Phagocytosis of Salmonella montevideo by Human Neutrophils ...
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Transcript of 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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
Myelopoiesis
Developing neutrophils can be divided into six subtypes Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cells Mature neutrophils
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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)
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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)
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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)
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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)
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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.
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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−)
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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.
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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.
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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)
Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson
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
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