Respiratory burst by Phagocytosis


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Respiratory burst by Phagocytosis

  1. 1. Speaker P.RAMESH Ph.D. SCHOLAR (ABC)
  2. 2. IMMUNITY Immunity refers to, reactions by an human body to foreign substances such as microbes and various macro molecules ( Abbas et al.,1991)
  4. 4. Anatomical Barriers Physiological Barriers Inflammatory Barriers Phagocytic/endocytic Barriers
  5. 5.  Phagocytosis is an Innate defense mechanism is ingestion of extracellular particles It is conducted by specialized cells such as Blood Monocytes Neutrophils and Tissue Macrophages
  6. 6.  Recognition  Adherence  Ingestion using Pseudopodia  Phagosome  Phagolysosome  Destruction of Microbes
  7. 7. Oxygen Dependent Killing Mechanisms Oxygen Independent Killing Mechanisms
  8. 8. Activation of Macrophages synthesizes Lysozyme Defensins Tumor necrosis factor-α (TNF-α) and Other hydrolytic enzymes
  9. 9. Activated phagocytes produce a number of Reactive Oxygen Intermediates & Reactive Nitrogen Intermediate When exposed to certain stimuli, phagocytes (Neutrophils, Eosinophils & Macrophages) Oxygen uptake increase greatly, some times more than 50 fold; undergoes a series of changes “Respiratory Burst”
  10. 10. “Respiratory Burst” occurs during:  Activation of macrophages during phagocytosis  Abrupt rise in Oxygen consumption  Increase Glucose consumption (HMP pathway)  Large amount of ROI  Activation of NADPH oxidase/Phagocyte oxidase
  11. 11. “NADPH Oxidase: It is also called as Respiratory burst oxidase/Phagocyte oxidase Present in membrane associated of phagocytic cells Catalyzes one-electron reduction of oxygen to O2202 + NADPH 202- + 2H+ 202- + NADP+ + H+ H202- + 02 Glucose is metabolized through HMP to generate NADPH
  12. 12. “NADPH Oxidase:  Originally it is discovered by Babior in 1973  It is having five major components in its structure  2 Membrane components  3 Cytosolic components  Guanine nucleotide binding proteins
  13. 13. Membrane Components: It is having 2 subuints: (p22PHOX & gp91PHOX) Distributed in membrane of secretory vesicles & specific granules, associated with a heterodimeric flavohemoprotein FlavoCytochromeb558 (1 FAD & 2 Hemes) Rac2 in resting cell is located in cytoplasm in a dimeric complex with Rho-GDI & Rac1 located in membrane PHagocyte Oxidase
  14. 14. Cytosolic Components: It is having 3 subuints: p40PHOX, p47PHOX & p67PHOX Guanine nucleotide binding proteins: Rac2 & Rac1 Rac2 in resting cell is located in cytoplasm in a dimeric complex with Rho-GDI Rac1 located in membrane Guanine nucleotide Dissociation Inhibitor PHagocyte Oxidase
  15. 15. Sequences of Events: External Stimuli: LPS in Bacteria Phosphorylation of p47PHOX p47PHOX:p67PHOX:p40PHOX migrates to membrane Association with Cytochromeb558 to assemble active Oxidase
  16. 16. Cell membrane gp91 p22 gp91 Rap1A p22 Rap1A Rac2 Rac2 OPO3 p67 OH p67 p47 p47 OPO3 OH p40 OH p40 ACTIVATED RESTING Cytosol OPO3
  17. 17. Action of NADPH oxidase during
  18. 18. Properties & Functions of Oxidase components:  Cytochromeb558:  It is a heterodimer containing one of each kind of subunit & contains 1 FAD & 2 Heme groups  In enzyme bound FAD having Isoalloxazine act as electron carrier/donar  Cytosolic components:  p40PHOX is responsible for transporting cytosolic components from cytosol to membrane during Oxidase activation
  19. 19.  Function of p67PHOX has been mystery  p67PHOX facilitates e- transfer from flavin of cytochromeb558 in absence of P40phox  In the presence of p40PHOX, p67PHOX transfer ebeyond the flavin centre to heme in cytochrome & then transfers to oxygen  p67PHOX in oxidase shows it is having catalytically essential binding site for NADPH
  20. 20. Small Guanine nucleotide binding proteins:  Rac2, Rap1A are low m.w of G-proteins  Rac2 is a member of Rho family where as Rap1A Ras family, it regulates cell proliferation  Rac2 having effector region (residues 26-45) & insert region (residue 125-145) is bind to p67PHOX but not p47PHOX
  21. 21.  Superoxide (O2¯• ) –  No direct effects on targets  Penetrates important sites  Subsequently converted to other ROI  Hydrogen Peroxide (H2O2) –  Dismutation of superoxide radical 2H + + 2O2¯• SOD H2O2 + O2 (SuperOxide Dismutase)  Reacts with thiols  Bacteriocidal only at higher concentrations  Secondary oxidants from H2O2 responsible for killing
  22. 22.  Hydroxyl Radicals (OH•) – Fenton Reaction Fe 2+ +H2O2  Fe 3+ + OH¯ + OH• OH• as a major component of neutrophil bacteriocidal arsenal is controversial  Limited radius of action  Secondary radicals from bicarbonate and chloride, which may have biological activity  Singlet Oxygen (O21) –  Electronically excited state of oxygen  Thought to be produced from reaction of H2O2 with HOCl
  23. 23. Myeloperoxidase (MPO) mediated Halogenation  Present in cytoplasmic granules at very high concentrations  Most H2O2 consumed by MPO  Heme Peroxidase, uses H2O2 to oxidize variety of compounds  Unique property – oxidizes Cl ¯to HOCl MPO H2O2 + HCl HOCl¯ + H2O
  24. 24.  Hypochlorous acid (HOCl)  Most bacteriocidal oxidant known to be produced  Bacterial targets – Fe-S proteins, membrane transport proteins, ATP generating system  Chloramines  Generated indirectly through reactions of HOCl with amines  Highly bacteriocidal  H+ + OCl¯ + R-NH2 RNHCl + OH¯
  25. 25.  Activated macrophages express high levels of Nitric oxide synthase (NOS)  NOS catalyzes: L-arginine + O2 + NADPH NO + L-citrulline +NADP+  NO has potent antimicrobial activity  Can combine with O2¯• to yield more potent antimicrobial substances (Peroxynitrites) NO + 2O2¯• ONOO¯
  26. 26.  Microbial killing mainly ROI dependent in phagocytic cells  RNI may play role in cells with deficiencies of NADPH oxidase/MPO pathways  NO can react with ROI to give more potent cytotoxic species  May facilitate migration of phagocytic cells from blood vessels to surrounding tissues by causing vasodilation
  27. 27.  O2- generated by oxidase, serves as a starting material for production of Reactive Oxygen Species (ROS) Production has to be tightly regulated to make sure they are only generated when & where required
  28. 28. Oxygen Dependent Myeloperoxidase Independent Reactions Glucose +NADP+ G-6-P-dehydrogenase NADPH + O2 Cytochrome b558 Pentose-P + NADPH + NADP + O2 - - 2O2 + 2H+ Superoxide dismutase - 2O2 + H2O2 H2 O2 + 1 O2 .OH + OH- + 1O2
  29. 29. Oxygen Dependent Myeloperoxidase dependent reactions H2 O2 + Cl- - OCl + H2O myeloperoxidase - OCL + H2O 1 O2 + Cl- + H2O - 2O2 + 2H+ Superoxide dismutase 2 H2 O2 catalase H2 O2 + 1 O2 H2 O + O 2