respiratory burst by phagocytosis
DESCRIPTION
TRANSCRIPT
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Respiratory burst by Phagocytosis
Speaker
P.RAMESH
Ph.D. SCHOLAR
(ABC)
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Immunity refers to, reactions by an human
body to
foreign substances such as microbes and
various
macro molecules ( Abbas et al.,1991)
IMMUNITY
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IMMUNITY
ACQUIRED IMMUNITYINNATE IMMUNITY
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Innate Immunity
Anatomical Barriers
Physiological Barriers
Inflammatory Barriers
Phagocytic/endocytic Barriers
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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
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Recognition
Adherence
Ingestion using Pseudopodia
Phagosome
Phagolysosome
Destruction of Microbes
Steps in Phagocytosis
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Phagocytic Killing
Oxygen Dependent Killing
Mechanisms
Oxygen Independent Killing
Mechanisms
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Activation of Macrophages synthesizes
Lysozyme
Defensins
Tumor necrosis factor-α (TNF-α) and
Other hydrolytic enzymes
Oxygen Independent Killing Mechanism
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Oxygen dependent Killing Mechanism
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”
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“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
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“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 O2-
202 + NADPH 202- + NADP+ + H+
202- + 2H+ H202
- + 02
Glucose is metabolized through HMP to generate
NADPH
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“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
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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
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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 membranePHagocyte Oxidase
Guanine nucleotide Dissociation Inhibitor
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ACTIVATION OF NADPH OXIDASESequences of Events:
External Stimuli: LPS in Bacteria
Phosphorylation of p47PHOX
p47PHOX:p67PHOX:p40PHOX migrates to
membrane
Association with Cytochromeb558 to
assemble active Oxidase
Transfer of e- from NADPH to Oxygen
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gp91 p22 Rap1A
Rac2
p67p47
p40
OH
OH
OH
gp91 p22 Rap1A
Rac2
p67p47
p40
OPO3
OPO3
OPO3
RESTING ACTIVATED
Cytosol
Cell membrane
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Action of NADPH oxidase during phagocytosis
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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
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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 e-
beyond the flavin centre to heme in
cytochrome & then transfers to oxygen
p67PHOX in oxidase shows it is having
catalytically essential binding site for NADPH
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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
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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
(SuperOxide Dismutase)
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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
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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
H2O2 + HCl HOCl¯ + H2O
MPO
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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¯
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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¯
Reactive Nitrogen Species
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RNI vs ROI
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
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THANKS
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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
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RESPIRATORY BURSTOxygen Dependent
Myeloperoxidase Independent Reactions
2O2
- + H2O2
.OH + OH- + 1O2
Glucose +NADP+
G-6-P-dehydrogenasePentose-P + NADPH
NADPH + O2
Cytochrome b558
NADP++ O2
-
2O2
- + 2H+
Superoxide
dismutase
H2O2 + 1O2
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RESPIRATORY BURSTRESPIRATORY BURST
Oxygen Dependent Myeloperoxidase dependent
reactions
H2O2 + 1O22O2
- + 2H+
Superoxide
dismutase
H2 O2 + Cl-
myeloperoxidaseOCl- + H2O
H2O + O22 H2 O2
catalase
OCL- + H2O1O2 + Cl- + H2O
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