Respiratory burst by Phagocytosis

Speaker

P.RAMESH

Ph.D. SCHOLAR

(ABC)

Immunity refers to, reactions by an human

body to

foreign substances such as microbes and

various

macro molecules ( Abbas et al.,1991)

IMMUNITY

IMMUNITY

ACQUIRED IMMUNITYINNATE IMMUNITY

Innate Immunity

Anatomical Barriers

Physiological Barriers

Inflammatory Barriers

Phagocytic/endocytic Barriers

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

Recognition

Adherence

Ingestion using Pseudopodia

Phagosome

Phagolysosome

Destruction of Microbes

Steps in Phagocytosis

Phagocytic Killing

Oxygen Dependent Killing

Mechanisms

Oxygen Independent Killing

Mechanisms

Activation of Macrophages synthesizes

Lysozyme

Defensins

Tumor necrosis factor-α (TNF-α) and

Other hydrolytic enzymes

Oxygen Independent Killing Mechanism

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”

“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

“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

“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

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

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

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

gp91 p22 Rap1A

Rac2

p67p47

p40

OH

OH

OH

gp91 p22 Rap1A

Rac2

p67p47

p40

OPO3

OPO3

OPO3

RESTING ACTIVATED

Cytosol

Cell membrane

Action of NADPH oxidase during phagocytosis

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

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

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

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)

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

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

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¯

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

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

THANKS

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

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

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