ROS/RNS is Host Defense Mechanisms

1. ROS/RNS is Host Defense Mechanisms Chem 464 Free Radical Biochemistry 02/23/’06 Linu Sara Abraham Chemistry Department University of Missouri-Rolla

2. Introduction Reactive Oxygen Species – forever a villain? No!! ROS critical for host defense mechanisms

3. Host Defense • Microorganisms can enter our body – skin, respiratory, GI tracts etc. • Protection? Epithelia forms physical barrier • Next life of defense? Phagocytes • Phagocytes respond to foreign invasion

4. Phagocytes • Phagocytes – WBCs that ingest microbes, other cells and foreign particles • Neutrophils, Eosinophils and Macrophages

5. Steps in Phagocytocis • Recognition • Adherence • Ingestion using pseudopodia • Phagosome • Phagolysosome • Destruction of microbe

6. Phagocytosis

7. Phagocytic Killing • Oxygen dependent killing • Oxygen independent killing mechanisms: • Lysozyme • Other hydrolytic enzymes • Defensins • Antimicrobial and cytotoxic peptides

8. Oxygen dependent killing mechanisms • Activated Macrophages produce ROI and RNI species Q. How are these ROI/RNIs made?? A. Respiratory Burst

9. Respiratory Burst • Occurs in macrophages during phagocytosis • Abrupt rise in oxygen consumption • Increased glucose consumption • HMP Shunt (Pentose phosphate pathway) • Large amounts of reactive oxygen intermediates • Enzyme – NADPH Oxidase

10. NADPH oxidase • Respiratory burst oxidase or phagocyte oxidase • Membrane associated • One electron reduction of oxygen at the expense of NADPH 2O2 +NADPH 2O2¯• + NADP+ + H +

11. NADPH oxidase • Originally described in 1973 by Babior • 5 major subunits • Membrane component • Cytosolic components

12. Membrane components • Flavocytochrome b558 • Core component • Distributed between plasma membrane and membrane of granules • Forms the electron transport system • 2 subunits: • gp91 phox • p22 phox phox – Phagocyte Oxidase • One FAD and 2 hemes

13. Cytosolic components • 3 subunits: • p47 phox • p67 phox • p40 phox • Guanine nucleotide binding proteins: • Rac 2 in cytoplasm • Rap1A located in membrane

14. Activation of NADPH oxidase • Sequence of events: Stimuli – eg. LPS in bacteria Heavy phosphorylation of p47 phox (p47 phox :p67 phox:p40 phox) migrates to membrane Associates with cyt b558 to assemble active oxidase Transfer of e¯s from NADPH to O2

15. NADPH Oxidase Ref-Babior,B.M;Blood (1999);93(5);1464-1476

16. Formation of phagocytic vesicle http://www.bioscience.org/2003/v8/s/1191/fig8.jpg

17. Free radicals formed by neutrophil • Superoxide (O2¯• ) – • No direct effects on targets • Penetrates important sites • Subsequently converted to other ROI • Hydrogen Peroxide (H2O2) – • Dismutation of superoxide radical 2H + + 2O2¯• H2O2 + O2 • Reacts with thiols • Bacteriocidal only at higher concentrations • Secondary oxidants from H2O2 responsible for killing SOD

18. Products of Oxidase: Role in Killing • 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 • Can react with a number of biological molecules

19. Myeloperoxidase (MPO) Mediated Halogenation • MPO – 5% of total neutrophil protein • Present in cytoplasmic (azurophilic) 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 H2O2 + HCl HOCl¯ + H2O MPO

20. MPO • Hypochlorous acid (HOCl) – • Most bacteriocidal oxidant known to be produced by neutrophils • 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¯

21. Reactive Nitrogen Species • 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¯

22. ROI vs RNI • Microbial killing mainly ROI dependent in normal neutrophils • 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 neutrophils from blood vessels to surrounding tissues by causing vasodilation

23. Chronic Granulomatous Disease (CGD) • Inherited immunodeficiency • Individuals have impaired or completely absent oxidative burst • Suffer from recurrent and life threatening infections • Incidence – 1 in 200,000 to 1 in 250,000 live births • Persistent infection of soft tissue, lungs, granulomas in multiple organs • Pneumonia and septicemia, sometimes leading to death • Organisms – S.aureus, salmonella, Pseudomonas, Candida albicans, aspergillus

24. Mutations causing CGD • Most common mutation – 75% is X-linked, almost exclusively in males. • Mutation in CYBB gene coding for gp91 phox • Autosomal recessive mutations: • NCF1 gene coding for p47 phox (25%) • NCF2 gene coding for p67 phox (<5%) • CYBA gene coding for p22 phox (<5%)

25. Myeloperoxidase deficiency • Incidence – 1 in 4,000 people • Not as serious as CGD • Occasional susceptibility to Candida infection • Possible explanation of CGD being more severe that MPO deficiency?? • Alternative oxidative killing mechanisms as backup • Increased non oxidative killing mechanisms • Increased peroxynitrite content

26. Non Phagocytic NADPH oxidase • NADPH oxidase components detected in non-phagocytic cells (eg.3 layers of aorta, vascular smooth muscles, endothelial cells etc) • Catalytic subunit of non-phagocyte NADPH oxidase genetically and structurally different from gp91 phox • Amounts of O2¯• produced are much less • May have role in intracellular signal transduction pathways, cell differentiation, cell proliferation, blood pressure regulation etc..

27. Conclusions • ROI/RNIs play an important role in our immune system • Consequences of lack of ROS production in neutrophils can be seen in CGD

28. Thank You Questions??