neutrophils

Biology Neutrophils

Introduction

PMN 50–75% of circulating leukocytes in humans

Important role in inflammatory responses that are critical for host defense against infection

first circulating cells to migrate to the site of infection

Phagocytosis, production of reactive oxygen intermediates (ROI), release of cytotoxic granule contents

Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson

Representing a major mechanism of innate immunity, release cytokines and chemokines that initiate and amplify inflammation, development of the acquired immune response


Neutrophil migration

Maturation PMN in the bone marrow and release into the bloodstream, migrate to airways under the influence of chemotactic factors and adhesion molecules

Mature PMN do not undergo cell division Generated continuously from the bone

marrow (∼1011 cells/day), can be greatly amplified in times of stress, e.g., infection


Myelopoiesis, where pluripotent stem cells divide and differentiate into myeloid precursors that follow a specific differentiation program

During maturation, PMN granules are formed, which contribute to the inflammatory response in the fight against microorganisms


PMN granules including serine and metalloproteinases, reactive oxygen species, lipid mediators, and defensins

Toxic molecules are released from activated PMN, and have ability to cause significant tissue damage to the lung and airways in asthma

Damage occurs when PMN accumulate in large numbers, and their activation is inappropriate or uncontrolled


Myelopoiesis

Developing neutrophils can be divided into six subtypes Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cells Mature neutrophils


Azophilic granules

Specific granule

Gelatinase granules

Many factors influence the development of PMN in the bone marrow Stromal cells - fibroblastoid cells,

endothelial cells, adipocytes, reticular cells and macrophages

Components of extracellular matrix - collagens, glycoproteins, and proteoglycans

Adhesion molecules - CD11b/CD18 Growth factors -G-CSF and GM-CSF


Important in this process are specific changes in gene expression patterns controlled by transcription factors such as C/EBPs and PU.1

Maturation in the bone marrow takes approximately 10–15 days, and depends on the detachment of the cells from the marrow microenvironment, and the mechanical ‘pumping’ of the cells into the bone marrow sinuses


Immature PMN can be released prematurely into the circulation in times of infection or inflammation, and these cells preferentially sequester into the lung microvessels

Exposure to inhalants, such as cigarette smoke, can decrease the transit time of PMN through the bone marrow, and cause the release of immature neutrophils into the bloodstream


Contact with cytokines (e.g., G-CSF, GM-CSF, IL-1) and chemokines (e.g., IL-8) can influence this process through the release of proteases (e.g., MMP-9) and the shedding of L-selectin

Released into the bloodstream, PMN half-life of 4–10 h, and can migrate into the tissues


NEUTROPHIL TRAFFICKING AND MARGINATION

Peripheral blood PMN are divided between Circulating pool, present in large and

small blood vessels Marginating pool that is arrested in

capillaries Margination in the systemic

circulation is regulated by selectin-mediated capture from the bloodstreamMiddleton 7th Jodie L. Simpson, Katherine J. Baines, Peter

G. Gibson

Rolling adhesion of PMN to the endothelium is mediated by L-selectin on the PMN and P- and E-selectin on the endothelium.

The pulmonary capillary bed is the main site containing marginating PMNs and measuring 20–60 times that of the concentration of large systemic blood vessels


Most PMN deform and elongate to travel through the pulmonary capillaries due to the vast network of the capillary bed, and the vessels being of a smaller diameter in comparison to spheric PMN

The requirement of PMN to deform to travel through the pulmonary capillaries increases their transit time, resulting in a higher concentration of PMN in this space


PMN sequestration, which is defined as amplified intravascular PMN numbers induced by inflammatory mediators and complement factors

Prolonged sequestration of PMN requires CD11b/CD18 (Mac-1)

The migration of PMN into tissues PMN rolling activation Firm adhesion to endothelial cells Migration through the endothelial cell layer, the

basement membrane and the epithelial interface and accumulation in the airway lumen


CELLULAR ADHESION MOLECULES

Rolling adhesion of PMN to the endothelium is mediated by L-selectin on the PMN and P- and E-selectin on the endothelium

Rolling allows interaction between CXC chemokines such as IL-8 presented on the surface of endothelial cells, which activates β2 integrin expression


Interaction between the integrins CD11a/CD18 and CD11b/CD18 and the endothelial immunoglobulin (Ig) superfamily members, intercellular adhesion molecule (ICAM)-1 and (ICAM)-2, are required for effective PMN transmigration and firm adhesion to the endothelium


Integrins

The integrins are a family of heterodimeric transmembrane glycoproteins that mediate direct cell–cell, cell–extracellular matrix, and cell–pathogen interactions

Contain two functional units: α and β chains β2 integrins are expressed on PMN and consist

of four different heterodimers: CD11a/CD18 or leukocyte function associated

antigen-1 (LFA-1); CD11b/CD18 or Mac-1 CD11c/CD18 or p150,95 CD11d/CD18


Leukocyte adhesion deficiency (LAD) results from a mutation in the gene for CD18 and is associated with recurrent bacterial infections due to an inability to recruit these cells to a site of infection

Functional state and presence of integrins on PMN is regulated by lipid, cytokine, and chemokine signaling molecules as well as ‘cross talk’ from other adhesion molecules


Integrins exist in predominately inactive states on circulating immune cells

Multiple mechanisms, including conformational change (affinity regulation) and clustering associated with the cytoskeleton (avidity regulation), are responsible for integrin activation, arising from or caused by ligand binding

Ability of the extracellular domains of integrins to bind ligands can be activated in <1 s via signals from within the cell (inside-out signaling)


Inflammatory stimulus

In the pulmonary circulation, PMN migration occurs through at least two pathways: CD11b/CD18 dependent CD11b/CD18 independent Dependent on the inflammatory stimulus

Inflammatory stimuli that invoke CD18-dependent PMN migration include Escherichia coli lipopolysaccharide (E. coli LPS), Pseudomonas aeruginosa immunoglobulin G (IgG), IL-1, immune complexes, and phorbol myristate acetate (PMA)


Stimuli that induce CD11b/CD18-independent PMN migration include Streptococcus pneumoniae; group B streptococcus, Staphylococcus aureus, hydrochloric acid, hypoxia, and C5a

Bacterial-derived chemoattractant fMLP stimulates CD18-dependent PMN migration,

whereas the host-derived chemoattractants IL-8 and LTB4 stimulate CD18-independent neutrophil migration


Endothelial cell interaction

First three steps of PMN migration (rolling, activation, and adhesion), the mechanisms that underlie transendothelial migration remain unclear

Leukocytes traverse the endothelial barrier through the cleft between two to three adjacent cells


Transendothelial migration, but also acquisition of cell polarity of the PMN, is thought to be mediated by platelet/endothelial cell adhesion molecule (PECAM)-1 and junction adhesion molecules (JAMs) expressed at intercellular tight junctions of endothelial and epithelial cells

The binding of JAM-C to Mac-1 was found to be of importance in neutrophil transendothelial migration


Epithelial cell interaction process involves three stages

epithelial adhesion migration post-migration

PMN firm adherence to the basolateral epithelial membrane is mediated exclusively by Mac-1

Transepithelial migration of neutrophils involves both cell–cell interactions that include adhesion molecules and signaling events to open the epithelial tight junctions, allowing the passage of cells without disturbance of the epithelial barrier


Interaction between CD47 and signal regulatory protein-a (SIRPa) enhances the migration rate of PMN through the epithelium

JAMs are likely to be important in the migratory process, as well as the formation of a seal around migrating cells to preserve barrier function.

After migration through the epithelium, PMN can adhere to ICAM-1 present on the apical surface of the epithelial cells


CHEMOTACTIC MEDIATORS

Through the endothelial basement membrane, PMN migrate along a chemotactic gradient

PMN chemotactic proteins include chemokines (e.g., IL-8), bacterial products (e.g., N-formyl methionyl peptides), lipid mediators (e.g., LTB4) and complement split products (e.g., C5a)


http://en.wikipedia.org/wiki/File:IL8_Solution_Structure.rsh.png

Chemokines are produced by inflamed tissues and activate signal cascades in the PMN that lead to increase in cell motility, adhesion and survival

IL-8 is a potent chemotactic factor for PMN

Blocking of IL-8 with a neutralizing antibody resulted in a 75–98% inhibition of its chemotactic activityMiddleton 7th Jodie L. Simpson, Katherine J. Baines, Peter

G. Gibson


IL-8 ( CXCL8 )is a member of the CXC subfamily, is produced by several cell types, in particular epithelial cells, macrophages and PMN themselves, and released upon proinflammatory stimulation

CXCR1, CXCR2 receptor Other members of this family include epithelial

cell-derived neutrophil activator-78 (ENA-78), growth regulatory gene (Gro)-α, Gro-β; neutrophil-activating peptide-2 (NAP-2), and granulocyte chemotactic protein-2 (GCP-2)



Chemokines

2 main subfamilies of chemokines, CXC and CC, which are classified according to the position of the first two cysteines in their amino acid sequence (separated by one amino acid – CXC, or adjacent CC), other (C , CX3C )

Many chemokines can bind to more than one receptor and most chemokine receptors can bind more than one chemokine


Leukotriene B4

Leukotrienes (LTs) are potent lipid mediators that have been implicated in the pathogenesis of airway diseases including asthma

LTs are synthesized from arachidonic acid via the actions of 5-lipooxygenase (5-LO), along with 5-LO-activating protein and terminal LTA4 hydrolase


Biosynthesis of leukotriene B4. 5-HPETE, 5-hydroperoxy-eicosatetraenoic acid; FLAP, 5-lipoxygenase-activating protein

Bing K. Lam* and K. Frank Austen

Classified into two classes; leukotriene B4 (LTB4) cysteinyl LTs

LTB4 is a potent chemoattractant and activator as well as enhances PMN adhesion and migration

LTB4 exerts its action through 2 seven-transmembrane G-protein receptors: the high-affinity BLT-1 and the low-affinity BLT-2Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter

G. Gibson

Innate immune activation

Activation of the innate immune system involves the detection of pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) including the toll-like receptor (TLR) family

Activation of TLRs results in an activation of a signalling cascade involving MyD88 and NF-κB that results in the release of chemokines and cytokines to further recruit neutrophils


Toll-like receptors (TLRs)

Currently the TLR family contains 10 members, and at the mRNA level, PMN appear to express all of these receptors except for TLR3

TLR4 is the major endotoxin receptor, and TLR2 recognizes PAMPs from Gram-positive organisms

TLR2 agonists include lipoteichoic acids (LTAs) and peptidoglycans


http://research4.dfci.harvard.edu/innate/index.html

Activation of both TLR2 and TLR4 regulates several important proinflammatory PMN functions through the activation of the NF-κB pathway, and these include PMN activation, migration, and survival

Exposure to LPS increases PMN expression of TLR2 and CD14 but does not change expression of TLR4


Upon stimulation with PAMPs including peptidoglycan, zymosan and araLAM (a component of Mycobacterium tuberculosis) PMN produce IL-8 and superoxide and also have increased phagocytosis

Rate of PMN apoptosis is delayed by the presence of bacterial lipoprotein and LPS to result in the augmentation of PMN inflammation

Anti-TLR2 monoclonal antibody prevents the delay in apoptosis in peripheral blood PMN


Mediators released by activated neutrophils

PROTEASESREACTIVE OXYGEN SPECIES

(ROS)DEFENSINS

Protease

Proteolytic enzymes play an important role in tissue remodeling and repair in the airways.

Levels of proteolytic enzymes, including active neutrophil elastase (NE) ) and matrix metalloproteinase-9 (MMP-9) increased in asthma and thought to indicate an imbalance in the protease/antiprotease system.


Courtesy of Dr Elizabeth Cramer, INSERM U474, Cochin Hospital, Paris

Granule Azurophil Specific Gelatinase Secretory vesicles

Marker enzyme Myeloperoxidase Lactoferrin GelantinaseAlkaline phosphatase

Membrane

CD 63 granulophysin

CD15,CD66,CD67 CD11b/CD18CD10,CD13,CD45, CD35 (CR1)

CD 68 CD11b/CD18 CD11b/CD18

Oxidase receptors (R)

Cytochrome b Cytochrome b Cytochrome b

Rap1A Rap1A Rap1AFMLP R FMLP R FMLP RC3bi R C3bi R CR 1R

Fibronectin R

C3bi R(CR 3R)

Laminin R CR 4R

Vitronectin R C1q R

FcGammaIII R

Plasminogen activator R

Signal transduction

Thrombospondin R Diacylglycerol deacylating enzyme

Decay accelerating factor

Gi2 protein subunit

Others

NB antigen

9 kd and 155 kd proteins

Granule Azurophil Specific Gelatinase Secretory vesicles

Matrix

Microbicidal MyeloperoxidaseNitric oxide synthase

LactoferrinLysozyme

Lysozyme

Lysozyme

BPI protein

Defensins

Serprocidins

Elastase

Cathepsin G

Proteinase 3

Azurocidin(CAP 37)

Hydrolases

Acid beta glycero-phosphatase alpha

Gelatinase Gelatinase

Mannosidase beta Collagenase Acetyltransferase

Glucuronidase beta Histaminase

Glycerophosphatase Heparanase

N-acetyl-beta glucosaminidase

NGAL

Sialidase Sialidase

Other

Acid mucopolysaccharide

Beta-2 microglobulin Beta-2 microglobulin Plasma proteins

Heparin binding protein

Plasminogen activator

Vitamin B12 binding protein

Neutrophil elastase (NE) is a 30 kDa serine protease can attack a number of proteins including lung elastin

High concentrations within the azurophilic granules of PMN and is important in host defence, specifically for the intracellular killing of Gram-negative infections


http://upload.wikimedia.org/wikipedia/commons/d/d1/1B0F_Human_Neutrophil_Elastase.png

Presence of extracellular active NE results in tissue destruction and is also a potent secretagogue, contributing to increased mucus production

Presence of extracellular active NE may indicate a protease/antiprotease imbalance

Neutrophil elastase can upregulate IL-8 gene expression and protein production in bronchial epithelial cells via a MyD88-dependent NF-κB signalling pathway



This upregulation is inhibited when cells are pre-treated with a TLR4 neutralizing antibody

Indicating that upregulation of IL-8 production is via an innate immune pathway

Neutrophil elastase is increased in asthma and also prominent in other airway diseases including COPD, cystic fibrosis, and bronchiectasisMiddleton 7th Jodie L. Simpson, Katherine J. Baines, Peter

G. Gibson


Secretory leukocyte protease inhibitor (SLPI) is a broad-spectrum inhibitor of mast cell and leukocyte serine proteases and produced by epithelial cells and submucosal glands

SLPI levels are increased in pneumonia and in the peripheral airways of subjects with emphysema, but decreased in chronic diseases such as diffuse pan-bronchiolitis and in COPD subjects with frequent exacerbations


S. Schneeberger Drug News Perspect 2002, 15(9): 568

SLPI inhibits NF-κB activation and LPS-induced TNF-α and IL-6 production in monocytes and macrophages

In vitro experiments have shown that once SLPI is inactivated, either by oxidation or complexed by NE, both antiprotease activity and antiinflammatory capacity are lost


α1-Antitrypsin (α1AT) is the major endogenous serine protease inhibitor produced by hepatocytes and is also expressed by PMN, epithelial cells, and macrophages

Both α1AT and SLPI counterbalance NE activity

Airway levels of α1AT are increased in the sputum of subjects with asthma Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter

G. Gibson

Levels of active NE are increased in asthma, so is the inhibitor α1AT, suggesting that the presence of antiproteases either are not sufficient or not functionally capable of inactivating the free enzyme


MMP-9 is a member of a family of zinc-containing enzymes that degrade extracellular matrix, modulate cytokine activity, and alter the activity of other proteases

MMP-9 has been identified in cells such as bronchial epithelial cells,PMN, mast cells, eosinophils, and macrophages


http://upload.wikimedia.org/wikipedia/en/7/71/PBB_Protein_MMP9_image.jpg

Tissue inhibitor of metalloproteinase (TIMP-1) is the major tissue inhibitor of MMP-9, secreted in association with MMP-9, and binds with both the pro- and active-forms of MMP-9 to cause inactivation

Levels of MMP-9 are increased in asthma compared to healthy controls and also in severe asthma compared to mild asthmaMiddleton 7th Jodie L. Simpson, Katherine J. Baines, Peter

G. Gibson


P Michaluk and L Kaczmarek

MMP-9 is expressed in the sub-basement membrane (SBM) in asthma and increased with increasing severity

The presence of MMP-9 in the SBM has been associated with the presence of PMN in the submucosa and also TGF-β positive cells

BAL levels of MMP-9 are also inversely related to FEV1 suggesting a relationship between this mediator and airflow obstruction



MMP-9 can inactivate α1AT to further NE-mediated tissue destruction

α1AT is a potent activator of PMN Oxidant radicals, including the hydroxyl

radical, peroxide, and hypochloride, can modify α1AT to a form that has no inhibitory capacity against proteases

TIMP-1 can be inactivated upon exposure to hypochlorous acid, which is released by activated neutrophils



REACTIVE OXYGEN SPECIES (ROS)

The respiratory burst involves the activation of NADPH oxidase, which is an enzymatic complex composed of cytosolic (p40phox, p47phox, and p67phox) flavocytochrome b558 which is composed of

membrane proteins (p22phox and gp91phox) Flavocytochrome b558 is located between the

plasma membrane and the membrane of the specific granules, and is incorporated into the phagocytic vacuole, where it pumps electrons from NADPH in the cytosol to oxygen in the vacuole


Reactive oxygen species (ROS) are generated as a result of NADPH oxidase activity to produce superoxide (O2

−) Superoxide can be rapidly converted

into hydrogen peroxide (H2O2) by the enzyme superoxide dismutase

Superoxide and hydrogen peroxide can also form to create the highly reactive hydroxyl radical (HO−)


hyperchlorous acid

Myeloperoxidase (MPO), a constituent of the azurophilic granules, generates hyperchlorous acid (HOCl) from hydrogen peroxide

Exposure to ROS can result in pulmonary injury Superoxide can activate granule proteins

through the recruitment of K+ to the phagosome, thus allowing cationic proteases of the azurophilic granules such as neutrophil elastase (NE) and cathepsin G (CG) to go from a highly organized intragranule structures into solution where they can kill ingested microbes.


ROS can inhibit a variety of protein tyrosine phosphatases through oxidation of key residues, allowing the phosphorylation of other molecules to proceed.

ROS can disrupt intercellular tight junctions, increase the permeability of the endothelial barrier via the phosphorylation of focal adhesion kinase in endothelial cells, and modulate PMN function by inducing apoptosis through a caspase-8 dependent manner


DEFENSINS

6 identified human defensins Small, arginine-rich peptides play an

important role in host defense to infections

Four defensins that are present in PMN are the human neutrophil peptides (HNP-1 to HNP-4).

Peptides kill pathogens by causing permeabilization of the bacterial membraneMiddleton 7th Jodie L. Simpson, Katherine J. Baines, Peter

G. Gibson

Mature defensins are present in high concentration in the azurophilic granules and constitute 5–7% of the neutrophil's total protein

Defensins also modulate the inflammatory response, as they can bind to protease inhibitors such as α1-antitrypsin


Neutrophil clearance and death After killing and digesting invading microbes,

PMN at the inflammatory site undergo programmed cell death (apoptosis) and are cleared by macrophages (efferocytosis)

Regulation of neutrophil apoptosis is crucial to maintain PMN numbers in the blood, as well as for the effective removal of invading pathogens, the resolution of inflammation, and the prevention of a necrotic cell death resulting in the release of the neutrophil's toxic cellular contents.


In vivo, this process may limit PMN, destructive capability.

Within a few minutes, PMN apoptosis results in irreversible chromatin condensation, nuclear collapse, cytosolic vacuolation, and cell shrinkage.

During this time the cell is unable to respond to agonists, is immobilized and inert.

Apoptotic neutrophils become instantly recognizable to alveolar macrophages, which result in cell removal via efferocytosis


Defined inflammatory stimuli, such as growth factors (e.g. GM-CSF and G-CSF), cytokines (e.g. IL-1 and IL-6), chemokines (e.g. IL-8), and even bacterial products (e.g. LPS), can delay PMN apoptosis

TNF-α and Fas-ligand (Fas-L) can increase the rate of PMN apoptosis

Corticosteroids delay PMN apoptosis, thus increasing their survival time, and influencing the persistence of neutrophilic inflammation


PMN apoptosis is induced by activation of cellular caspases and can occur through two main pathways. death receptor (DR) pathway, where the

clustering of TNF and Fas-receptors activates the caspase cascade beginning with cleavage of pro-caspase 8

intrinsic pathway consisting of mitochondrial cytochrome c and members of the Bcl-2 family that forms an apoptosome activating caspase 9


Cell stress due to exposure to ROS, DNA damage, or lack of growth factors can result in apoptosis, induced by the release of cytochrome c

Activated caspase 8 and 9 can then activate caspase 3 to cleave proteins essential for cell survival


D. Scheel-Toellner, Biochemical Society Transactions (2004) 32, (461–464)

CYTOKINE SYNTHESIS

The PMN is both a target and source of various proinflammatory cytokines (e.g., TNF-α and IL-1), chemokines (e.g., IL-8), and growth factors (e.g., GM-CSF and G-CSF), and hence has the ability to create a positive feedback loop on its own proinflammatory functions

Cytokine production by PMN is increased by inflammatory stimuli, bacterial endotoxin (LPS) being the most potent


Secretion of cytokines is varied and dependent on the agonist, and for some cytokine production, stimulation with more than one agonist is required, such as stimulation with IFN and LPS is needed for IL-12 production

Neutrophil cytokine expression can be modulated by T cell-derived cytokines: positively by Th1 cytokines (e.g., IFN) and negatively by Th2 cytokines (e.g., IL-4 and IL-13)


INTERACTIONS BETWEEN MICROBES AND

NEUTROPHILS

Opsonic factors principal Ig opsonins are IgG1 and IgG3,

while IgA1 and IgA2 also serve this function in the respiratory tract

complement components, C3b and C4b

Robert L Baehner

http://www.uptodateonline.com/online/content/author.do?topicKey=whitecel/7400


Phagocytosis and opsonic receptors Engulfment occurs via the advancing pseudopod

as the neutrophil surrounds the microbe Fc binding site is recognized by three classes of

opsonic receptors▪ Fc gamma RIII (CD16) - binds IgG subclasses 1 and 3 with

intermediate and low affinity▪ Fc gamma RII (CD32) - low affinity receptor with the

following subclass affinities: IgG1 = IgG3 >> IgG2 = IgG4 ▪ Fc gamma RI (CD64) - not expressed on basal stage,

found after exposed IFNg, binds IgG1 and IgG3 with high affinity, promoting phagocytosis of particles or bacteria opsonized with IgG

Robert L Baehner



Phagocytosis and opsonic receptors IgA antibody receptor - Fc alphaR (CD89),

signal transduction via G-protein linked phospholipase C activation, leading to phagocytosis and stimulation of the respiratory burst

Complement receptors▪ CR1 (CD 35) binds dimeric C3b▪ CR3 recognizes C3bi but not C3b, designated

CD11b/CD18 C1q receptor - C1q, the recognition subunit of

the classical complement pathwayRobert L Baehner



Conclusion

PMN migration- Myeloid development PMN trafficking - Rolling, Adhesion,

Migration Cellular adhesion mulecules – integrins

(CD11b/CD18) , inflammatory stimulus, endothelial and epithelial cell interaction

Chemotactic mediator – chemokines (IL8), LTB4,

Innate immune activation - TLR

Mediators released by activated PMN Proteases – Neutrophil elastase, MMP-9 Reactive oxygen species Defensin

PMN clearance and death Cytokine synthesis – proinflmmatory

cytokine (TNFa, IL-1), chemokine ( IL-8), growth factor (GM-CSF, G-CSF)

Interaction between microbe and PMN

neutrophils

Health & Medicine

peter g

katherine j

pmn granules

development of pmn

immature pmn

tissues pmn

migration of pmn

pmn halflife