Neutrophils

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Neutrophils

  1. 1. Biology Neutrophils<br />
  2. 2. Introduction<br />PMN 50–75% of circulating leukocytes in humans<br />Important role in inflammatory responses that are critical for host defense against infection<br />first circulating cells to migrate to the site of infection<br />Phagocytosis, production of reactive oxygen intermediates (ROI), release of cytotoxic granule contents<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  3. 3. Representing a major mechanism of innate immunity, release cytokines and chemokines that initiate and amplify inflammation, development of the acquired immune response<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  4. 4. Neutrophil migration<br />Maturation PMN in the bone marrow and release into the bloodstream, migrate to airways under the influence of chemotactic factors and adhesion molecules<br />Mature PMN do not undergo cell division<br />Generated continuously from the bone marrow (∼1011 cells/day), can be greatly amplified in times of stress, e.g., infection<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  5. 5. Myelopoiesis, where pluripotent stem cells divide and differentiate into myeloid precursors that follow a specific differentiation program<br /> During maturation, PMN granules are formed, which contribute to the inflammatory response in the fight against microorganisms<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  6. 6. PMN granules including serine and metalloproteinases, reactive oxygen species, lipid mediators, and defensins<br />Toxic molecules are released from activated PMN, and have ability to cause significant tissue damage to the lung and airways in asthma<br />Damage occurs when PMN accumulate in large numbers, and their activation is inappropriate or uncontrolled<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  7. 7. Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  8. 8. Myelopoiesis<br />Developing neutrophils can be divided into six subtypes<br />Myeloblast<br />Promyelocyte<br />Myelocyte<br />Metamyelocyte<br />Band cells <br />Mature neutrophils<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  9. 9. Azophilic granules<br />Specific granule<br />Gelatinase granules<br />
  10. 10. Many factors influence the development of PMN in the bone marrow<br />Stromal cells - fibroblastoid cells, endothelial cells, adipocytes, reticular cells and macrophages<br />Components of extracellular matrix - collagens, glycoproteins, and proteoglycans<br />Adhesion molecules - CD11b/CD18<br />Growth factors -G-CSF and GM-CSF<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  11. 11. Important in this process are specific changes in gene expression patterns controlled by transcription factors such as C/EBPs and PU.1<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  12. 12. Immature PMN can be released prematurely into the circulation in times of infection or inflammation, and these cells preferentially sequester into the lung microvessels<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  13. 13. 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<br />Released into the bloodstream, PMN half-life of 4–10 h, and can migrate into the tissues<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  14. 14.
  15. 15. NEUTROPHIL TRAFFICKING AND MARGINATION<br />Peripheral blood PMN are divided between <br />Circulating pool, present in large and small blood vessels<br />Marginating pool that is arrested in capillaries<br />Margination in the systemic circulation is regulated by selectin-mediated capture from the bloodstream<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  16. 16. Rolling adhesion of PMN to the endothelium is mediated by L-selectin on the PMN and P- and E-selectin on the endothelium. <br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  17. 17. 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<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  18. 18.
  19. 19. PMN sequestration, which is defined as amplified intravascular PMN numbers induced by inflammatory mediators and complement factors<br />Prolonged sequestration of PMN requires CD11b/CD18 (Mac-1)<br />The migration of PMN into tissues <br />PMN rolling activation <br />Firm adhesion to endothelial cells<br />Migration through the endothelial cell layer, the basement membrane and the epithelial interface and accumulation in the airway lumen <br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  20. 20. Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  21. 21. CELLULAR ADHESION MOLECULES<br />Rolling adhesion of PMN to the endothelium is mediated by L-selectin on the PMN and P- and E-selectin on the endothelium<br />Rolling allows interaction between CXC chemokines such as IL-8 presented on the surface of endothelial cells, which activates β2integrin expression<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  22. 22.
  23. 23. Interaction between the integrinsCD11a/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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  24. 24. Integrins<br />The integrins are a family of heterodimerictransmembraneglycoproteins that mediate direct cell–cell, cell–extracellular matrix, and cell–pathogen interactions<br />Contain two functional units: α and β chains<br />β2integrins are expressed on PMN and consist of four different heterodimers: <br />CD11a/CD18 or leukocyte function associated antigen-1 (LFA-1); <br />CD11b/CD18 or Mac-1<br />CD11c/CD18 or p150,95<br /> CD11d/CD18<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  25. 25. 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<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  26. 26. Integrins exist in predominately inactive states on circulating immune cells<br />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<br />Ability of the extracellular domains of integrins to bind ligands can be activated in &lt;1 s via signals from within the cell (inside-out signaling)<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  27. 27. Inflammatory stimulus<br />In the pulmonary circulation, PMN migration occurs through at least two pathways: <br />CD11b/CD18 dependent <br />CD11b/CD18 independent<br /> Dependent on the inflammatory stimulus<br />Inflammatory stimuli that invoke CD18-dependent PMN migration include Escherichia colilipopolysaccharide (E. coli LPS), Pseudomonas aeruginosa immunoglobulin G (IgG), IL-1, immune complexes, and phorbolmyristate acetate (PMA)<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  28. 28. Stimuli that induce CD11b/CD18-independent PMN migration include Streptococcus pneumoniae; group B streptococcus, Staphylococcus aureus, hydrochloric acid, hypoxia, and C5a<br />Bacterial-derived chemoattractantfMLP stimulates CD18-dependent PMN migration, <br />whereas the host-derived chemoattractants IL-8 and LTB4 stimulate CD18-independent neutrophil migration<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  29. 29. Endothelial cell interaction<br />First three steps of PMN migration (rolling, activation, and adhesion), the mechanisms that underlie transendothelial migration remain unclear<br />Leukocytes traverse the endothelial barrier through the cleft between two to three adjacent cells<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  30. 30.
  31. 31. 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<br />The binding of JAM-C to Mac-1 was found to be of importance in neutrophiltransendothelial migration<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  32. 32. Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  33. 33. Epithelial cell interaction<br />process involves three stages<br />epithelial adhesion<br />migration <br />post-migration<br />PMN firm adherence to the basolateral epithelial membrane is mediated exclusively by Mac-1<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  34. 34. Interaction between CD47 and signal regulatory protein-a (SIRPa) enhances the migration rate of PMN through the epithelium<br />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.<br />After migration through the epithelium, PMN can adhere to ICAM-1 present on the apical surface of the epithelial cells<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  35. 35.
  36. 36. CHEMOTACTIC MEDIATORS<br />Through the endothelial basement membrane, PMN migrate along a chemotactic gradient<br />PMN chemotactic proteins include chemokines (e.g., IL-8), bacterial products (e.g., N-formylmethionyl peptides), lipid mediators (e.g., LTB4) and complement split products (e.g., C5a)<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  37. 37. Chemokines are produced by inflamed tissues and activate signal cascades in the PMN that lead to increase in cell motility, adhesion and survival<br />IL-8 is a potent chemotacticfactor for PMN<br />Blocking of IL-8 with a neutralizing antibody resulted in a 75–98% inhibition of its chemotactic activity<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  38. 38. 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<br />CXCR1, CXCR2 receptor<br />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)<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  39. 39. Chemokines<br />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 )<br />Many chemokines can bind to more than one receptor and most chemokine receptors can bind more than one chemokine<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  40. 40.
  41. 41. Leukotriene B4<br />Leukotrienes (LTs) are potent lipid mediators that have been implicated in the pathogenesis of airway diseases including asthma<br />LTs are synthesized from arachidonic acid via the actions of 5-lipooxygenase (5-LO), along with 5-LO-activating protein and terminal LTA4hydrolase<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  42. 42. Biosynthesis of leukotriene B4.<br /> 5-HPETE, 5-hydroperoxy-eicosatetraenoic acid;<br /> FLAP, 5-lipoxygenase-activating protein<br />Bing K. Lam* and K. Frank Austen<br />
  43. 43. Classified into two classes; <br />leukotriene B4 (LTB4) <br />cysteinyl LTs<br />LTB4 is a potent chemoattractantand activator as well as enhances PMN adhesion and migration<br />LTB4 exerts its action through 2 seven-transmembrane G-protein receptors: the high-affinity BLT-1 and the low-affinity BLT-2<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  44. 44. Innate immune activation<br />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<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  45. 45. Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  46. 46. Toll-like receptors (TLRs)<br />Currently the TLR family contains 10 members, and at the mRNA level, PMN appear to express all of these receptors except for TLR3<br />TLR4 is the major endotoxin receptor, and TLR2 recognizes PAMPs from Gram-positive organisms<br />TLR2 agonists include lipoteichoic acids (LTAs) and peptidoglycans<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  47. 47. http://research4.dfci.harvard.edu/innate/index.html<br />
  48. 48. 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<br />Exposure to LPS increases PMN expression of TLR2 and CD14 but does not change expression of TLR4<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  49. 49. 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<br />Rate of PMN apoptosis is delayed by the presence of bacterial lipoprotein and LPS to result in the augmentation of PMN inflammation<br />Anti-TLR2 monoclonal antibody prevents the delay in apoptosis in peripheral blood PMN<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  50. 50.
  51. 51. Mediators released by activated neutrophils<br />PROTEASES<br />REACTIVE OXYGEN SPECIES (ROS)<br />DEFENSINS<br />
  52. 52. Protease<br />Proteolytic enzymes play an important role in tissue remodeling and repair in the airways. <br />Levels of proteolytic enzymes, including active neutrophilelastase (NE) )and matrix metalloproteinase-9 (MMP-9) increased in asthma and thought to indicate an imbalance in the protease/antiprotease system.<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  53. 53. Courtesy of Dr Elizabeth Cramer, INSERM U474, Cochin Hospital, Paris<br />
  54. 54.
  55. 55.
  56. 56. Neutrophilelastase (NE) is a 30 kDa serine protease can attack a number of proteins including lung elastin<br />High concentrations within the azurophilic granules of PMN and is important in host defence, specifically for the intracellular killing of Gram-negative infections<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  57. 57. Presence of extracellular active NE results in tissue destruction and is also a potent secretagogue, contributing to increased mucus production<br />Presence of extracellular active NE may indicate a protease/antiprotease imbalance<br />Neutrophilelastase can upregulate IL-8 gene expression and protein production in bronchial epithelial cells via a MyD88-dependent NF-κBsignalling pathway<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  58. 58. This upregulation is inhibited when cells are pre-treated with a TLR4 neutralizing antibody<br />Indicating that upregulation of IL-8 production is via an innate immune pathway<br />Neutrophilelastase is increased in asthmaand also prominent in other airway diseases including COPD, cystic fibrosis, and bronchiectasis<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  59. 59. 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<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  60. 60. S. Schneeberger Drug News Perspect 2002, 15(9): 568<br />
  61. 61. SLPI inhibits NF-κB activation and LPS-induced TNF-α and IL-6 production in monocytes and macrophages<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  62. 62. α1-Antitrypsin (α1AT) is the major endogenous serine protease inhibitor produced by hepatocytes and is also expressed by PMN, epithelial cells, and macrophages<br />Both α1AT and SLPI counterbalance NE activity<br /> Airway levels of α1AT are increased in the sputum of subjects with asthma<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  63. 63. 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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  64. 64. 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<br />MMP-9 has been identified in cells such as bronchial epithelial cells,PMN, mast cells, eosinophils, and macrophages<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  65. 65. 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<br />Levels of MMP-9 are increased in asthma compared to healthy controls and also in severe asthma compared to mild asthma<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  66. 66. P Michaluk and L Kaczmarek<br />
  67. 67. MMP-9 is expressed in the sub-basement membrane (SBM) in asthma and increased with increasing severity<br />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<br />BAL levels of MMP-9 are also inversely related to FEV1 suggesting a relationship between this mediator and airflow obstruction<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  68. 68. MMP-9 can inactivate α1AT to further NE-mediated tissue destruction<br />α1AT is a potent activator of PMN<br />Oxidant radicals, including the hydroxyl radical, peroxide, and hypochloride, can modify α1AT to a form that has no inhibitory capacity against proteases<br />TIMP-1 can be inactivated upon exposure to hypochlorous acid, which is released by activated neutrophils<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  69. 69. REACTIVE OXYGEN SPECIES (ROS)<br />The respiratory burst involves the activation of NADPH oxidase, which is an enzymatic complex composed of <br />cytosolic (p40phox, p47phox, and p67phox) <br />flavocytochrome b558which is composed of membrane proteins (p22phox and gp91phox)<br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  70. 70.
  71. 71. Reactive oxygen species (ROS) are generated as a result of NADPH oxidase activity to produce superoxide (O2−)<br /> Superoxide can be rapidly converted into hydrogen peroxide (H2O2) by the enzyme superoxide dismutase<br /> Superoxide and hydrogen peroxide can also form to create the highly reactive hydroxyl radical (HO−)<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  72. 72. hyperchlorous acid<br />
  73. 73. Myeloperoxidase (MPO), a constituent of the azurophilic granules, generates hyperchlorous acid (HOCl) from hydrogen peroxide<br />Exposure to ROS can result in pulmonary injury<br />Superoxide can activate granule proteins through the recruitment of K+ to the phagosome, thus allowing cationic proteases of the azurophilic granules such as neutrophilelastase (NE) and cathepsin G (CG) to go from a highly organized intragranule structures into solution where they can kill ingested microbes.<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  74. 74.
  75. 75. ROS can inhibit a variety of protein tyrosine phosphatases through oxidation of key residues, allowing the phosphorylation of other molecules to proceed. <br />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<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  76. 76. DEFENSINS<br />6 identified human defensins<br />Small, arginine-rich peptides play an important role in host defense to infections<br />Four defensins that are present in PMN are the human neutrophil peptides (HNP-1 to HNP-4). <br />Peptides kill pathogens by causing permeabilization of the bacterial membrane<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  77. 77. Mature defensins are present in high concentration in the azurophilic granules and constitute 5–7% of the neutrophil&apos;s total protein<br />Defensins also modulate the inflammatory response, as they can bind to protease inhibitors such as α1-antitrypsin<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  78. 78. Neutrophil clearance and death <br />After killing and digesting invading microbes, PMN at the inflammatory site undergo programmed cell death (apoptosis) and are cleared by macrophages (efferocytosis)<br />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&apos;s toxic cellular contents.<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  79. 79. In vivo, this process may limit PMN, destructive capability.<br /> Within a few minutes, PMN apoptosis results in irreversible chromatin condensation, nuclear collapse, cytosolicvacuolation, and cell shrinkage. <br />During this time the cell is unable to respond to agonists, is immobilized and inert. <br />Apoptotic neutrophils become instantly recognizable to alveolar macrophages, which result in cell removal via efferocytosis<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  80. 80. 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<br />TNF-α and Fas-ligand (Fas-L) can increase the rate of PMN apoptosis<br />Corticosteroids delay PMN apoptosis, thus increasing their survival time, and influencing the persistence of neutrophilic inflammation<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  81. 81. PMN apoptosis is induced by activation of cellular caspases and can occur through two main pathways. <br />death receptor (DR) pathway, where the clustering of TNF and Fas-receptors activates the caspase cascade beginning with cleavage of pro-caspase 8<br />intrinsic pathway consisting of mitochondrial cytochrome c and members of the Bcl-2 family that forms an apoptosome activating caspase 9<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  82. 82. 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<br />Activated caspase 8 and 9 can then activate caspase 3 to cleave proteins essential for cell survival<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  83. 83. D. Scheel-Toellner, Biochemical Society Transactions (2004) 32, (461–464)<br />
  84. 84. CYTOKINE SYNTHESIS<br />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<br />Cytokine production by PMN is increased by inflammatory stimuli, bacterial endotoxin (LPS) being the most potent<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  85. 85. 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<br />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)<br />Middleton 7th Jodie L. Simpson, Katherine J. Baines, Peter G. Gibson<br />
  86. 86. INTERACTIONS BETWEEN MICROBES AND NEUTROPHILS <br />Opsonic factors <br />principal Igopsonins are IgG1 and IgG3, while IgA1 and IgA2 also serve this function in the respiratory tract<br />complement components, C3b and C4b<br />Robert L Baehner<br />
  87. 87. Phagocytosis and opsonic receptors <br />Engulfment occurs via the advancing pseudopod as the neutrophil surrounds the microbe<br />Fc binding site is recognized by three classes of opsonic receptors<br />Fc gamma RIII (CD16) - binds IgG subclasses 1 and 3 with intermediate and low affinity<br />Fc gamma RII (CD32) - low affinity receptor with the following subclass affinities: IgG1 = IgG3 &gt;&gt; IgG2 = IgG4 <br />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<br />Robert L Baehner<br />
  88. 88.
  89. 89. Phagocytosis and opsonic receptors<br />IgA antibody receptor - FcalphaR (CD89), signal transduction via G-protein linked phospholipase C activation, leading to phagocytosis and stimulation of the respiratory burst<br />Complement receptors<br />CR1 (CD 35) binds dimeric C3b<br />CR3 recognizes C3bi but not C3b, designated CD11b/CD18<br />C1q receptor - C1q, the recognition subunit of the classical complement pathway<br />Robert L Baehner<br />
  90. 90. Conclusion<br />PMN migration- <br />Myeloid development<br />PMN trafficking - Rolling, Adhesion, Migration<br />Cellular adhesion mulecules – integrins (CD11b/CD18) , inflammatory stimulus, endothelial and epithelial cell interaction<br />Chemotactic mediator – chemokines (IL8), LTB4,<br />Innate immune activation - TLR<br />
  91. 91. Mediators released by activated PMN<br />Proteases – Neutrophilelastase, MMP-9<br />Reactive oxygen species<br />Defensin<br />PMN clearance and death<br />Cytokine synthesis – proinflmmatory cytokine (TNFa, IL-1), chemokine ( IL-8), growth factor (GM-CSF, G-CSF)<br />Interaction between microbe and PMN<br />

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