This document summarizes innate immunity and the process of phagocytosis. It describes how phagocytic cells like macrophages, neutrophils, and monocytes recognize, engulf, and destroy foreign particles and microbes. A key mechanism is the respiratory burst during phagocytosis, where phagocytes increase oxygen consumption and produce reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) to kill engulfed microbes. This is achieved through activation of the NADPH oxidase enzyme complex in the phagocyte cell membrane, which generates superoxide from oxygen and NADPH. Superoxide and other ROS/RNI are microbicidal or help generate microbicidal molecules like hypochlorous acid. Strict regulation

1. Speaker
P.RAMESH
Ph.D. SCHOLAR
(ABC)

2. IMMUNITY
Immunity refers to, reactions by an human body to
foreign substances such as microbes and various
macro molecules
( Abbas et al.,1991)

3. IMMUNITY
INNATE IMMUNITY
ACQUIRED IMMUNITY

4. Anatomical Barriers
Physiological Barriers
Inflammatory Barriers
Phagocytic/endocytic Barriers

6.  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

7.  Recognition
 Adherence
 Ingestion using Pseudopodia
 Phagosome
 Phagolysosome
 Destruction of Microbes

9. Oxygen Dependent Killing Mechanisms
Oxygen Independent Killing
Mechanisms

10. Activation of Macrophages synthesizes
Lysozyme
Defensins
Tumor necrosis factor-α (TNF-α) and
Other hydrolytic enzymes

11. 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”

12. “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

14. “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

15. “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

16. 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

17. 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

18. Sequences
of Events:
External Stimuli:
LPS in Bacteria
Phosphorylation of p47PHOX
p47PHOX:p67PHOX:p40PHOX migrates to
membrane
Association with Cytochromeb558 to assemble
active Oxidase

19. 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

20. Action of NADPH oxidase during

21. 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

22.  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

23. 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

24.  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

25.  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

26. 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

27.  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¯

28.  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¯

29.  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

36.  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

38. 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

39. 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