations where hematopoiesis is downregulated from normal levels by factors other
than stem ceil exhaustion, and 2) situations where removal of a factor results in higher
îhan normal constitutive levels of hematopoiesis. Evidence for situations in which
hematopoiesis is downregulated from normal levels cornes from studies of bone marrow
transplantation, acute leukernia, and gr&-vs-host disease.
First, syngeneic bone marrow transplantation studies have indicated the existence
of a phenomenon known as syngeneic host resistance. Syngeneic donor BMC do not
engrafi unless the recipient is conditioned by rernoval of most of its hematopoietic cells.
The resistance to engraftment is not caused by antigen-specific or NK-mediated rejec-
tion of donor cells, since the effect is apparent even in fully syngeneic sixain combina-
tions. The effects of the conditioning regime can be explained aitemately as creation of
space (Blazar et al. 1988) or as removal of regulatory cells (Sadelain et al. 1989). Since
it cannot be demonsvated that there is a lack of hematopoietic space in BM, the most
reasonable explanation of host resistance is that the recipient's hematopoietic cells nega-
tively regulate the entry of donor stem cells into cycle (Sadelain et al. 1990a).
Second, leukernic cells have been shown to negatively regulate hematopoiesis. In
hurnans, acute leukemia is often accompanied by severe neutropenia (Loughran Jr., 1993).
The possible explanations are 1) hematopoiesis is inhibited by lack of space, and 2)
leukernic cells directly inhibit growth of hematopoietic cells. In support of the latter
hypothesis, it has been shown that human acute leukemia cells can produce unidentified
factors which directly inhibit hematopoiesis in vitro (Loughran Jr., 1993; Najman et al.
199 1).
Third, graft-vs-host disease is a disease of transplant recipients (especiaily of BM
transplants) which consists of donor graft-derived ceUs attacking and killing recipient
tissues (Parkman, 1991). Paradoxical to this immune activation is a general imrnuno-
suppression of the recipient including lower than normal NK and CTL responses (Lum,
1990). Immunosuppression of the recipient is partially due to inhibition of hematopoiesis
by unidentifïed regulatory factors present in GVHD States (Hakim and Shearer, 1990).
1.3.3 -2 Isolation of Hematopoietic Negative Regdatory Factors
The most direct evidence for the existence of negative regdators of hematopoiesis
cornes from the isolation of molecules with these activities. The isolation and charac-
terization of several hematopoietic regulatory molecules resulted from research on mi-
totic inhibitors called "chdones". The chalone theory stated that each tissue has its own
regulatory chalone which originates from the mature cells of the population and is a
feedback inhibitor of growth of the immature cells of the population (Bullough and
Laurence, 1968). For exarnple, Rytomaa and Kiviniemi (1968) described a granulocytic
chalone partially isolated fiom mature granulocytes, which regulated granulocytopoi-
esis.
Lord and coIleagues (1976) extensively characterized a BM-derived factor with
inhibitory activity for proliferation of CFU-S. The 50-100 kDa protein factor was iso-
lated from sdine supernatants of normal BMC but could not be isoIated from BMC of
irradiated mice. They found the BMC producing the inhibitor to be low density, adher-
ent to plastic, phagocytic, radioresistant, negative for the cell surface marker Thy-1, and
positive for the Fc receptor (Wright et al. 1980). The cells producing the granulocytic
chalone were found in a different density fraction than the CFU-S (Lord and Wright,
1980).
Graham et al (1990) later purified and sequenced the CFU-S inhibitor described
by Lord which they termed SC1 for stem cell inhibitor. This 8000 Da peptide molecule
was identical to the chemokine-family protein macrophage infiammatory protein- 1-a
(MIP-la) (Graham et al. 1990; Oppenheirn et al. 1991). The stem cell inhibitory activ-
ity of MIP-la operates in the picomolar range while its more widely cited inflammatory
properties operate in the micromolar range (Graham et al. 1990). MIP-la acts as a
inhibitor for early stem cells including the CFW-S, but does not act on the earliest long-
term reconstituting stem cells or later cells such as granulocyte-macrophage progenitors
(Quesniaux et al. 1993). It is not clear what the role of MIP-la is in vivo as a stem cell
regulator since MP-la-knockout rnice have apparently normal hematopoiesis (Cook et
al. 1995).
The original work by Bullough and Laurence (1968) on mitotic chalones led to
the purification and sequencing of two peptide chdones with regdatory activity for
hematopoiesis. A peptide with the amino acid sequemeAc-Ser-Asp-Lys-Ro (AcSDKP)
was purified from fetal calf bone marrow on the basis of its ability to inhibit CN-S
(Frindel and Guigon, 1977; Lenfant et al. 1989). This peptide is apparently derived from
the N-temùnal end of thymosin-@ (Lenfant e l al. 1989). Synthetic AcSDKP inhibits
CFU-S but does not afTect CF'U-GM or proliferation of ce11 lines (Monzepat and Frindel,
1989).
A second peptide with the amino acid sequence pGlu-Glu-Asp-Cys-Lys-OH
(pEEDCK) was purified from mature human granulocytes on the basis of its ability to
inhibit CFU-S (Paukovits and Laerum, 1982). The sequence pEEDCK is apparendy
derived from the alpha subunit of the inhibitory G-protein Gi (Moser and Paukovits,
'99 1). Besides inhibition of CFU-S, pEEDCK inhibits CF'U-GM, LPS-induced B ce11
proliferation, and proliferation of various cell lines (Laemm et al. 1990). Because of its
CFU-S inhibitory properties pEEDCK is being used in clinical trials as an agent that
might protect stem cells from darnage by chemotherapeutic agents (Paukovits er al. 1993 ;
Moser and Paukovits, 199 1). Interestingly, rnice immunized against pEEDCK have higher
than normal levels of CFU-S and myeloid progenitor ceii proliferation, indicating that
this peptide functions in vivo as a negative regulator of hematopoiesis (Moser and
Paukovits, 1991).
TGFB is a 25 kDa protein dimer which was purified and cloned on the basis that it
was a growth factor foi fibroblasts (Sporn et al. 1986). TGFB inhibits many in vitro
colony forming assays including CFU-GM, BFU-E and CFU-Meg but does not affect
CFLT-ET CFU-G or C m - M (Keller et al. 1988). There is evidence that TGF, inhibits
proliferation of early stem cells since it can protect stem cells from radiation (Bonewald,
1992). Mice wiîh the genes forTGFB, deleted have hyperproliferation of blood cells and
develop multifocal inflammatory disease, suggesting that this protein may function in
vivo as a hematopoietic regulator (Christ et al. 1994).
Several other purified and characterized molecules are inhibitory for myelopoi-
esis in vitro and in vivo. These include acidic isofemtins, lactoferrin, turnour necrosis
factor alpha (TNFa), lymphotoxin, interferon gamma (ENy), prostaglandin E,, - and
gIycosphingolipids (GSLs) (Broxmeyer et al., 1987; Kaucic et al. 1994). Each of these
have been proposed to be endogenous negative regulators of myelopoiesis, although
there is Little direct evidence they play a role in vivo (Broxmeyer et al., 1987).
1.3.3.3 S u m m a r y
Situations in which normal levels of hematopoiesis are suppressed provide evi-
dence for negative regulation of hematopoiesis. These situations include host resistance
in syngeneic BM transplantation, anemia in acute leukernia, and defective hematopoiesis
in acute GVHD. Furthermore, rnolecules with inhibitory activity for hematopoiesis
have been purified and characterized. There is evidence for several of these molecules
being required for hematopoietic regulation in vivo since their inactivation results in
higher than normal levels of hematopoiesis. It is therefore reasonable to hypothesize
that hematopoiesis is controlled by regulatory circuits involving both positive and nega-
tive regulatory factors.
1.4 Bone Marrow and Immune Remdation
The cellular architecture of BM suggests that its primary purpose is the support
of hematopoiesis. However, BM cm also function as an organ of the immune system. It
was shown shortly after the development of the PFC assay that murine BM is an irnpor-
tant site of Ab production in natural and artificially-induced immune responses
(Thorbecke and Keuning, 1953; van Furth et al. 1966; McMillan et al. 1972). BM may
produce as much as 75% of the Ig produced by all the lymphoid organs in the body,
especidy in older mice (&mer et al. 1981). However, BM does not mount a primary
antibody response d e r a single immunization (McMillan et al. 1972; Berner et al.
1981). In fact it has been shown that Ig-secreting plasma cells mature in secondary
lymphoid organs before rnigrating back to BM to produce Ig, predorninantly IgG @ilosa
et al. 1991; Benner et al. 198 1). Benner et al (198 1) concluded that "BM lacks the
appropriate microenvironment andlor quantity or quality of cells (T lymphocytes?
Macrophages?) [sic] required for the earlier steps of induction of immune responses".
It is also possible that developrnent of immune responses in BM is inhibited by
regulatory elements normaliy present in situ. In autoimmune disease States, when
imrnunoregulatory elements are presumably lost, BM becomes a major source of
autoantibody production (Smythe, 1990). Autoantibody production by NZBAV mice is
regulated by a type of suppressor celi found in normal but not autoimmune mice (Smythe,
1990). Furthemore, BMC are fully capable of MLR responses or PFC production when
cells with immune suppressor activity are first removed by various rnethods (Saffran et
al. 199 1; Ryser and Dutton, 1977).
In summary, BMC are capable of producing immune responses but may contain
imrnunoregulatory elements that hold these responses in check. The next two sections
of this review will describe the immunoregulatory properties of BM in greater detail.
These regulatory activities in BM are known respectively as veto and natural suppres-
sion.
1.4.1 Veto Activitv
Veto activity was originally described as the ability of stimulator-type bone mar-
row cells to inhibit a mixed lymphocyte reaction (Muraoka and Miller, 1980). For ex-
ample, if an MLR consisted of type A ceils responding to type B irradiated stimulator
cells, then type B but not type C (third party) bone rnarrow cells could inhibit the reac-
tion. The mechanism of veto activity was found to be the inhibition of development of
responder-type CTL precursors, possibly by the veto ce11 killing the responder ce11 rec-
ognizing it (Miller, 1986). Veto activity was therefore antigen-nonspecific, although
WC-resûicted and not mediated by soluble factors (Muraoka and Miller, 1980). Fig-
ure 1.1 shows a simple mode1 of veto activity.
The identity of the veto ce11 has been investigated by several groups. In early
studies, it was found that veto activity was a natural property of many cloned cytotoxic T
lymphocyte (CTL) lines (Fink et al. 1988). The CTL which could veto MLR responses
could themselves be of any antigen specificity as long as they were recognized by the
responder ce11 type (Fink et al. 1988). However, veto activity is a property of cells found
naturally in bone marrow, thymus and fetal liver (Miller, 1986). Takahashi et al (1990)
identified and cloned a mouse BM veto cell which effectively inhibited CTL generation
in the MLR. This cell was Thy- 1' CD3- CD4- CD8- Class I+ and TCR-gainma'. It was
negative for the T ce11 receptor subunit. alpha, beta or delta. The cloned veto cell also
functioned in vivo since it could prolong responder-type skin grafts in infused mice
(Takahashi et al. 1990).
The mechanism of veto activity has been further investigated in vitro by Miller
and colleagues. They found the following requirements for veto-mediated killing of a
responder cell: 1) The responder cell recognizes the veto cell in a class 1 MHC-restricted
fashion, presumably involving the T ceil receptor (TCR). 2) The responder cell receives
a signal through the a3 domain of its class 1 MHC binding to CD8 on the veto cell. 3) If
the signal is received at a particular stage dunng differentiation into CTL, the responder
ce11 dies by apoptosis (Sambhara and Miller, 1991). According to this model, the veto
ce11 must express CD8 and the responder cell must be MHC class I positive. It is not
clear why the highly effective veto cell cloned by Takahashi et al (1990) was negative for
CD8.
Figure 2.1
Veto Activity
Unidirectional recognition is the basis for veto cell function. An autoreactive T
ce11 recognizes the self antigens of the veto cell and is in tum suppressed. The antigen-
specific receptors of the veto ceil are not engaged (Reproduced from Fink et al., 1988).
There is some evidence ihat veto activity can occur in vivo in mice and humans,
and therefore might be a useful method to control immune responses. One example is
the anti-male (W-Y) antigen transgenic rnouse system. When male cells were injected
into transgenic anti-H-Y female mice, the number of anti-H-Y female T cells decreased
up to 80% after a few days (Zhang et al. 1992). The authors concluded there was "the
existence of functionally deleting antigen-presenting cells (veto celis)" (Zhang et al.
1992). It is difficult to conclude whether deletion of responder T cells was a conse-
quence of donor antigen being processed and presented, or whether intact veto cells in
the donor population played a role. Since not all ce11 types had equal veto activity,
simple infusion of donor-type antigen does not appear to be the explanation.
The existence of cells with veto activity provides a possible explanation for at
least three human in vivo phenornena. First, donor-type blood transfusion before kidney
transplants in humans has long been known to reduce the severity of graft rejection. In
one study it was found that donor-type blood transfusion induced a reduction in the
frequency of anti-donor cytotoxic T lymphocyte precursors, as long as donor and recipi-
ent were matched for one HLA-B and one HLA-DR antigen (van Twuyver et al. 199 1).
This phenornenon rnight be explained by veto activity in the donor-type blood. A second
example is the use of anti-lymphocyte serum (ALS) plus donor-type bone marrow before
transplant to prevent rejection, as pubiished extensively by Wood and Monaco (reviewed
in Monaco, 1991; Wood et al. 199 1 ; Markees et al. 1992). The donor-type marrow
contains veto activity which can prevent rejection of skin grafts in rnice in an MHC-
specific fashion (Wood et al. 199 1). In a third example, it has long been known that in
bone marrow transplantation, T cell depletion of the stem cell fraction being transplanted,
which is intended to lower the incidence of graft-versus-host disease, also reduces the
level of engraftmenf of the donor stem cells (Kaufman et al. 1994). Recently Ildstad and
colleagues used flow cytometry to isolate a donor-type "facilitating cell" which greatly
increased allogenic engraftment of BM in rnice (Kaufman et al. 1994). The ce11 had the
celi surface phenotype CD8+ CD3+ CD45R+ Thy- 1' class IIdidLntcrmediao ap TCR- y6 TCR-
, which they speculated may belong to the dendritic lineage. This cell could be inter-
preted as having veto activity for recipient T ceils, therefore permïtting better engraftment
of the donor-type BMC (Kaufinan et al. 1994). The facilitating cell is now being used in
hurnan bone marrow transplantation pre-clinical trials.
In surnmary, veto activity is ce11 contact-dependent, does not involve soluble fac-
tors, and is MHC-restricted. Veto activity must be carefully distinguished from natural
suppressor activity, which is not cell contact-dependent, is mediated by soluble factors,
and is always MHC-unrestricted. Veto and NS activities are often confused in the litera-
ture since both can be mediated by suppressor cells found naturally in BM. Many in
vitro suppressor activities mediated by other cells such as M( cells or IL-2 activated
CTL can be explained as veto, natural suppression, or a combination of both (Azuma and
Kaplan, 1988).
1.4.2 Natural Su~pressor Activity
1.4.2.1 Discovery of Murine BM Suppressor Activity
Singhal et al were the f ist to observe that syngeneic bone marrow cells added to
in vitm spleen ce11 anti-SRBC cultures inhibitecl the number of plaques produced (Shghal
et al. 1972; Dniry and Singhal, 1974). The cells responsible were low density, nonadherent,
and could not be killed by anti-theta semm and complement. The suppressor cells were
therefore considered not to be T cells. Later studies revealed additional characteristics
of these cells: they were not killed by anti-macrophage serum and complement, they did
nt? ingest carbonyl iron and they were positive for the Fc receptor (Duwe and Singhal,
1979b; Duwe and Singhal, 1979). These tests mled out the possibility of the suppressor
ceil being a mature macrophage.
In addition to Singhal et al a number of other groups published findings related to
mouse bone marrow suppressor celis. Dauphinee and Tala1 (1979) showed that a radio-
sensitive, theta-negative bone marrow cell could suppress primary anti-SRBC PFC in
vitro, while spleen or thymus did not contain cells with such acîivity. Corvese et al
(1980) also showed that mouse bone marrow could inhibit primary anti-SRBC PFC re-
sponses. Their cell resembled the ones described above but was found to digest carbonyl
iron and adhere to plastic and Sephadex G-10. As a result, they concluded that the sup-
pressor was an immature rnyeloid cell. Fmally, Dorshkind et al (1980; Dorshkind and
Rosse, 1982) showed that mouse bone marrow cells (but not red blood cells, thymus cells
or spleen cells) could suppress proliferation of mouse spleen cells in a one-way MLC.
The suppressor ceIls were large, low density, relatively radioresistant, and resistant to
lysis by antibodies against Thy-1, Ig, Ia, and asialo GMl plus complement. This group
called the suppressor ce11 a "natural regdatory cell"; the term natural suppressor (NS)
cell did not corne into use untii several years later.
NS activity has been defined as "the ability of unprirned 'null' cells to suppress
the response of lymphocytes to immunogenic and mitogenic stimuli (Maier et aL, 1986).
Cells with NS activity were described first in adult BM by Singhal et al (1972; 1974)
who must therefore be credited with the discovery of these celIs. Later, other sources of
cells with NS activity were descnbed, including from spleens of rnice which are new-
born, pregnant, undergoing graft-vs-host disease, or are treated with TLI, cyclophospha-
mide, "Sr, or adjuvants.
1 A2.2 Murine Neonatal Spleen Suppressor Activity
It was well known for many decades that while thymocytes from newborn mice
could respond to rnitogens and participate in MLR responses by 18 days of age, splenocytes
can take until2-3 weeks of age to give similar responses. Rodriguez et al (1979) offered
three possible explanations for this observation: 1) newborn spleen contains no func-
tional macrophages and therefore lacks antigen presentation ability, 2) newbom spleen
contains no mature T cells and therefore lacks help for immune responses, and 3) new-
bom spleen contains suppressor cells which block the activities of otherwise functional
macrophages and T cells. In this same paper they showed that T cells purified from
mouse spleen using glass bead-Ig columns were reactive in the MLR by 6-7 days after
birth. They concluded that newborn mouse spleen contains theta-negative suppressor
cells which block the participation of otherwise mature and functional T cells in immune
responses.
Further characterization of murine neonatal spleen suppressor cells was perfomed
by a nurnber of Iabs. Murine neonatal spleen cells suppressing the in vitro ana-SREK
PFC response (Piguet et al. 1981) as well as the mixed lymphocyte reaction (Argyris,
198 1 ; Peeler et al. 1983; Oseroff et al. 1984; Schwadron et al. 1985; Jadus and Peck,
1986; Jadus and Parkrnan, 1986; Hooper et al. 1986; Knaan-Shanzer and Van Bekkum,
1987) have been described. In general these cells were low-density, nonadherent to plas-
tic, not MHC restricted in activity, and negative for lineage-specific cefl surface markers
such as Thy-1, Ig, Ia, Lyt 1, and Lyt2. Piguet et al (198 1) concluded that suppression was
due to monoblastic precursors, since adult spleen is "essentially lymphoid" while new-
bom spleen is "predominantly hematopoietic" with a large proportion of actively prolif-
erating cells. Peeler et al (1983) purified the suppressive population from newbom spleen
by negative selection with anti-Thy-1, anti-Ly anti-Ia, and lectin followed by density
centrifugation with Ficoll. They found that the suppressive fraction consisted primarily
of monocytes and mast cells and therefore concluded that the suppressor ceU was mono-
cytic. Although the above studies did not result in a dennitive description of the lineage
of the neonatal spleen suppressor cell, it was clear from the consensus phenotype that it
was not a mature T cell, B cell or macrophage.
1.4.2.3 Murine TLI-Induced Spleen Suppressor Activity
Total lymphoid irradiation (KI) consists of selective exposure of lymph nodes,
thymus and spleen to graded irradiation while non-lymphoid organs are shielded with
lead. Slavin (1987) extensively studied the effects of TL1 in rodents, which inciuded
induction of "a state that mimics the immature, tolerance-susceptible, fetd or neonatal
immune system". TL1 induced suppressor cells in the spleen which in many ways re-
sembIed, in size, density, and phenotype, the suppressor celis observed in neonatd spleen
(Slavin, 1987; Strober, 1984; Strober et al. Z984a). The spleens of rnice given TL1 Iacked
normal architecture, presumably because the spleen becarne an active hematopoietic or-
gan (Oseroff et al. 1984).
The work of Samuel Strober and associates showed that TLI-induced splenic sup-
pressor cells were identical to neonatal spleen suppressor cells both in spectrum of ac-
tivities and in phenotype. TM-induced suppressor cells were large, low-density null
phenotype (negative forThy 1.2, Lytl, L N , Ig) cells that blocked allogeneic MLR (Oseroff
et al. 1984). Stroberet al cIonedTL1-induced suppressor cells by fusion with a hybridoma
partner and found that these cells had suppressor activity but lacked the original null
phenotype, now being positive for Thy 1.2, Asialo GM1 and H-2Kd while negative for
Lyt 1.2, Lyt2.2, Ig, Mac- 1, FMO, 2C2 and Ia (Hertel-WuH et al. 1984). They went on to
extensively characterize the in vitro and in vivo suppressive activities of these lines (Strober
et al. 1987; Palathumpat et al. 1992b; Hertel-Wulff and Strober, 1988) but is question-
able whether these lines can still be considered "natural" suppressor cells.
1.4.2.4 Murine @Sr-Induced Spleen Suppressor Activity
89Sr is an isotope of Strontium which upon injection is quickly incorporated into
bone and causes the destruction of bone marrow cells by high-energy B-irradiation
(Merluzzi et al. 1978). A consequence of this treatrnent is that hematopoiesis as well as
cells with nonspecific suppressor activity are up-regulated in the spleen. Several studies
showed that rnice treated with 89Sr had spleen cells which could suppress anti-SRBC
PFC responses as effectively as ceUs frorn adult bone marrow (Merluzzi et al. 1978;
Levy et al. 1981). The suppressor ce11 fraction contained many large, low-density ceIls
and was e ~ c h e d for CFU-C, leading to the conclusion that "myelopoiesis is associated
with the generation of suppressor cells" (Levy et al. 198 1 ) .
1.4.2.5 Murine Cyclophosphamide-hduced Spleen Suppressor Activity
Cyclophosphamide is a chemotherapeu tic agent which is cytotoxic for ac tively
proliferating cells. Studies in mice showed that, similar to 89Sr, hernatopoiesis was dis-
rupted by cyclophosphamide injection and moved from the bone rnarrow into the spleen.
Mice injected with 250-300 mglkg cyclophosphamide had by days 4-7 spleen cells highly
suppressive for Con A-stimulated spleen celi proliferation (Nikcevich et al. 1987; Maier
et al. 1989). The suppressive cells were negative for the markers Thy 1.2 and Ig and were
slightly adherent to Sephadex G- 10. Nikcevich et al (1987) found that the activity of
these cells was abolished by culture with leucine methyl ester or catalase plus indometh-
acin, leading to the conclusion that the suppressor ceUs were of the monocyte/macro-
phage lineage.
1.4.2.6 Murine Adjuvant-Induced Spleen Suppressor Activity
Adjuvants are natural or artificial agents which cm up-regulate immune responses
to antigens which might not otherwise provoke an immune response (Janeway Jr. and
Travers, 1994). Bacillus Calmette Guérin (BCG) is a live attenuated strain of bovine
Mycobacterium tuberculosis which is used widely in humans as a vaccine for tuberculo-
sis (Guérin, 1980). BCG has adjuvant properties in that it can stimulate immune resist-
ance against tumours in mice and humans (OId et al., 1959; Guérin, 1980). BCG has
dso been found to induce the generation of suppressor cells in mice which c m block in
vitro CTL generation (KLimpel and Henney, 1978). Bennett et al (1978) were the fkst to
introduce the term "natural suppressor cell" to descnbe the BCG-induced spleen sup-
pressor cell. They found these cells to be large, low-density, Thy-1-, and nylon wool or
plastic adherent. Interestingly they thought these cells to be comparable in phenotype to
horse serum-induced colony-forming progenitor cells (Bennett et al. 1978).
In addition to BCG, many adjuvants have been descnbed which induce suppres-
sor cells in murine spleen. Alum (Ai(OH),) induced spleen suppressor cells which blocked
mitogen responses, PFC responses and CTL generation (Hanna et al. 1980). Injection of
Corynebacterium parvum caused spleen cells to be refiactory to mitogen responses or
MLR (Scott, 1972). E. coli lipopolysaccharide (LPS) induced spleen cells suppressive
for rnitogen responses (Holda, 1992). Finally, complete Freund's adjuvant (CFA), an
emulsion in oil of extracts from Mycobacterium tuberculosis, induced spleen suppressor
cells which blocked the onset of diabetes in NOD mice (Sadelain et al. 1990b; Qin et al.
1993). Spleen suppressor cells induced by adjuvant have not been well-characterized.
1.4.2.7 Murine Pregnancy-Induced Spleen Suppressor Actinty
In allogeneic matings, the fetus represents an ailograft to which the rnother mounts
an immune response (Slapsys and Clark, 1983). The immune response of the mother
against the fetus is suppressed in the local area of the uterus. Suppressor cells have been
descnbed in the uterus (Slapsys and Clark, 1983; Clark et al. 1990), the lymph nodes
draining the uterus (Slapsys and Clark, 1983), and the spleen of pregnant female rnice
(Hoskin et al. 1983). These suppressor cells inhibit in vitro immune responses and may
therefore be part of the mechanism of materna1 tolerance to the fetal allograft (Slapsys
and Clark, 1983; Hoskin et al. 1983).
Splenic pregnancy-associated natural suppressor cells (SPANS) have been de-
scribed as similar in phenotype to neonatal spleen suppressor cells (see section 1.4.2.2).
SPANS are large, low density, are bound by soybean agglutinin, and have the pattern of
ce11 surface markers CD3+4-8- CD45R+ HSA' NK2.1- Asialo GM1- (Hoskin et al. 1983;
Brooks-Kaiser et al. 1992). SPANS are hypothesized to contribute to matemal tolerance
to the fetal allograft (Hoskin er al. 1983; Brooks-Kaiser et al. 1992). SPANS are ac-
tively cycling and therefore resemble an immature progenitor or immature T ce11 (Brooks-
Kaiser et al. 1992).
1 -4.2.8 Murine GVHD-Induced Spleen Suppressor Activity
Acute graft-vs-host disease (GVHD) continues to be one the major problems as-
sociated with allogeneic bone marrow transplantation in humans (Deeg and Atkinson,
1990). GVHD is a complex set of symptorns caused by graft-derived lymphocytes re-
sponding to and attacking recipient (host) tissues. The GVH reaction induces severe
immunosuppression which uicludes induction of suppressor cells as well as maturational
defects in lymphocytes (Lapp et al., 1985).
In mouse models of acute GVHD, the onset of disease has been found to be asso-
ciated with generation of splenic suppressor cells. Splenic suppressor cells develop in
minor (Holda et al. 1985; Holda et al. 1986a) and major (Parthenais and Lapp, 1978;
Sykes et al. 1990; Sykes et al. 1988; Cleveland et al. 1988; hamura et al. 1987) histo-
compatibility rnismatches. Suppressor cells inhibit splenic responses to mitogens and
MLR in an antigen-nonspecific manner (Holda et al. 1986a). Preliminary charactenza-
tion of these cells showed them to be large, low density, nonadherent to plastic, and
negative for the cell surface rnarkers Thyl, Lyt2, L3T4, and sIg. Generation of nonspe-
cific suppressor cells in GVHD mice may explain the generalized immunosuppression
seen in these rnice (Lapp et al., 1985; Hakim and Shearer, 1990). Suppressor cells may
be induced by growth factors produced by proliferating T cells (Holda et al. 1985).
1.4.2.9 Sllmmary
Natural suppressor cells were discovered in the bone marrow of adult mice but
have been described in many sites, including the spleens of mice which are newborn,
pregnant, undergoing graft-vs-host disease, or are treated with TLI, cyclophosphamide,
89Sr, or adjuvants. In general these cells suppress Nt vitro immune responses in a dose-
dependent manner independent of any MHC restriction. These cells are large, low-den-
sity, nonadherent, and generally devoid of the surface markers characteristic of mature
cells. NS cells are always found in sites of active hematopoiesis and without exception
are induced by agents which also induce hematopoiesis (Strober, 1984b). The remainder
of this review will describe in more detail the charactenstics, activities, and mechanism
of action of adult murine BM NS cells.
1.4.3 Characterization of Murine BM NS Cells
Most reports of NS activity in murine adult bone marrow since their initial de-
scription in 1972 have included attempts to assign a Lineage to NS cells, although none
have drawn definite conclusions. The earliest studies by Singhal et al reported a cell in
murine adult bone marrow that suppressed anti-SRBC PFC responses and was moder-
ately radiosensitive, i.e. sensitive to 1000 rad but resistant to 100 rad (Singhal et al. 1972).
Duwe and Singhal(1979a, 1979b) reported extensive characterization of the suppressor
celf: velocity sedimentation showed it to be large; rosetting with SRBC showed it to be
FcR'; and it was resistant to depletion by anti-theta serum or anti-T ce1 serum plus com-
plement, as weil as carbonyl iron plus removal of magnetized cells. The suppressor cell
was also found in the bone marrow of nude (ndnu) mice. They concIuded that the sup-
pressor celi was not a T cell or pre-T cell, but did not draw any further conclusions.
McGarry and Singhal (1 982b) M e r showed by Ab and complement depletion studies
that the ce11 was negative for the cell surface markers Lyt-1, Lyt-2, 1-J, anti-T cell serum
(ATS), Ig, and Ia. The cell was positive for H-2 and an anti-stem ce11 serum obtained fiom
the laboratory of Golub et al. They concluded that the suppressor ceU was non-T, non-B,
and non-macrophage. But the question still remained: to what lineage did it belong?
The work frorn various laboratories toward characterization of murine BM NS
cells was summarized in a 1986 review by Holda et al (1986b). They described a consen-
sus phenotype of NS cells: low density, Thy-1-, L3T4-, LyQ-, sIg, Ia-, Fcf and without
killing activity for the NK targetsYAC- l or K-562. Therefore NS cells were not mature T
cells, B cells, or macrophages. Since 1986, most studies have indicated that NS cells may
be an immature progenitor-type cell radier than a mature ce11 type. The evidence for NS
cells being an immature ce11 can be surnrnarized as follows:
1) NS cells are without exception found in sites of hematopoiesis. These sites
include adult bone marrow, neonatal spleen, and spleens of mice which are pregnant,
undergoing GVKD, or treated with TLI, 89Sr, cyclophosphamide, or adjuvants. Non-
hematopoietic sites have little or no NS activity (Strober, 1984b; Maier et al. 1986).
2) Progenitor cells are typicaiiy of the "blast" morphology, since they are actively
cycling; while stem cells are small, Low density and relatively quiescent (Brecher et al.
1993; Moser and Paukovits, 1991). NS cells have been described as large and low den-
sity (1.043-1.067 g ml-l) using countefflow cenaifugal elutriation (CCE) (Saffran and
Singhal, 1990; SafXran et al. 199 1) or density gradients (Sugiura et al. 1988; Palathurnpat
et al. 1992a; Moore er al. 1992a). NS cells are also actively cycling since biological
activity is abrogated by cytostatic agents such as 5-fi uorouracil (Sugiura et al. 1988) or y-
irradiation (Sugiura et al. 1990a).
3) Mouse progenitor cells are immature and do not have cell surface markers char-
acteristic of hlly differentiated cells; however, mouse stem cells are positive for the sur-
face markers Thy 1. l and Sca-l (Uchida and Weissman, 1992). Adult BM NS cells are
Sca-1- and therefore do not resemble stem ceLls (Sykes et al. 1990). On the other hand,
they do not possess T or B ce11 markers (Holda et al. 1986b); markers characteristic of
mature macrophages such as Mac-1 or plastic adherence (Saffran and Singhal, 1990;
Hoskin et al. 1992; Sykes et al. 1990; Sugiura et al. 1988); NK cell markers such as
Asialo GM1 (Saffran and Singhal, 1990; Sugiura et al. 1988; Hoskin et al. 1992): NKl. 1
(Saffran and Singhal, 1990); or veto ce11 markers such as Qa2/3 or QalfïL (Sdran and
Singhal, 1990). Therefore the phenotype of BM NS cells is between stem cells and h l ly
differentiated cells in relative maturity.
4) NS cells do in fact possess markers typical of myeloid lineage progenitor cells,
including wheat germ agglutinin receptor (Sugiura et al. 1988), soybean agglutinin receptor
(Hoskin et al. 1992), IL3 receptor-associated antigen (Sugiura et al. 1992) or the my-
eloid-specific marker ER-MP12 (Fong, 1994). Cytokine-activated NS activity is abro-
gated by treatment with leucine rnethyl ester, which neutralizes lysosome-rich cells such
as granulocytic progenitors (Moore et al. 1992a).
5) BM Cell preparations e ~ c h e d for NS activity proliferate in response to the
myeloid-specific colony-sàmulating factors IL-3 and GM-CSF (Sugiura et al. 1990a;
Sugiura et al. 1992).
6) In ail attempts to puri@ BM NS cells, myeloid progenitor colony-forming celis
have been CO-purified (Saffian et al. 199 1 ; Sugiura et al. 1992; Sugiura et al. 1988).
In conclusion Sugiura and colleagues offered the following hypothesis: "cells that
exert NS activity in rnurine bone marrow are hematopoietic progenitor cells that are com-
mitted to the myeloid heage, but they are not mature myeloid/monocytic cells nor cells
of lymphoid lineage" (Sugiura et al. 1992). This theory in large part accounts for the
reasons that purification and characterization of NS cells has been so elusive. Myeloid
progenitor cells actively proliferate and differentiate in vitro; any attempt to snidy these
cells must therefore take into account this factor of uncertainty.
1.4.4 AduIt BM NS Cells in Other Species
Rat. Noga et al (1988a, 1988b) published extensive studies of NS activity in BM
from adult rats. They reported cells from AC1 rat BM which suppressed the AC1 vs Lewis
MLR. These cells were large and low density (by both counterflow centrifugai elutriation
and percoll gradients), nonadherent to fibronectin, and could be stained with dansylated
cyclosponne A (shown by FACS sorting). NS cells partially purified by percoll gradients
proliferated and differentiated into monocytes in response to the colony stimulating fac-
tors M-CSF, GM-CSF or IL-3. Furthemore, these factors up-regulated the inhibitory
activity of the NS population. They concluded that their results "suggest that an early
progenitor cell is responsible for NSCA [natural suppressor ce11 activity]" (Noga et al.
1988).
Rabbit. Cells from adult rabbit BM have been descnbed which suppress constitu-
tive proliferation of rabbit BMC (Soderberg, 1984a). These cells were found to be Fc-
gamma-R+, complement receptor negative (Soderberg, 1984a), and slightly adherent to
plastic (Soderberg, l984b).
Monkey. Cells from adult monkey BM have been described which suppressed
monkey peripheral blood leukocyte proliferation to ConA or PWM. These cells were low
density, wheat gerrn agghtinin receptof, Leu 1 1 a-, Leu 1 1 b-, Leu 1 1 c-, CD2-, dg-, and
Sephadex G-10 nonadherent (Sugiura et al. 1990b).
Human. NS cells from human adult BM have been extensively charactenzed
(Bains et al. 1982; Bains et al. 1986; Mo& et al. 1986; Mortari and Singhal, 1988;
Schmidt-Wolf et al. 1992). Cells with suppressive activity for anti-SRBC human PFC
responses were large (Bains et al. 1982; Mortari and Singhd, 1988)- low density, FcR+,
OKM l', SSEA- 1+, KNK- I+, OKTT, OKT8-, CD 1 lb-, glycophorin-, and CD 16- (Mortari
et al. 1986; Schmidt-Wolf et al. 1992). The authors concluded that the results "would
suggest that these cells belong to the myeloid lineage" (Mortari et al. 1986).
1.4.5 In Vitro Biolopical Activities of Murine BM NS Cells
Murine BMC have been shown to have suppressive activity in a wide variety of in
vitro immune assays. As reviewed above, the eariiest reports indicated murine BMC were
suppressive for anti-SRBC plaque production in Mishell and Dutton cultures (Singhal et
al. 1972; Drury and Singhal, 1974; Duwe and Singhal, 1979b; Duwe and Singhal, 1979a;
McGarry and Singhal, 1982b; Dauphinee andTalal, 1979; Corvese et al. 1980; Levy et al.
198 1; Dorshlcind and Rosse, 1982). Since these early reports, NS cells in various states of
purity derived from mwine BM have been found to be suppressive for proliferative re-
sponses to the mitogen ConA (Maier et al. 1989; Sugiura et al. 2988; Sugiura et al. 1992;
Moore et al. 1992c; Holda, 1992; Saffran and Singhal, 1990); proliferative responses to
the endotoxin lipopolysaccharide (LPS) (Schreiber and Fonnan, 1993; Sugiura et al. 1988;
Sugïura et al. 1992); allogeneic mixed lymphocyte responses (Saffran and Singhal, 1990;
S a f h n and Singhal, 199 1 ; Saf'fran et al. 199 1; Dorshkind et al. 1980; Palathumpat et al.
1992a; Hoskin et al. 1992; Sugiura et al. 1988; Sugiura et al. 1992); and proliferation of
immortalized cell lines (Sugiura et al. 1990a). NS cells freshly purified from muine
BM have not been tested in vivo systems.
1 A.6 Evidence That Murine BM NS Cells Act Through a Soluble Mediator
Among the earliest reports of suppressor cens in murine BM was evidence that
the suppressive activity was mediated by a soluble factor. Duwe and Singhal (2979a,
1979b) were arnong the first to demonstrate that murine BMC suppressed anti-SRBC
PFC in vitro. In a seminal experiment they cultured the plaque-forming culture and
murine BMC on opposite sides of a 0.2 pm nucleopore membrane using a hand-built
double chamber system @uwe and Singhal, 1978). They found that mouse BMC were
highly suppressive for anti-SRBC PFC even across the membrane. This result indicated
that a soluble factor, which they named BSF for "bone marrow-derived suppressor fac-
tor", mediated the suppressive activity. Fractionation of murine BMC supernatants by
ultrafiltration and gel filtration chromatography indicated the MW range of the suppres-
sive activity to be 1000-3500 Da @uwe and Singhal, 1978; McGarry et al. 1982a). BSF
was found to be heat-stable to boiling and resistant to the enzymes bypsin, RNAse and
neuraminidase (McGarry et al. L982a).
Reports from other laboratones confirmed that murine BM NS cells suppressed
in vitro immune responses by acting through a soluble mediator. Sugiura et al (1990a)
found that murine BMC suppressed proliferation of leukernia cells when separated from
them by a 0.4 pm semipermeable filter. They found that the protein synthesis inhibitor
puromycin blocked production of this soluble factor while the colony stimulating factors
IL-3 and GM-CSF increased suppressive activity. The same laboratory reported that
they fused highly purified munne BM NS cells with a lymphoma, producing a hybridoma
with highly suppressive supernatants (Sugiura et al. 1992). Further characterization of
this soluble mediator has not been reported.
There are many published reports of soluble factors which mediate the suppres-
sive activity of murine neonatal spleen suppressor cells (Argyris, 198 1; Jadus and Peck,
1986; Knaan-Shanzer and Van Bekkum, 1987; Barton, 1988), TLI-induced spleen sup-
pressor cells (Hertel-Wulff and Strober, 1988; Van Vlasselaer et al. 199 1)- lymphokine-
activated spleen suppressor cells (Moore et al. 1992c; Moore et al. 1992a; Young et al.
1992;Yamamoto et al. 1994), or tumour cells (Dittmer et al. 1984; Tsuchiya et al. 1988;
Hayamim er al. 1993). Partial purification of some of these factors have been reported
but none have been purifÏed to homogeneity or sû-ucturally charactenzed (Jadus and
Peck, 1986; Knaan-Shanzer and Van Bekkum, 1987; Van Vlasselaer et al. 1991). Anti-
body to TGFB partially reverses the suppressive activity of lymphokine-activated spleen
suppressor cells, indicating some of the activity is due to TGFB secretion (Moore et al.
1992c; Moore et al. 1992a;Young et al. 1992;'Yamamoto et al. 1994). The identity of the
soluble factor mediating the suppressive activity of adult BMC has remained a mystery.
1.4.7 Regdation - of Murine BM NS Celis by Cytokines
There is evidence that murine BM NS cells are subject to the regulatory effects of
cytokines. These results must be interpreted with great caution, since suppressor cells
induced by non-physiological concentrations of factors in vitro or in vivo can hardly be
considered "natural" suppressor cells. This section will briefly review the evidence that
NS activity can be regulated by cytokines.
The cytokines which have been shown to have stimulatory effects for murine BM
suppressor activity are IL-2, IFN-gamma, I L 3 and GM-CSF. Supematants of Con A-
stirnulated Lewis rat spleen ceUs (Rat-CAS) contain IL-2 and IFN-y and have been shown
to enhance the ability of murine BMC to suppress proliferative responses to mitogens
(Holda et al. 1986a; Moore et al. 1992b; Moore et al. 1992a; Moore et al. 1992c; Amma
and Kaplan, 1988; Azuma et al. 1989). These effects have been confmed using
recombinant IL-2 (Holda et al. 1986a; Azuma and Kaplan, 1988; Azurna et al. 1989) and
recombinant IFN-y (Holda et al. 1986a).
Studies in which mice were inoculated with malignant tumourç showed that some
tumours produce colony-stimulating factors or cytokines which stimulate suppressor celIs.
The ErLich murine carcinoma cell line (Subiza et al. 1989) and the LLC-C3 or LLC-LN7
Lewis Lung carcinoma ceil lines (Young et al. 1987) were found to be imrnunosuppres-
sive for the tumour-bearing host because of induction of a bone marrow-derived, NS-like
ce11 (Vinuela et al. 199 1 ;Young et al. 1988;Young et al. 199 1 ;Young et al. 1990). Murine
BMC treated in vitro with the supernatants of these cells could also be induced to be
highly immunosuppressive for proliferative responses to mitogens (Young et al. 1992).
The cytokines found to be responsible for the induction of suppressor cells by these tu-
mour ce11 lines were IL-3 and GM-CSF (Young et al. 1991). Studies with recombinant
murine IL-3 and GM-CSF confirmed that these cytokines stimulated andor caused pro-
liferation of murine BM suppressor cells (Moore et al. 1992b; Moore et al. 1992a; Sugiura
et al. 1992).
The phenotype of cytokine-induced BM suppressor celis indicates that they are
probably expanded numbers of normal BM NS cells (Moore et al. 1992a; Subiza et al.
1989; Young et al. 1987; Young et al. 199 1 ; Sugiura et al. 1992). As reviewed above, the
responsiveness of NS cells to the colony-stimulating factors IL-3 and GM-CSF is evi-
dence in favour of a granulocyte/macrophage lineage for these cells.
Although the studies reviewed above indicated that IL-2, IFN-y, IL-3 and GM-
CSF are stimulatory for murine BM NS cells, there is little evidence that NS cells are
dependent on these cytokines in vivo. However, some in vitro studies have suggested that
NS activity may be dependent on the cytokine EN-y. Antibodies against IFN-y abol-
ished the ability of normal or cytokine-stimulated BM NS cells to suppress proLiferathe
responses to mitogens (Holda et al. 1990; Maier er al. 1989;Angulo et al. 1995). Interest-
ingly, the suppressive effects of BM NS cells on mitogen responses were blocked by
LMMA, a chernical antagonist of inducible nitric oxide synthase (Angulo et al. 1995).
Since IFN-y is required for inducible nitric oxide synthase expression (Angulo et al. 1995),
it is conceivable that the antagonistic effects of anti-IFN-y may be mediated by a deficit in
nitric oxide production. Conversely stated, suppression of mitogen responses by murine
BM NS cells may require the production of nitric oxide at some point in the signalling
pathway (Angulo et al. 1995).
1.4.8 in vivo BioIorrical Activities of NS(-like) Cells
Since a murine BM NS cell which retains the consensus phenotype has never been
cloned or fully isolated, the precise functional role of this cell type in vivo must remain a
matter of speculation. Strober (1984; Strober et al. 1984a) has theorized that NS cells are
associated spatidy with hematopoietic sites and temporally with "windows of tolerance",
i.e. points in the life cycle of the mouse which are associated with ease of tolerance to
antigens, such as in the newborn. It is difficult to reconcile the lack of antigen specificity
or MHC restriction in suppression by NS cells with the requirements for antigen-specific
tolerance, but Strober (1984) concluded "the NS ce11 may be part of a 'fail-safe' mecha-
nism in which the few clones that escape central inactivation may be effectively inhibited
in the periphery".
Despite their lack of antigen specificity NS cells might be useful in the prevention
of graft-vs-host disease (Sykes et al. 1988). Some experiments were performed with a
cloned ceIl line derived from spleens of TLI-treated BALB/c mice (Strober et al. 1987).
This ce11 Line did not resemble adult BM-derived NS ceIls since it was positive for the T
cell markers Thy 1.2 and the af3 T ce11 receptor, although it did have rnany NS-like charac-
teristics (Strober et al. 1987). This cetl line blocked graft-vs-host disease in the mode1 of
BALB/c BM injected into C57BV6 recipients; and therefore had the ability to block de-
velopment of CTL in vivo by an antigen-nonspecific mechanism, ruling out veto activity
(Strober et al. 1987).
Murine BM NS activity may account for another unexplained phenornenon in the
area of transplantation: that of host resistance to donor bone marrow transplants. Host or
"natural" resistance is the observation that when recipient mice are given large amounts
of syngeneic donor marrow, the host remains resistant to engraftment by the donor-type
stem cells (Sadelain et al. 1989). The traditional explanahon is lack of space; Sadelain et
al. (1989, 1990a) proposed a regulatory hypothesis in which BM NS cells are responsi-
ble. They found an inverse correlation between levels of host NS activity and ability of
donor stem cells to engraft, using agents which up-regulated (CFA) or down-regulated
(anti-MHC antibody) NS activity (Sadelain et al. 1989). They proposed that "NS regula-
tory activity, produced by either cychg hematopoietic stem cells or some specialized
progeny, maintains other stem cells in a slowly proliferating or quiescent state" (Sadelain
et al. l99Oa).
In summary, the in vivo activities of BM NS cells may include 1) antigen-nonspe-
cific tolerance maintenance, and 2) maintenance of stem ceus, including those of donor
marrow from BM transplants, in a quiescent state. Work in this area could be advanced if
a m e NS cell clone were available.
1.4.9 Conclusion: A Mode1 of Natural Su~~ression
A theoretical mode1 of naturd suppression is shown in Figure 1.2. The central
hypothesis of this model is that natural suppressor cells are in fact myeloid Lineage pro-
genitor cells, probably granulocyte-macrophage progenitors. The evidence which sug-
gests this model is summarized in section 1.4.3. This model is neither conclusive nor
exhaustive, and more work will be required to prove the identity of murine BM NS cells
and myeloid progenitor cells. However, this model best fits the available evidence at this
tirne.
Figure 1.2
A Mode1 of Natural Su~pression
Al1 available evidence indicates the Natural Suppressor (NS) cell is a
hernatopoietic progenitor cell of the myeloid Lineage. NS cells exert growth inhibitory
and differentiation-inducing signals by means of a soluble rnediator.
1.5 Scope of the Thesis
This thesis describes experimental work performed towards purifkation and char-
acterization of the putative low MW soluble mediator of BM NS cells. Chapter two
contains theoretical background to biochemical pucification of biologîcally active factors
from natural sources. Chapter three contains methods used and results obtained in
fication and characterization of the low MW inhibitory factor produced by rat BMC.
Chapter four contains discussion of the results obtained and possible future work on this
project. The data presented describe purification and partial characterization of an in-
hibitory factor which may account for the suppressive activity of BM supematants. Based
on the work performed this molecule could be distinguished from ali known negative
regulatory molecules. This molecule may be a soluble mediator specifically produced by
BM NS cells.
CRAPTER 2
THEORETICAL BACKGROUND
2.1 Introduction
As reviewed in detail above, there is evidence that murine adult BM suppressor
cells act through an inhibitory soluble mediator @uwe and Singhal, 1978; McGarry et
al. 1982; Sugiura et al. 1990; Sugiura et al. 1992). AU characterizatioo work performed
to date indicates that this molecule may be unique (McGarry et al. 1982; Saffran, 1990).
Therefore to better undentand the nature of murine adult BM naturaI suppressor activity,
the purification and characterization of this soluble mediator was undertaken. The fol-
lowing section provides a brief theoretical framework for the work perforrned for this
thesis. The major parameters for the purification of a particular substance from a natural
source include biological function, source of raw material, method of quantitication, and
purification method. The biological funciions of soluble mediators of NS ceUs have been
described in detail in Chapte; 1.
2.2 Source of Material
The source of tbe substance to be purified is one of the most important choices
that c m be made in the design of a purification method (Ersson et al. 1989). Factors
important in the choice of raw matenal include: the concentration of the molecule of
interest, availability and cost, stability, presence of interfering activities, and difficdty of
handling. Also critical to the success of later fractionation steps is the initial extraction
method, which may include precipitation, electrophoresis, or chromatography (Ersson et
al. 1989).
2.3 Method of Ouantification
For substances with biological activities, there are two typ asurernent th
are important after each purification step: biological activity, and concentration (Ersson
et al. 1989). Biological activity depends on the action of the molecule of interest on a
specifc biological system. In the most cornmon definition of biological activity, one
arbitrary unit is defined as the amount of the substance of interest required to cause half-
maximal response in a paaicular system (Ersson et al. 1989). For example, in enzyme
purification, the most comrnon measure of activity is the use of the enzyme to cleave its
substrate, followed by measurement of the amount of cleavage products. For substances
that induce or inhibit cellular proliferation, 50% or half-maximal stimulation or inhibi-
tion of proliferation is a convenient definition (Ersson et al. 1989). Concentration is
expressed as unit mass per unit volume and can be rneasured by various methods. A
detailed review of methods of physical quantitation is beyond the scope of this thesis.
2.4 minfication S trategies
The fractionation steps to be used in isolation of a pdcular molecule depend on
its chernical nature. In general, choice of fractionation strategy depends on the following
characteristics of the molecule of interest: (a) size and shape, @) net charge and distribu-
tion of charged groups, (c) isoelectric point, (d) hydrophobicity, (e) metal binding, (f)
content of exposed thiol groups, and (g) biospecific affinities (Ersson et al. 1989). A
variety of methods is available to fractionate mixtures of molecules based on any one of
these characteristics.
Chromatography refers to the group of methods in which fractionation of a mix-
ture of molecules utilizes interactions between two phases, a stationary phase and a mo-
bile phase (Janson and Jonsson, 1989). There are four basic modes of chromatographie
separation: liquid-solid (adsorption), liquid-liquid (partition), ion exchange, and size
exclusion (Szepesi, 1992). Most often the stationary phase is packed into a tubular col-
umn and the mobile phase is a liquid solvent pumped into one end and eluted €rom the
other (Janson and Jonsson, 1989). In al1 types of chromatography except partition chro-
matography the stationary phase usually consists of a porous maûix imbibed with sol-
vent. The substances to be fractionated are dissolved in the mobile phase and pumped
through the column; the time it takes for various substances to reach the other side de-
pends on interactions between the substances and the stationary phase (Janson and Jonsson,
1989).
In utilizing a series of steps to pur* a molecule based on the above parameters,
there is no logical reason for any particular order of steps (Ersson et al. 1989). However,
practical considerations usually dictate what the most efficient strategy for any one mol-
ecule will be. In general, the best approach is to use a sequence of difîerent types of
hctionation methods, starting with the most robust methods and ending with the least
robust. The robustness of a technique refers to whether it gives high concentrations, has
high chernical and physical resistance, and has low cost. The most common initial
fractionation steps in purification strategies are ion exchange, hydrophobie interaction,
and gel filtration chromatographies (Ersson et al. 1989).
2.4.1 Ion Exchange Chromatomaphy
The basic principle of ion exchange chrornatography is that separation of mol-
ecules is achieved on the bais of the charges carried on the solute molecules. Therefore
if a mixture of molecules cary a variety of charges at a particular pH, they cm be sepa-
rated using ion exchangers (Haddad and Jackson, 1990). An ion exchanger is an insolu-
ble matrix to which charged groups have been covalently bound. Examples of charged
groups are the cornmonly used diethylaminoethyl (DEAE, formula -
0C%CH2N(+)H(CyCHJ2) and carboxymethyl (CM, -0CYCOO-) (Himmelhoch, 197 1).
DEAE is positively charged and therefore becomes associated with negatively charged
counter ions, or anions. For this reason DEAE is referred to as an anion exchanger. CM
is negatively charged and therefore becomes associated with positively charged counter
ions, or cations. Counter ions c m be reversibly exchanged with other ions of the same
charge, such as die charged groups on the substance of interest. This allows a positively
or negatively charged substance to bind to an cation or anion exchanger (Haddad and
Jackson, 1990).
2.4.2 Gel Filtration Chromatomuhv
Gel filtration chromatography, also known as size exclusion chromatography, has
a simple basic principle: in a matrix or gel, elution of molecules is a Iinear function of the
log of the MW and the elution volume (Hirnmel and Squire, 1988). In a mairix of a
defined size, some molecu1es in a mixture rnay be larger than the pores in the matrix and
some may be smaller. Therefore the smaller the molecule, the larger the volume accessi-
ble to it within the matrix and the larger the elution volume must be to elute it from the
column (Hïmmel and Squire, 1988). For large molecules, especially globular proteins,
the MW is directly proportional to the size of the molecule. Gel fdtration is therefore a
good method to estimate the MW of a molecule based on its relative size. For smaller
molecules this relationship may not hold true but gel filtration c m still be an efficient
method for separation of mixtures of molecules (Himmel and Squire, 1988).
2.4.3 Adsorption Chromatomaphv
Adsorption chromatography refer to methods in which the principle of separation
is chernical interactions (other than by charge) of solutes with the mobile and stationary
phases. Normal-phase chromatography refers to the use of nonpolar eluant with a polar
stationary phase; revened-phase therefore refers to the use of a polar eluant with a nonpolar
stationary phase. Nomal phase chromatographic separations often depend on interac-
tion of the substance of interest with silanol groups, or other polar phases bonded to silica
such as cyclohexanol, phenyl, cyanopropyl, diol, or amine groups (Szepesi, 1992). Re-
versed-phase chromatographic separations depend on interaction of the substance of in-
terest with hydrophobic phases bonded to silica. The most commonly used hydrophobic
phase is octadecyl silica (ODS or C 18) (Szepesi, 1992).
2.4.4 High-Performance Liquid Chromatomaphy MPLQ
High-Performance Liquid Chromatography W L C ) is the combination of tradi-
tional chromatographie separation methods with the use of high pressures and carefuliy
controlled solvent flow rates. HPLC was made possible by the use of pumps which c m
deliver extremely stable flows of solvent at high pressures for long penods of tirne (Pryde
and Gilbert, 1979). The groundwork for the development of HPLC was laid by Martin
and Synge who won a Nobel prize in 1952 for introducing liquid-liquid partition chroma-
tography, a technique which proved to have more versatility than gas-Iiquid chromatogra-
phy. Arnong their findings was that the use of high pressures in liquid chrornatography
could overcome problems induced by the viscosities of liquid solvents. The use of high
pressures have enabled the particle size of vax-ious stationary phases to be reduced to 5 'lm
or less (Pryde and Gilbert, 1979).
2.4.5 Chromatogra~hic Parameters
There are several parameters which are important to the optimization of methods
in chromatography. Capacity Ratio (k') is the ratio of the retention volume (the volume
passed through the column until the emergence of a certain component) to the phase ratio.
The retention volume must be at least double the void volume for separation to occur.
Capacity factor is usually expressed as retention time or retention ratio (Szepesi, 1992).
Theoretical Plates CN) is a measure of band broadening within a given system. The higher
the number of theoretical plates, the narrower the peak width and therefore the better the
resolution (Szepesi, 1992). Selectivity Factor ta) or resolution is the difference between
two capacity factors. If there is no difference between the capacity factors of two peaks,
there is no resolution of these peaks (Szepesi, 1992).
2.4.6 Exarn~les of Purification from Naturd Sources
In the following section some examples are given of the purification of oegative
regdatory molecules based on bioIogical assays. These exarnples may be relevant to the
purification of the murine BM inhibitory factor.
2.4.6.1 'Itaosforming Growth Factor Beta (TGFp)
TGFB is a family of related 25 kDa protein dimers with both growth-promoting and
inhibitory functions (Sporn et al. 1986; Keller et al. 1988). The inhibitory effect of TGFB
on gowth of some cell types has been used as a basis for its purification. Ideda et al
(1987) used 50% inhibition of proliferation of the Mink lung epithelial cell Line MvlLu to
define one unit of activity of human TGFB, for purification. They were able to purify
TGFp, from PC-3 human prostatic adenocarcinorna cells using batch adsorption to glass,
Bio-Sil TSK-250 gel filtration, and reverse-phase HPLC (RP-HPLC) using pBondapak
C, , (Ikeda et a[. 1987). In another study, Wrann et al (1987) used 50% inhibition of
mouse thymocyte proliferation to Con A to define one unit of activity of a TGFB-related
protein. They were able to purify the TGFB-like protein from the human glioblastoma cell
line 308 using a combination of PTGC 00005 ultrafiltration, hydroxylapatite chromatog-
raphy, two steps of Pro-RPC RP-FPLC, Mono-S cation exchange FPLC, and one final
step of Pro-RPC RP-FPLC (Wrann et al. 1987).
2.4.6.2 Macrophage Infiammatory Protein-1-Alpha (MIP-la)
As descnbed earlier, MIP-la has been purified several times, including on the
bais of its inhibitory activity for entry of hematopoietic stem ceUs into cycle (Graham et
al. 1990; Oppenheim et al. 199 1; Quesniaux et al. 1993). Graham et al (1990) purified
SCI/MIP- la on the basis of inhibition of generation of macrophages in the in vitro CFU-
A assay. They purifïed SCVMIP- la from culture medium conditioned by the mouse ceil
Iine J774.2 using a combination of ion-exchange chromatography, heparin-Sepharose af-
fini ty chromatography, blue Sepharose atfinity chromatography, and haUy SDS-poly-
acrylamide gel electrophoresis (Graham et al. 1990).
2.4.6.3 AcSDKP
The peptide Ac-Ser-Asp-Lys-Pro (AcSDKP) has been described as a possible en-
dogenous inhibitor in BM for enhy of hematopoietic stem cells into cycle (Ledant et al.
1989; Monzepat and Frindel, 1989). Lenfant et al (1989) used inhibition of Ara-C-in-
duced CFU-S in mice as an in vivo biological assay in order to p w AcSDKP. On the
basis of this assay, they purifid AcSDKP from fetal calf bone rnarrow extracts using a
combination of ultrafiltration, Bio-gel P-2 gel fdtration, Sep-Pak C,, extraction, and
Hypersil ODS C,, RP-HPLC using a variety of solvent systems (Lenfant er al. 1989).
2.4.6.4 pEEDCK
The peptide pGlu-Glu-Asp-Cys-Lys-OH (pEEDCK), like AcSDICP, rnight be a
natural inhibitor of the entry of hematopoietic stem cells into cycle (Paukovits and Laerum,
1982; Paukovits et al. 1993; Tonegawa, 1983; Moser and Paukovits, 199 1). Paukovits
and Laerum (1982) used inhibition of in vitm CFU-C and in vivo CF'ü-S as biological
assays in order to purify pEEDCK to homogeneity. They purified pEEDCK from the
conditioned media of human leukocytes using a combination of Sephadex G- 10 gel filtra-
tion, thiopropyl-Sepharose 6B chromatography, Polygosil60-C,, chromatography, AG50
x 4 resin chromatography, and final confirmation of purity by thin layer chromatography
or thin layer electrophoresis (Paukovits and Laerum, 1982).
2.5 Mass Spectrometry
A detailed review of mass spectrometry is beyond the scope of this thesis. How-
ever, the following is a brief introduction to the operating principles of a quadrupole mass
spectrometer with an electrospray ionization interface. In generai a quadrupole mass
analyzer consists of a radiai array of four accurately positioned electrodes, in which adja-
cent rods have opposite polarities (Wysocki, 1992). When a combination of a direct
current O C ) and radio frequency (RF) voltage is applied to the electrodes, the quadrupole
acts as a tunable bandpass mass filter. By varying the DC voltage and RF frequency, the
quadrupole will select whether an ion of a particular mass and charge can foIlow a stable
trajectory from one end of the quadruple to the other. Quadnipoles generally give only
unit mass resolution but have very fast scan rates through a mass range of 0-3000 Da
(Wysocki, 1992).
Mass spectrometers do not directly measure m a s of molecules but actually meas-
ure mass over charge (dz) ratio. There are two major challenges in the analysis of polar
molecules by mass spectrometry: 1) to evaporate molecules so they may enter the mass
analyzer in the gas phase, and 2) to cause molecules to carry a charge, also known as
ionization (Wysocki, 1992). Most biomolecules are highly polar and therefore not vola-
tile enough to enter the gas phase without pnor derivatization and high temperatures. A
second problem is that many biomolecules are heat labile and are therefore degraded
before they can be analyzed (Kebarle and Tang, 1993).
The invention of the electrospray ionization device helped to overcome many of
the problems associated with analysis of polar molecules. In bnef, electrospray is the
pneumatic spraying of solvent containing the substance of interest from an electrically
charged capillary (Kebarle and Tang, 1993). The resulting electncally charged aerosol
quickly evaporates. Ions are ernitted from the charged droplets as they evaporate, and
enter the quadrupole cell which is at a pressure of IO4 Torr relative to the electrospray
interface (Kebarle and Tang, 1993). This technique is extremely useful for the study of
smail polar molecules since in general, ionization is extremely gentle. Electrospray ioni-
zation allows the observation of molecular ions with very Iittle fragmentation. Fragrnen-
tation of molecular ions can be performed under controlled conditions by collision-in-
duced dissociation (CID) using a collision gas (Wysocki, 1992).
2.6 Parameters of the Pro-iect
Our laboratory and others have reported that the immunosuppressive activity of
BM NS cells is mediated by a low MW soluble factor (see chapter 1 section 1.4.6). Work
has been perforrned in our laboratory towards purification and characterization of this
factor. Experiments using dialysis and ultrafiltration indicated the factor to be 1000-3500
Da MW @uwe and Singhal, 1978; McGarry et al. 1982); practical experience with ulûa-
filtration indicated the MW might be less than 1000 Da (unpublished observations). The
factor was found to be heat-stable to boiling and resistant to trypsin, RNAse and neurami-
nidase (McGany et al. 1982). Since the factor appeared soluble in chloroform-methanol
the most reasonable conclusions were the that factor was a glycolipid or a carbohydrate
(McGarry et al. 1982; Mortari and Singhal, 1988). These atternpts to pur@ and charac-
terize the soluble inhibitory factor provided important background information for the
work presented in this thesis.
Many in vitro biologicd activities of the partially purified inhibitory factor have
been reporteci including suppression of PFC responses @uwe and Singhal, 1978; McGarry
et al. 1982; Mortari and Singhal, L988), MLR (Saffran and Singhal, 199 1; Fong, 1994),
myeloid CFU (Parsons, 1992; Fong, 1994), and proliferation of ce11 lines (SaEran, 1990;
Parsons, 1992). heparations of the inhibitory factor also inhibited some in vivo responses
including autoimmunity (Smythe, 1990; Weingust et al. 1989) and growth of bladder
carcinomas (unpublished observations). These biological activities of the inhibitory fac-
tor were considered in designing a biological assay.
CHAPTER 3
METHODS AND RESULTS
3.1 Methods
3.1.1 Animals
Wistar Fuah (WF), Dark Agouti (DA), Sprague Dawley (SD) or Lewis rats were
obtauied from Charles River and Harlan Sprague Dawley Inc. (Indianapolis, Indiana).
Male rats 250-350 g or male retired breeders were used in all experiments. BALBIc or
C57BV6 mice were obtained from Charles River Breeding Laboratories (S t. Constant,
PQ). Mice were bred and maintained at the University of Western Ontario animal care
facilities. Fernale mice of ages 6-12 weeks were used in ali experiments.
3.1.2 Cell Culture Media
Uniess otherwise indicated, complete RPMI medium consisted of RPMI- 1640 sup-
plemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin G, 100
pgh l streptomycin, and 25 pg/d gentamicin (Gibco-BRL, Burlington ON).
3.1.3 Ce11 Lines
Cell lines used in th is snidy were the human histiocytic Iymphoma U-937 (Ameri-
cm Type Culture Collection [ATCC], Rockviile Maryland), the human histiocytic lym-
phomaTHP- 1 (MCC), the human promyelomonocytic leukemia HL-60 (provided by Dr.
I. Harris, London Regional Cancer Centre), the human chronic myelogenous leukemia K-
562 (AïCC), the mouse rnonocyte/macrophages P388D, and J774a (ATCC and Dr. M.
Huff, University Hospiral, London ON), the mouse myeloblast M l (ATCC), and the mouse
myelomonocytic leukernia WEHI-3 (ATCC). Cell lines were maintained by regular pas-
sage in 25 cm2 tissue culture flasks (Nunc plastics, Roskilde, Denmark) in complete RPMI
medium. Cells were in log phase growth at the time of each experiment.
3.1.4 Pre~aration of Bone Marrow Cells
Male adult rats were used as a source of bone marrow cells- Femurs and tibias
were removed aseptically fiom animais sacrificed by CO, - asphyxiation. Bone marrow
was flushed fiom the bones using a 10 ml syringe and 18G needle (Terumo Medical
Corp., EIkton, Maryland) into 10 ml plastic petri dishes (Nunc) containing sterile Hanks'
balanced salt solution (HBSS, Gibco BRL). Single ce11 suspensions were obtained by
flushing BM cells through progressively higher gauge needles, then washed once in HBSS
before transfer to 20 ml glas vials. Erythrocytes and other debris were removed by lay-
ering 6 ml of Lympholyte-Rat (Cedadane Laboratories, Homby ON) under each BMC
suspension and cenaifuging at 1500 rpm for 30 minutes. Cells remaining at the interface
were collected. BM cells were washed three times in HBSS before use.
3.1.5 Counterflow Centrifu~al Elutnation
Counterflow centrifuga1 elutriation (CCE) was performed using a Beckxrian JE-6B
elutriation rotor (Beckrnan, PaIo Alto, California). Single cell suspensions of rat BMC
were prepared as described above in Ca2+ and Mg2+-free HBSS (Gibco BRL) containing
0.1% bovine serum alburnin (Sigma-Aldrich Canada Ltd., Mississauga ON). Widi the
rotor speed held constant at 3000 rpm cells were loaded at a buffer flow rate of 13 rnllmin.
The flow rate was increased to collect the different celi populations: lymphocytes were
collected at 13-18 mVmin, monocytes at 18-30 ml/min, and blast cells collected at rotor-
off as previously descnbed (Noga et al. 1988). Light scatter of each population was
measured using a Coulter counter and Channelyzer (Coulter electronics Inc., Hialeah
Florida).
3.1.6 Ce11 Line Proliferation Assavs
For each ce11 line, 2 x 104 cells were cultured per well in 96-well flat bottom cul-
ture plates (Nunc) dong with various dilutions of substances to be tested dissolved in
complete FWMI medium. The cultures were incubated at 37" C in 5% CO, for a total of
48 hours, including a four-hour pulse with 0.5 pCi/well 3H-thymidine (ICN Pharmaceuti-
cals Inc., Mississauga ON). Cultures were harvested ont0 glass fibre filter paper (90 x
120 mm printed filtermat "A", LKB/Wallac, Turku, Finland) on an automatic 96-weU ce11
harvester (Tomtec 96, Orange, Connecticut) and counted on a Microbeta 1450 liquid scin-
tillation counter (LKBIWallac) using an IBM PS/2 mode1 30 286 computer.
3.1 -7 Cell Viabilitv Assav
WEHI-3 cells were incubated under the same conditions as described above for 24
hours with the addition in the final three hours of 25 ng/well 3,(4,5-dimethythiazol-2-
yl)2,5-diphenyl-tetrazolium bromide (MïT? Sigma-Aldrich). Formazan blue crystals were
solubilized with developing reagent as previously described (Hansen et al. 1989). The
amount of conversion of MTT to formazan blue correlates with the number of intact rnito-
chondria in a population of viable ceUs. Absorbance was measured at 590 nrn in a 96-well
plate reader (Titertek MuIüskan Plus, Flow Labs, Mississauga ON).
3.1.8 Mixed Lymphocyte Reaction
Mouse MLRs were generated by culture of 2 x IO5 C57BY6 (H-23 responder spleen
cells with 2 x 1W BALB/c (H-2d) stirnulator spleen cells in a total culture volume of 200
p1 in 96-well round bottom plates (Nunc). Stimulators were inactivated with 2500 rad
irradiation using a Gamma Cell (Atomic Energy of Canada Ltd., Ottawa ON). Culture
medium consisted of complete RPMI with 2.5% fetal bovine serum. Cultures were incu-
bated for 96 hr, including a 4-hour pulse with 0.5 pCi/well tntiated thymidine (ICN).
Plates were harvested and counted as described above for cell line proliferation assays.
3.1.9 Pre~aration of Bone Marrow Su~ernatants
Rat BMC were plated in sterile 150 x 20 mm petri dishes (Nunc) at 1 x IO7 celIs/ml
in serum-free HBSS supplemented with 100 U/ml penicillin G, 100 p g M streptomycin,
0.25 pg/ml fungizone, and 25 pglml gentamîcin (Gibco BRL). Supematants of cultured
BM cells, i.e. HBSS conditioned by culture with BMC, were collected at 24 hours, cells
were removed by cenagat ion at 1500 rpm for 7 minutes, fresh HBSS was added, and
supernatants were collected again at 48 hours. Pooled supernatants were centrîfûged at
2000 rpm for 10 min to remove large debris, then filtered through a 0.8 pm Acrodisc
syringe filter (Gelman Sciences, Rexdale ON) and stored at -70" C.
3.1.10 Solid Phase Extraction
C 18 cartridges (Mini-Spe-ed C 18- 14%, Applied Separations, Allentown, Pennsyl-
vania) were prepared by washing with 10 ml of methanol followed by 10 ml 18 megohm-
cm deionized-distilled water. Up to 100 ml of rat BM cell supernatants were passed through
each cartridge before elution. After washing the cartridges with 5 ml of deionized-dis-
tilled water, matenal adsorbed to the C, , cartridge was eluted with 3 ml HPLC-grade metha-
no1 (Fisher Scientific, Unionville ON) evaporated under a Stream of nitrogen, redissolved
in a small volume of methanol, and stored at 4" C. Before testing the eluate for activity in
vitro, the methanol was evaporated under nitrogen and replaced by sterile culture medium.
3.1.1 1 Dialvsis
Cl8 extracts of Rat BM cell supematants were dialyzed for 48 hr against 1 litre 1X
HBSS (Gibco BRL) using 1000 Da cutoff SpectraPor dialysis tubing (Spectrurn Medical
Industries inc., Los Angeles, California). Following dialysis the contents of the tubing
were re-extracted using Cl8 cartridges and tested for biologicd activity in the WEHI-3
bioassay.
3.1.12 SoIvent Extractions
Cf 8 extracts of culture medium as a control or rat BM-conditioned medium were
dissolved in 3 ml phenol-red-free RPMI- 1640 (Gibco B K ) and acidified with either ace-
tic acid or HCl (Fisher). Extracts were then shaken with 6 ml HPLC-grade hexane (Fisher)
or 6 ml redistilled "AnaIR" grade methylene chloride (BDH Inc., Toronto ON). The aque-
ous and organic phases were allowed to separate upon standing. Aqueous fractions were
concentrateci by freeze-drying while hexane was evaporated under a Stream of nitrogen.
Methylene chloride was dried with NaSO, pellets overnight before evaporation on a Buchi
Rotavap "R" (Cadab Scientific Products, Mississauga ON).
3.1.13 Enzvmatic Digestion
Cl8 extracts of culture medium as a control or rat BM-conditioned medium were
dissolved in 500 pL Tris-HCI buffer (Sigma-Aldrich) with either papain, pepsin, subtili-
sin, or carboxypeptidase B diluted according to the manufacturer's instructions (Sigma-
Aldrich). After incubation at 3 7 O C for 90 minutes enzyme was inactivated by boiling for
5 minutes. Enzyme was removed by filtration through a Microcon-lO 10 kDa centrifuge
microconcentrator (Amicon Canada Ltd., Oakville ON) at 13000g for 30 minutes. The
filtrate was re-extracteci by C 18 carüidges, dissolved in sterile culture medium and tested
in the WEHI-3 bioassay.
3.1.14 Ion Exchanrre Chromatomphv
DEAE-52 (diethylaminoethyl cellulose, Cl- form, Whatman Biosystems Ltd., Maid-
stone, Kent, England) was washed and equilibrated in 10 n M Tris-HC1 buffer pH 7.4
(Sigma-Aldrich). DEAE-52 was packed into a 1.5 x 3.0 cm glass colum and equifibrated
with 30 ml of the same buffer. Rat BM supernatant C 18 extracts were dissolved in 3 ml
buffer and loaded ont0 the column. 5 ml were collected and designated as fraction one.
After washing with 15 ml buffer, 5 ml buffer containing 1M NaCl was added to elute all
bound material, designated fraction two. Each fraction was re-extracted using Cl8 car-
tridges and redissolved in sterile culture medium before testing in the WEHI-3 bioassay.
Ion exchange chromatography with CM (carboxymethyl cellulose, Whatmm) was carried
out by an identical procedure using pH 6.0 10 nM phosphate buffer.
3.1.15 Gel Filtration
Bio-Gel P-2, nominal exclusion limit 1800 Da (Bio-Rad Laboratones (Canada)
Ltd., Mississauga ON) was washed and pre-swelled in 20 rnM Tris-HC1 buffer, pH 7.2
(Sigma-Aldrich) and thoroughly degassed by gentle swirling under vacuum. Bio-Gel P-2
was packed into a g las column and equïiibrated with 3 colurnn volumes of the same
buffer. Cl8 cartridge-extracts of Rat BM ce11 supernatants were evaporated to dryness
under a Stream of nitrogen and dissolved in 20 mM Tris-HC1 buffer, pH 7.2. SampIes
were carefully applied to the column and the flow rate was adjusted to 7.8-8.2 mVhr.
Fractions were collected from the column using a mode1 21 12 Redirac fraction collector
( L K B ~ d l a c ) and the absorbance of these fractions was measured at 2 15 nrn using a DU-
8B spectrophotometer (Beckman). Material in each fraction was recovered using C,,
cartridges, as described earlier. The biological activity was tested using the WEHI-3 cell
bioassay.
In prelirninary experirnents, a 1.0 x 42 cm g las column (Pharrnacia Biotech AB,
Uppsala, Sweden) was used. Samples were applied in volumes of less than 300 pl and
fractions of 2.1 ml were collected. For preparative experirnents, a 2.6 x 89 cm column
(Pharrnacia) was used. Samples were applied in volumes of less than 2.5 ml and fractions
of 15 ml were coilected.
3.1.16 Anion Exchange FPLC
C,, cartrïdge and gel filtration-purified, biologically active fractions were concen-
trated and redissolved in Tris-HCl buffer, pH 8.4 (Sigma-Aldrich). The FPLC system
consisted of an LCCdOO CI liquid chromatographie controiler with a 80286 PC running
"FPLC Manager", 2 P-500 pumps, an MV-7 injection motor valve, W - M absorbance
monitor, and Frac- 100 fraction collector (LKBIWallac). Samples were injected and loaded
on a Fast-Q Sepharose FPLC column (Pharmacia) ninning Tris-HC1 buffer pH 8.4 (Sigrna-
Aldrich). Matenal was eluted from the colurnn with a 0-0.5 M NaCl gradient; peaks were
monitored at 280 m. Material in each fraction was recovered using C , , cartridges, as
described earlier. The biological activity was tested using WEHI-3 cells as described
below.
3.1.17 High Performance Li uid Chromatomaphy (HPLC)
Liquid chromatography was performed on a Waters HPLC system consisting of a
mode1 720 system cootroller, model 730 data module, two M 4 5 pumps, U6K injector and
model 48 1 LC spectrophotometer (Millipore Waters Chromatography, Mississauga ON).
3.1.18 Amino M.$ HPLC I
Biologically active fractions purified by C,, cartridge extraction, gel filtration and
anion exchange were fractionated using a Waters Carbohydrate Analysis column, 3.9 mm
x 30 cm (Millipore). n i e mobile phase consisted of 80% redistilled acetonitrile in HPLC-
grade water. The flow rate was I mVmin and absorbance was measured at 2 15 nm. Frac-
tions were collected using a model 221 1 Superrac fraction collector (LKWWdlac). The
volume of each fraction was significantly reduced by evaporation under a Stream of nitro-
gen before recovenng the activity on C,, cartridges, as described above, and tested for
biological activity.
3.1.19 Reverse-Phase HPLC
Biologically active fractions purified by gel filtration, anion exchange and Amho
(iWJ HPLC were fractionated using a Brownlee Aquapore RP-300 C8 carûidge colurnn,
2.1 mm x 22 cm (Chromatographic Specialties Inc, Brockville, ON). The mobile phase
consisted of 25% redistilled HPLC-grade acetonitrile (Fisher) and O. 1% ûifluoroacetic
acid (Sigma-Aldrich) in deionized-distilled water. The flow rate was 0.2 mVmin and ab-
sorbance was measured at 2 15 nm. Fractions were collected using a model 221 1 Superrac
fraction collecter (LKB/W'ac). Coilected fractions were dried directiy under a stream
of nitrogen and redissolved in methanol before testing for biological activity.
3.1.20 Liauid Chrornatomph~/Mass Spectrometry CLC-MS)
Ionspray mass spectrometry was perfomed at the University of Toronto at the
Carbohydrate Centre, Department of Medical Genetics. The LC-MS equipment con-
sisted of an AppIied Biosystems 140B solvent delivery system eluting into the ionspray
ionization interface of a Sciex API DI triple quadrupole mass spectrometer (Perkin-Ehed
Sciex Instruments, ThomhiU ON). The stream was split such that 1/10 of the sample
eluted directly into the ionspray interface of the mass spectrometer; 9/10 of the sarnple
was collected into fractions and dried directly under a stream of nitrogen before redissolving
in methanol and teshg for biological activity. BM-derived inhibitory activity purified by
Cl 8 extraction, gel fdtration, anion exchange and Arnino (NY)-HPLC was injected into - the LC and fractionated onA BrownleeAquapore RP-300 C8 cartridge column. 2.1 mm x
22 cm (Chromatographie Specialties) which eluted into the mass spectrometer. in sorne
experiments, BM-denved inhibitory activity purified by C 18 extraction, gel filtration,
anion exchange, Amino (NY) and C8 RP-HPLC was injected directly into the mass - spectrometer. The mobile phase consisted of 25% redistilled HPLC-grade acetonitrile
(Fisher) and 0.1% trifluoroacetic acid (Sigma-Aldrich) in HPLC-grade water, at a flow
rate of 0.2 ml/rnin. Data analysis was perfomed on an Apple Macintosh Quacira 640
computer.
3.1.21 Unit ofActivity
One unit of activity is defined as the amount of BM-denved inhibitory activity
which produces 50% inhibition of 3H-thymidine incorporation in one standard culture of
the WEHI-3 cell line. The standard culture is described in section 3.1.6 above. Based on
assays of inhibitory activity purified by C,, cartridge, it was estimated that BMC
supernatants contain an average level of about one unit of activity per 0.1 ml of supernatant.
3.1.22 Statistics
Unless otherwise indicated, ail results shown are based on the means and standard
deviations of triplicate cultures.
3.2 Results
3.2.1 Effect of Murine BMC Su~ernatants on Ce11 Lines
As reported in section 2.6, previous results from this laboratory showed that ultra-
filtration-concentrated mouse BM supematants were suppressive for various biological
responses including an ti-SRBC PFC (Duwe and Singhal, 1 W8), MLR (Saffran and Singhal,
199 l), myeloid colony formation (Parsons, 1992) and proliferation of ce11 lines (Saffian,
1990; Parsons, 1992). In order to develop a rapid and simple in vitro biological assay for
BM-derived inhibitory activity, various ce11 lines were tested for sensitivity to suppres-
sion by ultrafiltration-concentrated mouse BM supematants. Murine lines tested included
the myeloblast M 1, the myelomonocyte WEHi-3, the myelomonocyte NFS-60. the mono-
cyte J774a, the monocyte P388D,, the mastocytoma P-8 15, the erythroleukemia BB88,
the lymphoma EL4, the LymphomaYac- 1, the T-lymphoma LBRM-33, the myeloma SP2/
0, and the B hybridoma 1 lB l 1 (data not shown). Human Iines tested included the lym-
phorna Jurkat, the promyelocyte HL-60 and the myelocyte K-562 (data not shown). From
the cell lines tested the mouse myelomonocyte WEHI-3 was selected for use in a biologi-
cal assay because it had high constitutive proliferation and repeatable sensitivity to sup-
pression by mouse BM supematants.
3.2.2 C 1 8 Cartrid~e Extraction of Rat Tissue Su~ernatants
In preliminary experiments ulû-afiltration-concentrated rat BM supematants sup-
pressed proliferation of -HI-3 cells to a degree equivalent to ultrafiltration-concen-
trated mouse BM supernatants (data not shown). Furthemore, an average of 1 x 108 BMC
could be obtained per rat compared to 1 x IO7 BMC per mouse. Since quantity of staaing
material was important for purification, Rat BMC supernatants were therefore selected as
a source of inhibitory activity. As described in chapter 2, it is important for purification
protocols to have an efficient step for concentration of the original source matenal. Solid-
phase extraction using Cl8 cartridges is a technique commonly used for concentration
and desalting of low MW substances (S wartz et al- 1987; Wehr, 1987). Relahvely hydro-
phobie molecules bind to the Cl8 packing matenal under aqueous conditions and can be
eluted with solvents such as methanol or acetonitrile (Wehr, 1987). To test whether this
approach was applicable to concentration of rat BM-derived inhibitory activity, the
supematants prepared from rat BM, thymus or spleen cells were passed through C 18 car-
tridges and eluted wiîh methanol, as described in methods section 3.1.10. The extracts
were tested on WEHI-3 cells. As shown in Figure 3.1, extracts of rat splenocyte or thymo-
cyte supernatants had sorne inhibitory activity for proliferation of WEHI-3 cells but much
less than extracts of BMC supernatants. At a concentration of 1.0 relative to the original
supematants (the sarne concentration as the original supernatant), BM extracts suppressed
WEHI-3 proliferation up to 80% while thymus and spleen extracts had no significant
effect.
3.2.3 Effect of Rat BM C 18 Extracts on WEHI-3 Viabilitv
To exclude the possibility that rat BM supernatant extracts inhïbited WEHI-3 pro-
liferation by killing the cells, an M?T viability assay was performed. M?T is a tetrazolium
salt whose conversion to the coloured product blue formazan can be measured spectro-
photornetncally. Reduction of M?T is performed by cellular dehydrogenases; therefore
the amount of conversion is directly related to the number of cells present in culture (Hansen
et al. 1989). Figure 3.2 shows the results of a 24-hour M'IT assay performed with C 18
Figure 3.1
Cl 8 Extracts of Rat BM Supematants are More Suppressive
for WEHI-3 Proliferation thm Extracts frorn Other Tissues
Single celï suspensions of rat BMC, thymocytes or splenocytes were cultured
to produce conditioned media as described in methods section 3.1.9. C 18 extracts of
conditioned media were prepared and tested for biological activity in the WEHI-3
bioassay as described in methods sections 3.1.10 and 3.1.6. Constitutive proliferation
of WEHI-3 cells is indicated (@). Various concentrations of Cl8 exaacts were tested,
including that of medium dont as a control (W), thymus (V), spleen (A) and BM (+).
Data are expressed as means (syrnbols) and standard deviations (vertical enor bars) of
iriplicate cultures.
O 2 4 6 8 1 O
Relative Supernatant Concentration
Figure 3 -2
C 18 Extracts of BM-derived Inhibitorv Activity
Are Not Cytotoxic for WEHI-3 Cells
Rat BMC supernatant inhibitory activity was purified by Cl8 extraction as de-
scribed in methods section 3.1.10. Extracts were added to standard WEHI-3 cultures
for 20 hours before a 4-hour pulse with either 3H-thymidine or M m , as described in
methods section 3.1.7. )H-thymidine incorporation is indicated (O) on the left vertical
ais . M m conversion is indicated (CI) on the right vertical axis. Constitutive prolif-
eration of WEHI-3 cells measured by 3H-thymidine incorporation is indicated (O). Data
are expressed as means (symbols) and standard deviations (vertical error bars) of trip-
licate cultures.
1 2 3 4
Relative Supernatant Concentration
extracts of rat BM supernatant added to WEHI-3 cells. W~thin 24 hours of culture, prolif-
eration of WEHI-3 cells was significantly suppressed while MTT conversion was not
significantly affected. These results indicate that the BM Cl8 extract was not cytotoxic
for WEHI-3 ceus.
3.2.4 Effect of Elutriated Rat BMC Supematants on WEHI-3 Proliferation
Rat BM NS cells have been described as large and low density using counterflow
centrifugal elutriation (Noga et al, 1988% 1988b). These characteristics are similar to
those descnbed for mouse BM NS cells (SaEran and Singhal, 1990; Saffran et al. 199 1).
To test whether inhibitory activity was produced by large, low density rat BMC, rat BMC
were fractionated using counterflow centrifugal elutriation (CCE) using previously de-
scnbed methods (Noga et al., l988a). BMC were fractionated into small (F 1, lymphocytes),
medium (F2, monocytes) and large (F3, blasts) ce11 fractions as shown in Figure 3.3.
Each fraction was cultured at 1 x IO7 cellsfml in serum-free media as described in meth-
ods section 3.1.9; conditioned media were tested directly on WEHI-3 cells for inhibitory
activity. As shown in Figure 3.4, supematants produced by the blast ce11 fraction were the
most suppressive for WEHI-3 proliferation. This result confirmed that large, low density
BMC were responsible for release of inhibitory activity into the culture media. As shown
in Figure 3.5, the inhibitory activity could be extracted fiom the supematants of the vari-
ous cellular fractions using C 18 cartridges. The biological activity of the C 18 extract was
iess than that of raw supernatant, since a relative supernatant concentration of 1.25-fold
suppressed WEHI-3 proliferation by 80% while raw supematants suppressed by up to
90% at 0.5-fold.
3.2.5 PreIinGnary Characterization of the Inhibitory Factor
3.2.5.1 Dialysis
Previous work in this laboratory indicated that the mouse BM-denved suppressor
Figure 3.3
Countefflow Centrifugal Elutriation of Rat BMC
4.18 x 1 Os WF rat BMC were prepared as described in methods section 3.1.4
and diluted in elutriation buffer. BMC were fractionated by counterflow centrifugai
elutriation (CCE) using a Beckman JE-6B rotor as described in methods section 3.1 S.
Unfractionated BMC or separated fractions were analyzed for light scatter using a
Coulter Counter. (A) shows unfractionated marrow while (B) shows the various frac-
tions collected. Results are expressed as the relative number of cells observed at each
relative cell volume.
Figure 3.4
Supematants of Elutriated Rat BMC are DirectIy
Suppressive for WEHI-3 Ce11 Proliferation
Small (FI, Lymphocytic), medium (F2, monocytic) and large (F3, blast) cell
fractions of WF rat BMC were collected by CCE as descnbed in methods section 3.1.5
and culnired to produce conditioned medium as described in methods section 3.1.9.
Conditioned media were added to standard cultures of WEHI-3 cells to measure their
suppressive activity for cellular proliferation as described in methods section 3.2 .o. Supematants were diluted in culture to relative concentrations of 0.5-fold (open bars),
0.25-foId (filled bars) or 0.125-fold (hatched bars). Per cent suppression refers to per
cent suppression of constitutive WEHI-3 proliferation. Per cent suppression was cal-
culated fkom comparing the average values of triplicate cultures.
Elutriation Fraction
Figure 3.5
C 18 Extracts of Elutriated Rat BMC Su~ematants are
Sup~ressive for WEHI-3 Cell Proliferation
Conditioned media from small (FI, lymphocytic), medium (F2, monocytic)
and large (F3, blast) ceil fractions of WF rat BMC were extracted using C l 8 car-
tridges as described in methods section 3.1.10. Extracts were tested for suppressive
activity on WEHI-3 ce11 proliferation as described in methods section 3.1.6.
Supernatant extracts were tested at relative concentrations of 1.25-fold (open bars),
0.625-fold (filled bars) or 0.3 13-fold (hatched bars). The results are expressed as per
cent suppression of WEH-3 cell constitutive proliferation. Per cent suppression was
calculatecl fkom comparing the average values of triplicate cuitures.
Elutriation Fraction
factor was low MW, most Likely in the 1000-3500 Da range @uwe and Singhal, 1978;
McGarry et al. 1982; Mortari and Singhal, 1988; Saffian and Singhal, 199 1). To CO-
the MW range of the rat BM-derived inhibitory factor, C 18 extracts of rat BM supernatants
were dialyzed for 48 hours using 1000 Da cutoff membranes, then re-extracted and tested
for biological activity in the MLR. Figure 3.6 shows the following results: 1) Rat BM
C 18 extracts were suppressive for rnouse MLR, as reported for mouse BM extracts (Safnan
and Singhal, 199 1). 2) Rat spleen C 18 extracts were suppressive for MLR but not to the
same degree as BM extracts. This result confirmed in the MLR what was found in the
WEHI-3 assay s h o w in figure 3.4. 3) 48-hour didysis removed all inhibitory activity
from Cl8 extracts of spleen or BM supematants. In fact, dialyzed spleen extracts signifi-
cantly stirnulated proliferation in the MLR. These results confirmed that the BM-derived
inhibitory factor was low MW in nature. Although didysis does not provide precise MW
information, it is conceivable that the MW of the factor is less than 1000 Da.
3.2.5.2 Solubility
Rat BM-derived inhibitory activity is apparently soluble in water, since it was
recovered in aqueous supematants of BMC. The factor was also fully soluble in metha-
nol, since it was eluted h m Cl8 cartridges and stored using methanol as a solvent as
descnbed in methods section 3.1.10. Revious attempts at characterization of the mol-
ecule responsible for mouse BM-derived inhibitory activity led to the conclusion that it
was lipid in nature (McGarry et al. 1982; Mortari and Singhal, 1988).
It is possible for lipids to be soluble in aqueous medium if they are bound to
proteins (Christie, 1982); therefore several types of lipid extraction were performed on
Cl8 extracts of rat BMC supematants. Preliminary experiments using the Folch extrac-
tion method to extract lipids from Cl8 extracts (Folch et al. 1957) were unsuccessful,
since even low concentrations of dried solvent were highly cytotoxic for WEHI-3 ceus
(data not shown). Therefore several liquidfiquid partitions of aqueous media and solvent
were perfonned as previously described (Kates, 1986). Partitions were performed at
Figure 3.6
Inhibitory Activity in BM and Suleen C 18
Extracts is Dialyzable
50 ml condltioned media from rat BM or spleen cells were prepared and ex-
tracted with C 18 cartridges as described in methods sections 3.1.9 and 3.1.10. Ex-
tracts were tested for activity in the rnixed lymphocyte reaction as described in meth-
ods section 3.1.8 before and after 48-hour dialysis on 1000 Da membranes as de-
scribed in methods section 3. l. I l. Positive (A, C57BY6' --> BALB/cs) and negative
(+, C57BU6' --> C57BW) controls are indicated. BM extracts (solid Iine) were added
to the MLR at various concentrations before (m) and d e r (0) dialysis. Spleen ex-
tracts (dashed line) were added to the MLR at various concentrations before (O) and
&er (O) dialysis. Results are expressed as means (symbols) and standard deviations
(vertical error bars) of triplicate cultures.
1 2 3 4
Relative Supernatant Concentration
neutral and low pH to eliminate pH-dependent Lipid-protein interactions (Christie, 1982).
Tables 3.1 and 3.2 show the results of a hexane/water partition of Cl8 extract at pH 3.0
and 7.0. At both pH levels, inhibitory activity was recovered in the aqueous phase after
partitioning. Therefare rat BM inhibitory activity was insoluble in hexane at low or neu-
tral pH.
Table 3.3 shows the result of a methylene chloride/water partition of Cl8 extract
at pH 3.0. Inhibitory activity was recovered equdy in aqueous and organic phases. There-
fore it is possible that the inhibitory activity was sparingly soluble in methylene chlonde.
However, this technique could not be used for purification since high concentrations of
dried solvent were cytotoxic for WEHI-3 cells (data not shown). These results showed
that inhibitory activity was not freely soluble in highly hydrophobic solvents such as
hexane or methylene chloride, and therefore suggested that the inhibitory molecule was
not a lipid.
3.2.5.3 Susceptibility to Proteolytic Enzymes
To test whether the rnolecule responsible for inhi'bitory activity was a peptide or protein, C 18
extracts were subjecied to digestion by four broad-specifïcity pteolytic enzymes. The enzymes
used were papain, pepsh, subtibin, and carbOxypeptidase. Papain is a member of the family of
plant sulfliydryl proteolytic enzymes which hydrolyses the amide bonds of a-substituted arginine,
lysine, glutamine, histidine, glycine and tyrosine (Boyer, 1971). Pepsin is a mammidian gasûic
protease which at low pH cleaves bonds of phenylalanine, methionine, leucine or ûyptophan to other
hydrophobic residues (Barrett and McDonald, 1980). Subtilisin is a bacterial exûacellular alkaline
pmteinase which cleaves neutrai and nonpolar residues bound together, both intemal and terminal
(Boym 197 1). Finally, carboxypeptidase is amammalian zinc<ontainuig pmteolytic enzyme which
cleaves C-teminal L-arnino acids @arrea and McDonald, 1980).
As show inTable 3.4, incubation with papain, pepsin, subtilisin or carboxypeptidase did not
remove any q p s i v e activity h m rat BM Cl 8 exûacts. This result suggested tbat the molecule
responsible for inhibitory factor was not a protein or peptide.
Table 3.1
HexanelWater Partition of C 18 Extracts, pH 3.0.'
Condition Concentrationb CPM (mean i S . D . I c % Suppressiond
Medi a Water e x t r a c t 1.25
0.63
Hexane e x t r a c t 1.25 0.63
BM Water e x t r a c t 1.25
0.63
Hexane e x t r a c t 1.25 0.63
" C 18 extracts of 50 ml of RPMI- 1640 medium as a control or 50 ml rat BM-condi- tioned medium were prepared. A srnall amount of each was set aside for a control and the rest acidified and extracted with hexane as described in section 3.1.10.
Concentration refers to relative concentration of supernatants.
' Counts per minute were obtained in die WEHI-3 bioassay.
Per cent suppression of WEHI-3 celIs by BM Cl8 extracts was calculated using the media control. Suppression less than 5% was considered not active.
Hexanewater Partition of C 18 Extracts, pH 7.0"
Condi t i on Concentrat ionb CPM (mean f S.D. I C % Suppressiond
Media Water e x t r a c t
Hexane e x t r a c t 1.25 0.63
BM Water e x t r a c t 1.25
0.63
Hexane e x t r a c t 1 .25 0.63
a Cl8 extracts of 50 ml of RPMI-1640 medium as a control or 50 ml rat BM-conditioned medium were prepared. A small amount of each was set aside for a control and the rest extracted with hexane as described in section 3.1.10.
Concentration refers to relative concentration of supematants.
' Counts per minute were obtained in the WEHI-3 bioassay.
* Per cent suppression of WEHI-3 cells by BM CL8 extracts was calculated using the media control. Suppression less than 5% was considered not active.
Table 3.3
Methylene ChioridelWater Partition of C 18 Extracts, pH 3.0"
Condi t ion Concentrat ionb CPM (mean f S.D.Ic % Suppressiond
Medi a Water e x t r a c t 1.25
0.63
CH2C12 e x t r a c t 1.25 0 -63
BM Water e x t r a c t 1.25
0.63
CH2C12 e x t r a c t 1.25 0.63
a Cl 8 extracts of 50 ml of RPMI- 1640 medium as a control or 50 ml rat BM-condi- tioned medium were prepared. A small amount of each was set aside for a control and the rest extracted with hexane as described in section 3.1.10.
Concentration refers to relative concentration of supernatants.
Counts per minute were obtained in the WEHI-3 bioassay.
Per cent suppression of WEHI-3 cells by BM Cl8 extracts was calculated using the media control. Suppression less than 5% was considered not active.
Table 3.4
Enzymatic Digestion of BM Cl8 ExtracP
Condi t i o n b CPM (mean I S . D . I c % Suppressiond
Medi a c o n t r o l 164391 * 5114
BM Cl8 e x t r a c t 43501 * 4352 7 4 . 3 - - --
Enzymes + Media c o n t r o l
Papain 158414 k 6371 6 . 5
Pepsi n 158085 * 3066 6 . 7
S u b t i l i s i n 169284 I7723 - -
Carboxypept i dase 144501 f 1225 1 4 . 7
Enzymes + BM Cl8 e x t r a c t
Papain 44037 * 4744 7 4 . 0
Pepsin 44281 I 1988 7 3 . 9
S u b t i l i s i n 50896 * 1360 7 0 . 0
Carboxypept idase 35038 * 3661 7 9 . 3
a Cl8 extracts of media or BM supernatant were incubated with enzymes as described in section 3.1.1 1.
Digested extracts or controls were tested in the WEHI-3 bioassay at a relative supernatant concentration of 5.0-fold.
Counts per minute were obtained in the WEHI-3 bioassay.
* Per cent suppression of WEHI-3 cells by BM Cl 8 extracts was calculated using the media control. Suppression less than 5% was considered not active.
3.2.5.4 Ion Exchange Chromatography
To test whether the molecule responsible for rat BMderived inhibitory activity
could carry a charge and bind to a charged matrix, BM C 18 extracts were passed through
DEAE-cellulose equilibrated at pH 7.4 or through CM-cellulose equilibrated at pH 6.0.
The columns were washed with bufTer to test if the activity was adsorbed before elution
with NaCl, as descnbed in methods section 3.1.14. The result is shown in Table 3.5.
Activity was not washed out of the DEAE column with buffer alone, but NaCl was effec-
tive in eluthg the activity. Buffer aione eluted most of the biological activity from the CM
column; very little additional activity was eluted with NaCI. These results suggested that
the inhibitory molecule was negatively charged at pH 7.4. Other experiments using ion
exchange Sep-Paks and anion exchange HPLC confirmeci that the inhibitory activity bound
to anion exchangers but not cation exchangers at neutral pH (data not shown).
3.2.5.5 Gel Filtration Chromatography
Results obtained using dialysis as described in section 3.2.5.1 indicated that the
molecule responsible for rat BM-derived inhibitory activity was low MW, possibly less
than 1000 Da. Therefore gel filtration media with a nominal exciusion limit of 100-1800
Da was chosen for further fractionation of rat BM Cl8 extracts. Figure 3.7 shows the
results of fractionation of rat BM C 18 extracts using Bio-Gel P-2. Myoglobin (MW 17000)
eluted in fraction 8, indicating fractions 1-8 to be the void volume. As shown on the
graph, most biological activity eluted in fractions 12-14 while most UV-absorbing mate-
rial eluted in fractions 14-20. No biological activity was found in the void volume (data
not shown). The MW 1356 standard compound vitamin B,, eluted in fractions 10-1 1.
These results suggested: 1) the MW of the inhibitory molecufe is less than vitamin BI?,
since it eluted after B12. 2) the fractions containing the biological activity were well-
separated from the fraction containing most UV absorbent material, indicating that Bio-
Gel P-2 gel filtration was a good purification step. The inhibitory activity in BM
supematants purified by Cl8 extraction and Bio-Gel P-2 gel filtration was found not to be
Table 3.5
Fractionation of BM Cl8 Extract by Anion and Cation Exchangea
Exchanger E l u t i on w i t h Concen t ra t i onb % SuppresssionC
B u f f e r
B u f f e r
1 M NaCl
a In each experiment 0.15 ml BM C 18 extract was evaporated, and diluted in buffer before addition to the column.
Concentration refers to relative concentration of supematants.
Per cent suppression of WEHI-3 cells by various fractions was calculated using the media control (data not shown). Suppression less than 5% was considered not active.
Figure 3.7
Gel Filtration Chrornatorrraphv of BM Cl8 Extracts
100 ml rat BM-conditioned medium was concentrated by Cl8 extraction as
described in methods 3.1.10. The extract was dissolveci in 0.2 ml 20 mM Tris-HC1
buffer pH 7.2 and applied to a 1 .O x 42 cm column of Bio-Gel P-2 equilibrated in the
same buffer, as described in methods section 3-1-15 Various fractions were collected,
re-extracted with Cl8 cartridges and tested for activity in the WEHI-3 bioassay. Re-
sults are indicated (hatched bars) on the left vertical axis as the per cent suppression of
constitutive WEHI-3 proliferation aven by each fraction. Each fraction was tested at
a 1/64 dilution of 0.5 ml extract, equivalent to a relative supernatant concentration of
3.13-fold. W absorption of each fraction at 215 nrn is indicated (O) on the right
vertical axis.
16 18 20 22 24 Fraction Number
due to cornpetition for 3H-thymiduie incorporation by cold thymidine, for several rea-
sons: 1) Thymidine alone eluted in fractions 2 1-23. 2) The suppressive fractions had no
W absorbance at 267 nm, where thymidine absorbs most strongly. 3) Unlike thymi-
dine, the inhibitory activity was not destroyed by hydrazine and piperidine treatment
(data not shown).
3.2.6 Establishment of a Standard Curve for Rat BM-Derived Inhibitory Activity
Inhibitory activity was purified by Cl8 extraction and Bio-Gel P-2 gel filtration
of rat BM supernatants, then tested over a wide concentration range in the WEHI-3
bioassay. Figure 3.8 shows that the pattern of suppression caused by the gel filtration-
pwified inhibitory activity was not significandy different than that caused by Cl8 ex-
tract, as shown in Figure 3.4.
In order to measure relative purification it is necessary to quantZy the arnount of
material recovered after each purification step (Ersson et al. 1989). Since the chernical
nature of the molecule responsible for rat BM-derived inhibitory activity was unknown,
a quantitative biological assay was used. Following the precedent of the purification of
other negative regulatory factors (Ikeda et al. 1987; Graham et al. 1990; Lenfant et al.
1989; Paukovits and Laemm, 1982) it was decided to use the following definition: One
unit of the rat BM-derived inhibitory activity is defined as the mount necessary to sup-
press proliferation of one standard culture of WEHI-3 cells by 50%. A standard WEHI-
3 culture is descnbed in methods section 3.1.6.
To sirnpliw calculations of units of activity the data shown in figure 3.8 was used
to generate a standard curve of biological activity. Figure 3.9 shows the dilution cuve
plotted as per cent suppression versus units per culture. The dilution curve was linear
when plotted on a natural log scale. 50% suppression is by definition 1 unit per culture,
i.e. e0 units per culture.
Figure 3.8
Inhibition of WEHT-3 Ce11 Roliferation b~ BM-Denved
Inhibitorv Activitv Pre~ared bv Cl 8 Extraction
and Gel Filtration Chrornatomaphv
1 litre rat BM-conditioned medium or HBSS as a control were extracted by Cl8
c d d g e s and hctionated by gel filtration over 5 separate runs as described in meth-
ods section 3.1.15. The biologically active fractions from BM (e) or control (m) ex-
tracts (fractions 12- 14) were re-extracted using C 18 cartridges, then tested at various
concentrations in the WEHI-3 bioassay. Constitutive proliferation of WEHI-3 cells is
indicated (A). Results are expressed as means (symbols) and standard deviations (ver-
tical error bars) of triplicate cultures.
O 10 20 30 40 50 60
Relative Supernatant Concentration
Figure 3.9
Standard Curve of BM-Derived Inhibi torv Activitv
The data from suppression of WEHX-3 cells by BM extracts purified by C 18
cartridges and gel fdtration shown in Figure 3 -7 was used to generate a standard curve.
One unit per culture was defined as described in methods section 3.1 -2 1. 50% inhibi-
tion of proliferation of WEHI-3 cells is indicated as a heavy iine on the vertical axis.
50% inhibition corresponds by definition to 1 unit per culture, or e0 U/culture on the
horizontal axis.
=-5-0 = 4 . O e-3.0 =-20 e-l.O e O - O el.O = 2 0 =3.O e4.0 e5.O
Log UnWCulture
3.2.7 Effect of BM-derived Inhibitorv Activitv on Proliferation of Mveloid Ce11 Lines
According to the mode1 of natural suppression proposed in chapter 1, NS cells are
myeloid lineage progenitor ceus. NS cells may regulate proliferation and differentiation
of myeloid stem and progenitor cells. NS cells and their soluble mediators may also
generally inhibit cell proliferation in hematopoietic sites. To test whether rat BM-derived
inhibitory activity equally inhibits a l l cell lines or has specificity for certain ceU types,
material purified by Cl8 extraction and Bio Gel P-2 gel filtration was tested for inhibition
of proMeration of various rnyeloid cell lines, including the human histiocytic lymphoma
U-937, the human histiocytic leukemia THP- 1, the human promyelocytic leukemia HL-
60, the human chronic myelogenous leukemia K-562, the mouse monocyte-macrophages
P388D, and J774a, the mouse rnyeloblast Ml, and the mouse myelornonocyte WEHI-3.
Figure 3.10 shows that at a concentration of 1 .O units/culture, P-2-purified material sup-
pressed U-937 cells by 13%, THP-1 cells by 48%, HL-60 cells by 30%, P388D, cells by
45%, and J774a cells by 36%. K-562 and Ml cells were not significantly affected. Fur-
ther experiments confïrmed that M l and K-562 cells were insensitive to BM-derived in-
hibitory activity even at higher concentrations (data not shown). These results confirmed
that rat BM-derived inhibitory activity did not suppress proliferation of all celi lines equally.
3.2.8 Scale-Up of Production of BM-derived Inhibitorv Activity
In order to isolaie the molecule responsiile for BM-derived inhibitory activity it was neces-
sary to maxhke production of BManditioned medium. In one set of expiments mnditioned
media were prepared h m young bovine rib rnarrow. Aithough a large number of ceUs codd be
obtained h m bovine BM, the inhiiitory activity for WEHI-3 cells by gel-filtration purified Cl8
extmcts was connstently ten-fold l e s that control preparatons h m rat BM (data not shown). The
use of swine BM was also explored but the bones were unsuitable for -on ofmarrow ells due
to hi& fat content (data not shown). It was decided to continue to piinfy inhibitory activity h m rat
BM supem;itants due to their high biological activîty.
Figure 3.10
BM-Derived Inhibi tory Activity
Su~~res ses Proliferation of Myeloid Ce11 Lines
BM-derived inhibitory activity partially purified by Cl8 extraction and gel fil-
tration chromatography was tested for activity on proliferation of various myeloid celi
lines as described in methods section 3.1.3 and 3.1.6. Inhibitory ac tivity was added to
cultures at 2.0 unitslculture (filled bars), 1.0 unit/culture (hatched bars) or 0.5 unitsf
culture (open bars). Results are expressed as per cent suppression of constitutive pro-
liferation of each ce11 line. Constitutive proliferation in for each cell line, expressed as
means and standard deviations of triplicate cultures, was as follows: 70045 i 2603 (U-
937), 69006 + 3577 (THP-1), 28228 I 1538 (HL-60), 74073 1981 (K-562), 7163 +_
262 (P388D,), 8155 I 582 (J774a), 16047 + 904 (Ml) and 41486 I 2776 (WEHT-3).
I T I 1 I 1 f
O 10 20 30 40 50 68 70
Per Cent Suppression
To rnaxirnize collection of BM-conditioned media rat BMC were cuhred for four
days and checked for celi viability each day. Supematants were collected each day, ex-
tracted using Cl8 cartridges and checked for inhibitory activity in the WEHI-3 bioassay.
Results are shown in Figure 3.1 1. rat BM decreased in viability steadily from the begin-
ning of culture, as rnight be expected in serum-free media. However, the inhibitory activ-
ity in the supematants did not significantly decrease until day 4. Therefore it was decided
to use 24,48 and 72-hour supernatants of rat BMC for the final purification of the mol-
ecule responsible for inhibitory activity.
3.2.9 Purification of BM-derived Inhibitoxv Activity
Viu-ious steps towards the purification of the molecule responsible for BM-derived
inhibitory activity were undertaken. As described above, conditioned media were ini-
tiaily extracted and concentrated using C 18 cartridges. After each step, biological activity
was measured using the WEHI-3 bioassay. Where possible, relative purification achieved
at each step was measured by comparison of the area of irrelevant W absorbance peaks
with UV absorbance peaks in the area of biological activity. The results in the following
sections are representative exarnples drawn from steps which were repeated many times.
Results of a typical purification of BM-derived inhibitory activity are summarized in Ta-
ble 3.6.
3.2.9.1 Gel Filtration
Gel filtration chromatography was employed to purify BM-derived inhibitory ac-
tivity as described in results section 3.2.5.5, except that a larger column was used as
described in methods section 3.1.15. A typical column run is shown in Figure 3.12. Us-
ing ibis column rnyoglobin (MW 17000) eluted in fraction 14, indicating the void volume
was fractions 1-14. Vrtamin B,, (MW 1356) eluted in fractions 21-23. As shown in the
figure most biological activity was in fractions 22-25, which were associated with a minor
peak of UV absorbance at 215 MI; no biological activity was found in the void volume
Figure 3.1 1
Rat BMC Decrease in Viabilitv and in Inhibitory Activitv
Production Over Four Days of Culture
Rat BMC were prepared and cultured as described in methods sections 3.1.4 and
3.1.9. At day 0, 1,2,3 and 4 supernatants were collected, extracted by C 18 cartridges
and tested for biological activity (hatched bars and right vertical axis) in the WEHI-3
bioassay. At each tune point 100 pl of cell suspension was removed and tested for
viability in the MTï assay (*, lefi vertical axis) as described in methods section 3.1.7.
Data are expressed as means (symbols) and standard deviations (vertical error bars) of
triplicate cultures.
O 1 2 3 4
Time (Days)
Table 3.6
Purification Tablea
Step Vol urne T o t a l A c t i v i t y % Recovery of P u r i f i c a t i o n (ml (Uni t s I b T o t a l A c t i v i t y F a c t o r c
Supernatant 6000 I
Cl8 E x t r a c t i o n 30 6073 I
P Z Gel F i l t r a t i o n 3.0 4555 75
J. Fas t -Q I o n Exchange 3.0 1640d 27
J.+ Amino-HPLC 0.5 146 2.4
5 C8-HPLC O, 5 44 0.72
a This table is shown for a representative purification, batch 19, out of seven batches purifed using the same purification protocol. The purification steps shown are sequen- tial, starting with the ceIl-free supernatant of rat BMC.
Total activiw was caiculated using the WEHI-3 bioassay to test dilutions of extracts after each purification step and calculating total activity using the standard curver shown in Figure 3.9.
Purification factor was calculated according to the following formula: Area Under Curve In helevant Fractions Area Under Curve in BiologicaIly Active Fractions
Unless othenvise indicated, the area under the curve was calculated by integration of the UV peaks measured at 215 m.
W absorbance was measured at 280 nm for this step only.
Figure 3.12
Gel Filtration Chromato maphy
1 litre of rat BMC supernatant concentrated by Cl8 extraction was dissolved in
2.5 ml 20 mM Tris-HCl buffer pH 7.2 and a p p W to a 2.6 x 89 cm column of Bio-Gel P-
2 as descnbed in methods section 3.1.15. Various fractions were collected, tested for
W absorbance at 215 m, re-extracted using Cl8 cartridges, and tested for activity in
the WEHI-3 bioassay. W absorbance of each fraction is indicated (a) on the left ver-
tical axis. Biological activity of each fraction is indicated (hatched bars) on the nght
vertical axis.
Fraction Number
(data not shown). The average purification achieved at this step was 33-fold while the
average recovery of biological activity at this step was 75%. The biologicaily active frac-
tions were concentrated using C 18 cartridges and used for M e r purification.
3.2.9.2 Anion Exchange FPLC
Anion exchange chromatography using an FPLC system was used to further puriQ
BM-derived inhibitory activity after Cl8 extraction and Bio-Gel P-2 gel filtration. A Q-
sepharose column was used which utilizes quatemary aminoethyl groups [-
0C~C-N(+)(C2H,)2CH,CH(OH)CHJ - as the anion exchanger. Injected matenal was
adsorbed to the column at pH 8.4, then eluted using a 0-0.4 M NaCl gradient over 40
minutes. The results of a typical run are shown in Figure 3.13. Most biological activity
eluted at 12-20 minutes while the largest single peak of W absorbante at 280 nm was at
20-25 minutes. The average purification achieved using this step was 3.9-fold while the
average recovery of biological activity at this step was 36%. The biologically active frac-
tions were concentrated and desalted using Cl8 cartridges and used for m e r purifica-
tion.
3.2.9.3 Amino (NIZ) HPLC
For m e r purification of BM-denved inhibitory activity it was decided to use
normal phase high-performance liquid chromatography (NP-HPLC). BM-denved inhibi-
tory activity is retained on C 18 cartridges, yet is soluble in methanol and water, indicating
that it has both hydrophilic and hydrophobie characteristics. NP-IBLC using amino-
bonded silica packîngs has been successfully used to separate carbohydrates based on the
number and positions of hydroxyl groups in the molecules (Churms, 1990). Since hy-
droxyl groups might contribute to the polar aspect of BM-denved inhibitory activity it
was decided to use a bonded amino column for further purification.
Biologically active fractions of rat BM supematants purified by Cl 8 extraction,
Bio-Gel P-2 gel filtration, and Fast-Q anion exchange were fiactionated using NP-HPLC.
Figure 3.13
Anion Exchange FPLC
Inhibitory activity purified from 1 litre of rat BMC supernatant by C 18 extrac-
tion and gel filtration chromatography was dissolved in 0.5 rd 20 m . Tris-HCI buffer
pH 8.4 and applied to a Fast-Q anion exchange FPLC colurnn as descnbed in methods
section 3.1.16. Material was eluted from the column with a 0-0.4 M gradient of NaCl
(dotted line, lefmiost vertical axis). W absorption (solid h e , left vertical axis) was
monitoreù at 280 nm by an in-line spectrophotometer. Fractions were coilected as
indicated, re-extracted and desalted using Cl8 cartridges and tested for activity in the
WEHI-3 bioassay (hatched bars, right vertical axis).
The results of an average run are shown in Figure 3.14. The standard compound maltose
monopalmitate eluted at 3.0 minutes, glucose at 6.3 minutes, and maltose at 8.9 minutes.
Most biological activity eluted at 6-8 minutes while the largest peak of W absorbance
overlapped slightly at 7-12 minutes. The average purification achieved using this step
was 4.9-fold while the average recovery of biological activity was 8.9%. The most highly
biologically active fraction (6-8 minutes) was concentrated as described in methods sec-
tion 3.1.18 and used for further pudication.
3.2.9.4 Reversed-Phase HPLC
Reversed-phase HPLC (RP-HPLC) was selected as the find step of purification of
BM-derived inhibitory activity. The major p ~ c i p l e of reversed-phase chromatography
is partition based on hydrophobie interactions (Szepesi, 1992). RP-HPLC has been de-
scnbed as one of the most robust HPLC techniques, with excellent resolving power for a
wide variety of substances. RP-HPLC has been used successfully in the purification of
many thousands of compounds (Szepesi, 1992).
Conditions for the fractionation of BM-derived inhibitory activity were initially
worked out using a Waters ~Bondapak octadecyl silica (ODS, C 18) column. It was found
that a mobile phase additive, trifluoroacetic acid V A ) , was required to prevent peak
tailing (data not shown). Peak tailing was Likely caused by interactions of BM-derived
inhibitory activity and other contarninating substances with exposed silanol groups on
the Cl8 column (Szepesi, 1992). Conditions were later optimized for the use of a 2mm
i.d. C8 column with isocratic solvent conditions. This colurnn reduced solvent consump-
tion and significantly improved resolution (data not shown).
Rat BM supernatants purified by Cl8 extraction, Bio-Gel P-2 gel filtration, Fast-
Q anion exchange, and amino-HPLC were fractionated by C8-KPLC; a typical run is
shown in Figure 3.15. Biological activity eluted at 22-26 minutes while most W absorb-
ent material at 215 nm eluted at 0-10 minutes. Biologicd activity was not associated with
a significant W peak in any experiment (data not shown). The average purification
Figure 3.14
Inhibitory activity purified from 1 Litre of rat BMC supernatant by CI8 extrac-
tion, Bio-Gel P-2 gel filtration and Fast-Q anion exchange FPLC was dissolved in 150
pl 80% acetonitrile and injected at 50 pl per run using an Amino (NY) HPLC column
as described in methods sections 3.1.17 and 3.1.18. Virious fiactions were collected,
recovered as descnbed in methods section 3.1.18, and tested for biologicd activity in
the WEHI-3 bioassay. UV absorption at 215 nm is indicated (solid line) on the left
vertical axis. Biological activity is indicated (hatched bars) on the right vertical axis.
O 2 4 6 8 10 12 14 16 18 20 Fraction Collected (minutes)
Figure 3.15
Reversed-Phase HPLC
BM-derived inhibitory activity purified by Cl8 extraction, Bio-Gel P-2 gel fil-
tration, Fast-Q anion exchange FPLC and amino-HPLC was further fractionated by
RP-HPLC using a C8 column. Inhibitory activity p d e d from 6 litres of rat BMC
supernatant was dissolveci in 45 pl 25% acetonitrile + 0.1 T'FA and injected into the
HPLC system at 15 pl per run as descnbed in methods section 3.1.19. Fractions were
collected as indicated, concentrated as described in methods section 3.1.19 and tested
for biological activity in the WEHI-3 bioassay. W absorption at 215 nm is indicated
(soiid line) on the left vertical axis. Biological activity is indicated (hatched bars) on
the right vertical mis.
achieved using this step was 219-fold while the average recovery of biological activity at
this step was 30%. The single rnost biologically active fraction was concentrated as de-
scribed in methods section 3. l . 19 and used for characterization experiments.
3.2.10 Characterization of BM-derived Inhibitory Ac tivity usinp Mass Spectrometrv
After purification of BM-derived inhibitory activity using the steps descnbed above,
inhibitory activity could be detected using the WEHI-3 bioassay but no material could be
detected by any physical rnethod. There was no visible residue, no protein detectable by
protein assays, no visible spots on TLC plates, and no UV absorbance at any wavelength
above 2 15 nm (data not shown). Since BM-derived inhibitory activity could ody be
purified in small quantities it was decided to monitor purity and if possible perform pre-
Iirninary characterization using a mass specwometer.
3.2.10.1 Electrospray MS Analysis of Punned BM-derived Inhibitory Activity
For this snidy a Sciex API III triple quadrupole mass spectrometer with an ionspray
ionization interface was used, in collaboration with Dr. Henrianna Pang and Mary Cheung
of the Carbohydrate Research Group, University of Toronto. BM-derived inhibitory ac-
tivity was purifïed from rat BM supernatants using Cl8 extraction, Bio-Gel P-2 gel filtra-
tion, Fast-Q anion exchange, amino-HPLC and C8-HPLC, as described above. W e d
BM-denved inhibitory activity (batch 19) was injected directly into the ionspray interface
of the m a s spectrorneter, as described in methods section 3.1.20, and analyzed using the
first quadrupole as a mass analyzer. The mlz range 200-1000 was selectively monitored
since dialysis and gel filtration experirnents indicated the MW of BM-derived inhibitory
activity to be less than 1ûûO Da. The results shown in Figure 3.16 indicated various
significant peaks were observed, with the following m/z ratios (in order of abundance):
353.2, 367.2, 326.2, 210.0, 373.2, 317.2, 335.2, 295.0, 277.2, 251.0, 299.2, and 313.4.
This result indicated that various molecules were present in the most highly pwified prepa-
ration of inhibitory activity.
Figure 3.16
Mass Spectrometry of Purified Inhibitory Activity
Inhibitory activity was purified fiom 6 litres of rat BMC supernatant using Cl8
extraction, Bio-Gel P-2 gel filtration, Fast-Q anion exchange FPLC, amino-HPLC, and
C8 RP-HPLC. The biologicaily active fiaction was dissolved in 500 pl methanol and
injected at a flow rate of 20 pl per minute directly into a quadrupole mass spectrometer
equipped with an ionspray ionization interface, as descnbed in methods section 3.1.20.
Positively charged ions with mass over charge (rdz) ratios 200-1000 were monitored
by continuous scanning. Results are expressed as the per cent relative intensity of ions
detected at each m/z ratio.
3 -2.10.2 Identification of an IonAssociated Wïth BM-Derived Inhibitory Activity Using
LC-Ms
The results described above indicated that the molecde responsible for BMderived
inhibitory activity rnight be any one of several ions detected in the purifid sample. Since
more than one ion was detected in the RP-HPLC fraction containhg the inhibitory activity it
was decided to search for ions whose retention time correlated with the inhibitory activity.
Inhibitory activity was purifïed fiom rat BM supematants by Cl8 extraction, Bio-Gel P-2 gel
nitration, Fast-Q anion exchange and amino HPLC, t ' e n fiactionated using the C8 colurnn
with the HPLC system eluting directly into the ionspray interface of the mass spectrometer. A
flow splitter was used to direct 1/10 of the eluent into the mass spectrometer, 9/10 of the
eluent sample was collectai and tested for biological activity in the W W - 3 bioassay. AU
ions m/z 200- 1000 were monitored by continuous scanning through this mass range.
As described in the previous section, ions of many nilz ratios were observed in the
biologically active fiaction. However, when each of these d z ratios was extracted fiom the
total ion chromatogram and plotted as intensity vs. retention tirne, only m/z 373 correlated
with the retention time of the biological activ* (data not show). In a total of four experi-
ments, of which three are s h o w in Figure 3.17, m/z 373 was the only ion unique to the
biologically active fiaction. The retention time of mlz 373 and of the biological activity were
variable between 20-30 minutes in any particular experiment. These results suggested that d
z 373 was uniquely associated with BMderived inhibitory activity. Wz 373 might be related
to the biologically active molecule or might be the ionized biologically active molecule itself.
Analysis of isotopic peaks c o h e d that d z 373 was singly protonated; therefore the actual
MW of this compound was 372 (+ HC).
3.2.10.3 Daughter Ion Analysis of m/z 373
Triple quadruple MS was used to f i e r characterize the mlz 373 ion. Miibitory
activity was purified from rat BM supematants using C l 8 extraction, Bio-Gel P-2 gel
filtration, Fast-Q anion exchange, amino-HPLC and CI-HPLC, then was injected di-
Figure 3.17
CorreIation of m/z 373 with BioloPicaI Activity by LC-MS
In three separate experiments, BM-derived inhibitory activity was purified as de-
scribed above and analyzed by online LC-MS as described in methods section 3-1-20.
Inhibitory activity was purified fiom batches of n t BMC supernatant of increasing size:
2.8 litres (experiment l), 4.0 litres (experiment 2), and 6 litres (experiment 3). In each
experiment inhibitory activity was dissolved in 30 pl 25% acetonitrile + 0.1 % T'FA and
injected in 15 pl in two separate LC runs. Various fractions were collected, concentrated
as described in methods section 3.1.20, and tested for biological activity in the WEHI-3
bioassay. Positively charged ions of m/z 200-1000 were monitored by continuous scan-
ning. The ion mlz 373 was extracteci fiom the database containing the total ion chroma-
togram. Relative intensity of mlz 373 is indicated (solid line) on the left vertical axis.
Total biological activity recovered in each fraction is indicated (open bars) on the right
vertical axis.
- Experiment 1
Experiment 2
Experiment 3
O 2 4 6 B I O 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Fraction (minutes)
rectly into the ionspray interface of the mass spectrometer, as descnbed in methods sec-
tion 3.1.20. W z 373 was analyzed using the mass spectrometer in the following configu-
ration: 1) Quadnrpole 1 was used as a mass filter to selectively transmit only m/z 373. 2)
Quadrupole 2 was used as a collision celI as described in methods section 3.1.20. 3)
Quadrupole 3 was used as a mass analyzer, selectively scanning through the range m/z
25-400. M/z 373 was fragmented in the collision cell by collision-induced dissociation
(CID) using Argon as the collision gas. This configuration of triple quadrupole mass
spectrometry is referred to as daughter ion andysis, since ail ions observed in the thûd
quadrupole are derived from (i-e. are daughters of) m/z 373 (Wysocki, 1992).
As shown in Figure 3.18, daughter ions were observed at the following m,z ratios:
372.8, 340.8, 278.8, 217.0, 207.8, 200.0, 184.8, 171.6, 159.8, 130.0, 114.2, 90.2, 69.8,
and 55.0. Analysis of these ions did not reved any hierarchy or structure (data not shown).
Furthemore, the pattern of daughter ions did not resernble the pattern generated by the
fragmentation of peptides or simple carbohydrates (data not shown). A search of MW
372 using the U.S. National Institute of Standards (NISTEPANM) mass spectral data-
base revealed 156 compounds, none of which had fragmentation patterns sirnilar to m/z
373 or had other chemical characteristics sirnilar to BM-derived inhibitory activity. These
results suggested that the ion at d z 373 originated from, or itself was, a unique novel
molecule.
3.2.1 1 Surnmary of Results
The results presented in this thesis describe the purification and partial characteri-
zation of the putative soluble mediator of NS cells. Previous results showed that
supematants of mouse BMC were Uihibitory for various in vitro proliferative responses.
To establish a rapid and sensitive biological assay for this activity, the mouse myelo-
monocyte cell h e WEHI-3 was selected from a panel of cell lines. Inhibitory activity
was prepared from supernatants of rat BMC in order to scale up production. Preliminary
Figure 3.18
Dauehter Ion Analysis of the Ion 373*
Inhibitory activity was purifieci korn 4 litres of rat BMC supernatant as described
above, dissolved in 500 pl methanol, and anaiyzed by direct injection at 20 pl per
minute into the triple quadrupole mass spectrometer as described in methods section
3.1.20. Ions of d z 373 were selected using the £ k t quadrupole as a mass filter, fiag-
mented by collision-induced dissociation (CID) with argon in the second quadrupole,
and were analyzed by continuous scanning from m/z 25400 in the third quadrupole.
Results are expressed as per cent relative intensity of ions detected at each m/z ratio.
work indicated that inhibitory activity was produced by large, Iow density rat BM NS
cells. The molecule responsible for inhibitory activity was low MW, as described for the
mouse BM inhibitory activity; was not a protein; and was anionic. In order to puri@ the
inhibitory molecule the foIlowing purification scheme was used: solid-phase extraction
(Cl8 camidges), gel filtration chromatography (Bio-Gel P-2), anion exchange chroma-
tography (Q-sepharose), normal phase HPLC ~ - s i l i c a ) , and reversed-phase HPLC (Cg-
silica). Electrospray mass spectrometry of the purified, biologicalIy active fraction showed
that a limited number of ions were present in the final mixture. One of these ions, rn/z
373, was shown using online LC-MS to be uniquely and repeatably associated with bio-
logical activity. Structural information obtained on mlz 373 using triple quadrupole mass
spectrometry indicates it to be a unique molecule.
CHAPTER 4
DISCUSSION AND FUTURE WORK
4.1 Discussion
4.1.1 Preliminârv Work
Research in the area of natural suppression has concentrated on the phenotype,
biological activities and mechanism of action of the NS cell. For this project we chose to
purifj the soluble factor responsible for mediating BM NS activity from BM supematants.
Although most work on BM NS cells has been in the mouse system, rat BM NS cells have
been extensively characterized and are identical in phenotype to the mouse BM NS ceil
(Noga et al., 1988a, 1988b). Both mouse and rat BM NS cells are large, low density,
negative for most Lineage-specific markers, and suppressive for in vitro immune responses
in an antigen and MHC-unrestricted manner. Mouse and rat BM NS activities are medi-
ated by soluble factors.
Since the chernical nature of the rat BM-derived inhibitory activity was unknown
it was not possible to design a physical assay for its measurement. Therefore it was
decided to p m the rat BM-denved inhibitory activity using a biological assay. After
testing a panel of lymphoid, myeloid and connective tissue ce11 lines the mouse
myelomonocytic leukemia WEHI-3 was chosen because of its high promerative rate and
high sensitivity to inhibition by ultrafiltration-concentrated BM supematants. The WEHI-
3 myelomonocytic leukemia ceIl Line was onginally isolated by Donald Metcalf and col-
leagues as a paraffin oil-induced tumour in BALB/c mice (Warner et al. 1969). WEHI-3
was characterized as an early rnyeloid progenitor based on its appearance, lysozyme con-
tent, and the fact that it can be differentiated into mature granulocytic ceus by G-CSF or
endotoxin (Wamer et al. 1969; Nicola and Metcalf, 1984; Ratph and Nakoinz, 1977).
Agents which cause differentiation of WEHI-3 also inhibit proliferation; therefore inhibi-
tion of proliferation of WEHI-3 may be caused by a differentiation signal (Nicola and
Metcalf, 1984; Ralph and Nakoinz, 1977).
Roiiferation ofWEHI-3 cells was inhibited by C 18 extracts of rat BM supernatants
as compared to extracts of spleen or thymus supernatants. This indicated production of
inhibitory activity to be unique to bone rnarrow. Cl8 cartrïdge extracts were inhibitory, but not
cytotoxic for WEHI-3 cells, as shown by the MTT viability assay in Figure 3.2. Therefore rat
BM-derived inhibitory activity suppressed growth of WEHT-3 c e k by a cytostatic mecha-
nism.
Rat BMC were separated according to size and density using counterfiow centrifugai
elutriation (CCE) in order to confimi that the inhibitory activity was produced by cells with a
phenotype similar to that described for rat BM NS cells. The results shown in Fïbwes 3.3 and
3.4 confimied that most inhibitory activity was produced by large, low density BMC. These
results are consistent with the idea that NS cells specifically produce the inhibitory activity
found in supernatants of rat BMC. Prelimuiary characterization of the molecule responsi-
ble for inhibitoy activity revealed the following:
1) The molecule was of low MW since it was hlly dialysable on 1ûûû Da membranes,
and eluted b e b d vitamin B,, (MW 1356) in gel nitration experiments. It cannot be con-
cluded that the inhibitory activity was lower MW than vitamin BI2 due to the unpredictable
behaviour of low MW compounds on gel filtration columns. However, the inhibitory activity
was clearly in the 1OOO Da MW range.
2) The m o l d e was relatively polar, since it was soluble in methano1 and water but did not
dissolve in hexane or methylene chloride. These results suggest that the inhibitory molecule was not
a fatty acid or triglymide, wfiich are relatively soluble in hexane. However, the inhibitory activity
was highly soluble in rnethanol., which disthguished it h m hexose sugan like glucose which are
insoluble in methanof.
3) The molecule was likely not a peptide, since it was not digested by broad-qedïcity
proteolytic enzymes.
4) The molecule appeared to have a negaiive charge at neutrai pH, since it was adsorkd to
anion but not to cation exchangers. It is conceivabie that the negative charge is due to an acidic
chemicai group such as caffK)xyl or phosphate. These characteristics were different h m any previ-
ously charactenzed imrnun~~~pPressive facto~s and thmiore suggested it to be a novel molecule.
To quantify biological activity, one unit of inhibitory activity punfied by C 18 ex-
traction and gel filtration was defined as the amount necessary to suppress one standard
culture of WEHI-3 cells by 50%. This definition is similar to the definition of one unit of
TGFpsing inhibition of Mv 1 Lu ceils (Ikeda et al. 1987); and sirnilar to definitions used
in the purification of other negative regdatory molecules (Graham et al. 1990; Lenfant et
al. 1989; Paukovits and Laerum, 1982). When biological activity was plotted on a log
scale there was a Linear relationship between the log of the number of uni& and the per
cent inhibition of proliferation of WEHI-3 cells, as shown in Figure 3.9. This linear
relationship greatly simpiifïed the calculation of the number of units present in a given
batch.
4.1.2 Bioloeical Activity
Previous work from our laboratory indicated that mouse BM supernatants selec-
tively inhibited myeloid, but not lymphoid or erythroid colony formation (Parsons, 1992).
Since it was suggested that NS cells may be reIated to myeloid progenitor cells (Figure
1.2), it became important to investigate if NS cells could regulate the proliferation of other
rnyeloid progenitor cells by release of a soluble mediator. lnhibitory activity purified
from BMC supematants by Cl8 extraction and gel fiItration was tested on myeloid pro-
genitor cell lines immortalized at various stages of matunty. K-562 and Ml cells are
considered stem cell Lines (Leenen et al. 1986; Vainchenker et al. 198 l), while HL-60 and
WEHI-3 cells are considered relatively immature myeloid progenitors (Leenen et al. 1986;
Nangia-Makker et al. 1993; Warner et ai. 1969). THP-1, U-937, J774a and P388D, are al l
relatively mature macrophage-like cell lines (Tsuchiya et al. 1980; L-ck et al. 1980;
Mîshell 2nd Dutton, 1967; Koren et al. 1975). It is interesting that the BM-derived inhibi-
tory factor was most suppressive for the rnyeloid progenitor-like (HL-60, WEHI-3) and
mature macrophage-like (THP-1, U-937, J774a, P388DJ cell lines. The inhibitory activ-
ity did not affect the least mature cells (K-562 and Ml). These observations suggested
that BM-derived inhibitory activity may selectively inhibit proliferation of progenitor,
late progenitor and relatively mature myeloid cells. These experiments demonstrated that
the activity is not a nonspecific inhibitor of ai l cell types. In fact, the proliferation andlor
viability of K-562 cells was unaf5ected at any concentration of inhibitory activity tested.
4.1.3 Purification
Based on the preluninary characterization of the inhibitory molecule discussed
above, attempts were made to further puri@ inhibitory activity from larger quantities of
rat BM supematants. As discussed in results section 3.2.8, cow or pig BM proved to be
unsuitable for production of inhibitory activity due to the presence of very hi& fat content
and low inhibitory activity. Therefore rat BM supematants were used as a source for
purification. The viability of rat BMC in se--free HBSS indicated supematants couId
be collected up to 72 hours before there was a decline of biological activity. A total of 2 1
batches of an average of three litles of supernatant were produced and tested for the stud-
ies described in this thesis.
A sequence of steps for the purification of the inhibitory molecule was designed
using the parameters discussed in Theoretical Background (section 2.4). Initially, solid-
phase extraction using C 18 cartridges was used to concentrate rat BM supernatants and to
recover biological activity after each purification step. The use of Cl8 cartridges obvi-
ated the requirement to use separate steps for concentration and desalting.
Gel filtration using Bio-Gel P-2 was selected as the £kst purification step because
of the relative reliability of this technique. Supernatants could be applied to colurnns of
Bio-Gel P-2 many times without significant loss of resolution. As shown for a character-
istic batch in Table 3.6, Bio-Gel P-2 gel filtration produced a purification factor of 33 and
recovenes of inhibitory activity averaging 75%. Gel filtration with a large column further
confirmed the low MW nature of the activity, since activity eluted behind vitamin B,,
which has a MW of 1356.
Anion exchange chromatography was selected as the second purification step. The
strong anion exchanger Q-sepharose was used because this produced better recovery of bio-
l o g i d activity than the weak anion exchanger DEAE. Inhibitory activity eluted at a low
concentration of NaCl, collectively suggesting that the molecule responsible for inhibitory
activity was weakly charged.
NP-HPLC using an amino-bonded silica column was selected as the third purification
step. Amino-column HPLC of BMdenved inhibitory activity produced a purification factor
of 4.9, as s h o w in Table 3.6. The inhibitory activity had a retention t h e between that of
glucose and maltose. This suggested that the relative polarïty of the molecule responsible for
inhibitory activity, which is related to the number of hydroxyl groups and charged groups, rnay
be related to glucose and maltose, with carbhydrate-like characteristics.
RP-HPLC was chosen as the final purification step. As s h o w in Table 3.6, RP-HPLC
of pareially purified fractions using a Cl8 or C8 column provided a pudication factor of 2 19.
The retention time of the inhibitory activity was greater than those of hydrophobie peptides,
yet less than those of polar Iipids. Tnfluoroacetic acid mA) was required to prevent peak
tailuig in this system, suggesting that polar groups on the inhibitory molecule were interacting
with the silanol backbone of the reversephase colum. These resuits suggest that the inhibi-
tory molecule had both polar and nonpolar chernical groups. Recovery of biological activity at
this step was consistently poor, ranging from 5-30% in any given experiment. There are two
potential reasons for this low recovery: 1) the inhibitory rnolecule rnay be volatile under the
pH conditions used (pH 2-3 for 0.1% TFA) or 2) the inhibitory rnolecule might be chemically
altered during the purincation process.
Relative pur@ of the inhibitory activity was increased by a factor of 1.38 x 10S-fold by
using the purification steps described above. Although recovery of biological activity was low
at severai steps, specific activity (biologicai activity per ml) was increased. These resuits
suggest the molecule responsible for biological activity was highly purified using the steps
described in this thesis.
4.1.4 Characterization
The nature of the molecule responsible for inhibitory activity was investigated
further using electrospray mass spectrometry. Electrospray ionization is a "soft" ioniza-
tion technique, meaning that most ions generated are protonated intact molecular ions
with very little fragmentation (Keba.de and Tang, 1993). Therefore the purity of the final
biologically active fraction couid be monitored using electrospray mass spectrometry.
Observation of a number of peaks suggests relative irnpurity of the sample.
Inhibitory activity purified by Cl8 extraction, gel filtration, anion exchange, NP-
HPLC and RP-HPLC was shown by direct injection into an electrospray mass spectrometer
to contain a number of molecules. Therefore correlation of the retention time of various
ions with biological activity was used to identiQ the molecule responsible for inhibitory
activity. One ion at m,z 373 correlated very closely with biological activity in four con-
secutive experiments and was therefore the strongest candidate for the molecule responsi-
ble for the inhibitory activity. The slight shift in overlap of m/z 373 peaks with biological
activity suggests the possibility of two or more isomers of this molecule. At present it
cannot be ruled out that only one isomer may be responsible for the inhibitory activity.
Examination of isotopic peaks revealed that d z 373 was singly charged. In the
singly charged state the largest isotopic peak would be at (M + 1)f 1 = 374. If mlz 373
were doubly protonated the largest isotopic peak would be at (M + 1)/2 = 373.5. Since the
largest isotopic peak was in fact observed at m/z 374, m/z 373 was deduced to be singly
protonated. Therefore the actual MW of this molecule is 372 with a proton associated to
give m/z 373.
To further investigate its molecular structure, collision-induced dissociation (CID)
of d z 373 was used. The pattern of daughter ions resulting from CID of m/z 373 is
shown in Figure 3.18. Of the fraeoments generated, only mlz 114.2 corresponded to a
residue mass value of an amino acid, as observed in quacirupole mass spectrometry pep-
tide hgmentation studies (1 13.08, leucine or isoleucine). Therefore m/z 373 may not be a
peptide since CID of peptides results in a series of hgment peaks correspondhg to the resi-
due mass values of amino acids (Wysocki, 1992). Furthemore, the fragmentation pattem did
not resemble that of a hexose sugar or nucleic acid, which are known to give difTerent patterns
of base peaks. This suggested that the structure of mlz 373 was not that of a simple peptide,
simple sugar or nucleic acid and may represent a unique complex molecular structure. In
attempting to establish a hierarchy of daughter ions several possible losses of 18 were ob-
sewed, which cornespond to %O. This result M e r indicates that m/z 373 may possess
hydroxyl groups and could be a highly substituted complex carbohydrate.
As discussed earlier, a search of the NIST/EPA/NIH mass spectral database for the
MW 372 identifiecl 156 compounds. Most of these were cholesterol denvatives without a
match for the inhibitory activity in t e m of solubility and other characteristics. A search of
this database based on daughter ion peaks also did not produce any matches, which is not
supising since most of the database entries are denved fiom electron impact spectra At
present no database exists for CID daughter ion spectra using triple quadrupole mass
spectrometry.
4.1.5 Sipniflcance
This study represents the first successfid attempt to pur* and partially idenw the
soluble mediator responsible for the biological activities of BM NS celis. We have now PLI&
fied the inhibitory activity in rat BM supematants using Cl8 extraction, gel filtration, anion
exchange, NP-HPLC and RP-HPLC. A single ion observed by mass spectrometry at d z 373
was found to be uniquely associated with the inhibitory activity. The ion at m/z 373 may in
fact be the molecule responsible for the irnmunosuppressive activity of BM supernatants.
Ali work performed to date in the characterization of NS ceUs is sumrn-d in Figure
1.2. BM NS ceils may participate in 1) regulation of hematopoiesis, and 2) regulation of
immune responses. A soluble factor is known to be responsible for mediating this activity
between cells. The loss of this soluble factor by mutation or functional dysregulation rnay
lead to hematopoietic dysfunction, including Ioss of negative regulation of stem and pro-
genitor cell proliferation. Loss of normal control of stem and progenitor ce11 proliferation
rnay be part of the stepwise generation of leukemias (reviewed in Witte, 1994). It has
been suggested that NS cells rnay dso be a "fail-safe" mechanism for control of al10 and
autoimmune responses (Strober, 1984). Therefore the loss or abrogation of BM-derived
negative regulators rnay be a contributing factor for autoimmune conditions. The BM-
derived inhibitory molecule, when fuUy characterized and synthesized, rnay have clinical
potential for the treatment of leukemia and autoimmunity.
4.2 Future Work
Experiments are already under way to scale up production of BM-derived inhibitory
activity in order to elucidate its complete molecular structure using a variety of state of the
art techniques. Sorne of the approaches that will be used to solve the structure of the
inhibitory molecule are described as following.
4.2.1 Scale up of BM-Denved Inhibitorv Factor Production
To scale up production of the molecule both the amount of source material and the
capacity of various purification steps could be increased. The gel filtration step can be
adapted for an FPLC system using a P h m a c i a Superdex HR/30 column, which would
reduce the arnount of time per run by 600%. Anion exchange HPLC systems using qua-
ternary-ion bonded silica columns rnay also provide additional purification steps.
4.2.2 Final S tmctural Elucidation
As discussed earlier m/z 373 is a negatively charged molecule possessing both
polar and non-polar properties. It is not a simple peptide, carbohydrate or nucleic acid
structure. If it is a complex glycoconjugate-type structure, the following types of analyses
may be used: 1) purification to homogeneity, 2) sugar composition, 3) anorneric configu-
ration of each residue, 4) conformation of sugar rings, 5) sequence or arrangement of the
sugars, 6) conformation of the intersugar residue linkages, 7) identity, points of attach-
ment, stereochemistry of nonsugar substitutents, and 8) the secondary or three-dimen-
sional orientation of the carbohydrate chah (Sweeley and Nunez, 1985; Dwek et al. 1993).
4.2.2.1 MethyMon Analysis
If the molecule contains carbohydrate, methylation analysis cm be used to f'urther
elucidate its structure (Sweeley and Nunez, 1985; Bjomdal et al. 1970). In methylation analy-
sis the substance is first permethylated, then hydrolysed in 95% acetic acid and 0.5 M sulphu-
ric acid resulting in partiaily methylated (and possibly substituted) monosacch&des. These
are then acetylated with acetic anhydride and analyzed by gas chromatography (GC) and
electron impact m a s spectrometry (EI-MS). This approach has successfulIy been used to
solve the structures of many glycoconjugates (Sweeley and Nunez, 1985; Bjomdal et al. 1970).
4.2.2.2 Fast Atom Bombardment (FAB) Miss Spectrometry
The purified molecule could be analyzed by FAB-MS to provide a hi&-resolution
MW analysis. This technique aliows othenvise nonvolatile polar substances to give intense
rnolecular peaks with very linle hgmentation. Coupled with a double-sector mass spectrometer,
FAB could provide very precise MW that wouid help deduce a molecular formula (Sweeley
and Nunez, 1985; DeU et al. 1994).
4.2.2.3 Nuclear Magnetic Resonance Spectroscopy
Large arnounts of the purifid molecule would facilitate analysis by NMR to provide
direct structural information. Types of NMR analyses that are useful for complex stnictures
kclude correlation spectroscopy (COSY), total correlation specûoscopy (TOCSY), relayed
correlation spectroscopy (REMY), nuclear Overhauser effect spectroscopy (NOESY), and
mta t ing-he Overhauser effect spectroscopy (ROESY) (van Halbeek, 1990; Dwek et al.
1993).
4.2.2.4 Ion Trap Mass Spectrometry
Ion trap MS could be very useful for M e r analyses of the purified or partiaily
purified molecule. A quadrupole ion trap can be used to store and selectively fragment
ions. Many sequential rounds of daughter ion analysis (MSn) can be performed to pro-
duce a hierarchical analysis of the structure of a molecule (Todd, 1995). In fact this
technique has been used to solve the structures of smaU molecules including chalcones,
hydoxy- and methoxy indoles, and a and P hydroxycholesterol (Catinella and Traldi,
1995). The inhibitory molecule could be hlly silylated or methylated, then analyzed by
GC-ion trap MS to produce extensive daughter ion analyses. This approach may produce
extensive structural information without the requirement to pur* the molecule to homo-
genei ty.
4.2.2.5 Chernical Synthesis
Once the complete structure has been determined for the molecule it will be con-
firmed by synthesis. This will also provide larger quantities of the compound for further
snidies of its in vivo activity. As suggested, if the molecule is carbohydrate possessing an
anionic group, it will be possible to devise a synthetic route; for example, cornmercially
available carbohydrate precursors can be used if appropriate, and they will be denvatized
by applying hown procedures for carbohydrate derivatization.
4.2.3 Biological Studies
Larger quantities of synthetic material will dlow further in vitro and in vivo bio-
1ogica.l studies.
4.2.3.1 Cellular source and target
At present little is known about the precise identity of the cells producing the in-
hibitory molecule. Chernical precursors of the inhibitory molecule could be used to study
its metabolic pathway of synthesis and as probes to investigate the ceLi type involved in its
production. Synthetic inhibitory molecules could be labelied to study the possible nature
a cell surface and/or interna1 receptor. Labeiled molecules could also facilitate studies of
the inhibitory cell signal transduction pathway.
4.2.3.2 in v&o Myelopoiesis
Previous results from our laboratory suggested that inhibition of proliferation of
myeloid progenitor cens by BM supernatants was accompanied by the induction of difîer-
entiation in these ceils (Parsons, 1992). The inhibitory molecule could be tested for in-
duction of dinerentiation on a panel of myeloid ceil lines identified according to cell
surface markers and functional abilities (Leenen et al. 1986). These ce11 Iines could be
treated with the inhib.itory rnolecule in vitro and then monitored for changes in ce11 surface
markers. The inhibitory molecule could also be tested for effects on proliferation andor
lineage differentiation in normal hematopoietic progenitor cell assays including Cm-M,
CFU-G, CFU-GM, BFU-E, CFU-GEMM, CFU-B, LTC-IC, and ES cells.
4.2.3.3 Stem ceiI protection
Negative regdators of hematopoiesis have also been determined to block the entry
into cycle of CFU-S and pluripotent stem cells. These agents may therefore be usefiil in
protecting stem celis from chemotherapeutic agents, an approach that would reduce the
need for bone marrow transplantation foilowing chemotherapy (Moser and Paukovits,
1991). Agents which have been found to be protective of mouse stem cells include TGFB
(Bonewald, 1992), M'Ela (Quesniaux et al. 1993), IL-l (Zucali et al. 1994), TNF-a
(Zucali and Moreb, 1993), AcSDKP (l3ogden et al. 1991), and pEEDCK (Moser and
Paukovits, 199 1; Paukovits et ai. 1993). The inhibitory molecule could also be tested for
its ability to block entry of stem cells into cycle. It should be first tested in mice to see if
it inhibits exogenous or endogenous CN-S before testing in a preclinical stem ceil protection
assay (Ploernacher and Brons, 1989; Migliorati et aL 1989; Moser and Paukovits, 199 1).
4.2.3.4 Cancer Therapy
The inhibitory molecule has been shown to suppress proliferation of a variety of my-
eloid and other cell lines, as described in Results section 3.2.1 and 3.2.1 1. Unpublished pre-
Ziminary results have also shown that rat BMderived inhibitory activity slows the growth of
bladder carcinoma in vivo. Therefore preparations of the synthetic inhibitory molecuie should
be evaluated for anti-cancer potential using various mouse cancer models, including leukemias
and solid tumours.
4.2.3.5 Autoimmunity and Inhnmation
Preliminary results in this laboratory showed that BM extracts delayed the onset of
autoimmunity in NZB/W mice (Smythe, 1990). Therefore preparations of the synthetic in-
hibitory molecule shodd be tested for anti-innammatory effects in various mouse autoimmunity
models, including SLE in NZBN mice and EAE in SJL rnice.
4.3 Conclusions
Murine adult BM NS cells inhibit various cellular immune responses through elabo-
ration of a soluble factor. A novel inhibitory moIecule has been purified from BM
supernatants by solid-phase extraction, gel filtration, anion exchange FPLC, nomd-phase
HPLC and reversed-phase HPLC. LC-MS analysis indicates an ion at m/z 373 is uniquely
associated with biological activity and therefore may be the inhibitory molecule. Based
on MW, solubiiïty, charge, resistance to proteases, chromatographie retention times, and
MS daughter ion analysis, m/z 373 c m be distinguished from al1 known negative
immunoregulatory molecules. Further andysis, including chernical synthesis, is needed
to conclusively prove that m/z 373 is the molecule responsible for the observed biological
activity. This molecule may be an important regulator of hematopoiesis and immune
responses o c c u r ~ g in hematopoietic sites.
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