A four-year-old boy comes with the H/O of several

"boils" on his arm. The informant being his mother

tells us that the boy has had similar lesions on several
previous occasions that were treated successfully
with antibiotics. She denies any history of eczema or
typical childhood illnesses such as measles or chicken
pox. The child has had all of his immunizations.
Laboratory examination

CBC: N
Ig levels : N
B & T cell counts : N
Complement levels: N
Serum Ca+2 & PTH levels : N
The NBT test : - ve
CBC= Complete blood count , N=normal , PTH=parathyroid
hormone , NBT : nitroblue tetrazolium.
The child had a defect of the phagocytosis
called the Chronic granulomatous disease
What is Phagocytosis?

 It is defined as
ingestion of particles
of >0.5µm by cells.
Who discovered phagocytosis?
 Ellie Ilya Metchnikoff
discovered it in 1882.
 Received Nobel prize
for the same in 1906
 Carl Fredrich Claus
coined the term
Phagocytosis
WHAT IS THE MAIN
PURPOSE OF
PHAGOCYTOSIS ?
infection
Innate no
immunity disease

x adaptive
immunity

disease

An Extensive cross talk is seen between the adaptive and innate immunity
 What are the cells involved in
phagocytosis ?

Professional phagocytes
1. Neutrophils
2. Monocytes
3. Macrophages
4. Dendritic cells
5. Mast cells
6. Eosinophils
 Cells Function
Neutrophils Phagocytose antigens coated with
antibodies and complement,
intracellular killing, inflammation and
tissue damage
Monocytes Phagocytosis and acts as APC,
Precursors of macrophages and
dendritic cells
Macrophages Phagocytosis intra & extracellular
killing, acts as APC, tissue repair
,stores iron in bone marrow , play a
potent role in tumor cell destruction,
Dendritic cells Major role in phagocytosis and AP in
viral infections
Mast cells Allergic responses , Phagocytose and
act as APC against gram negative
bacteria like salmonella
Eosinophils Immunity against parasites , weakly
phagocytose and kill the parasite
Other features
Short life span , have primary and
secondary granules in cytoplasm,and
segmented nucleus, main station is
blood,CD66 membrane marker.
Longer life span , cytoplasm has
granules , main station is blood
Longer life span , no granules in
cytoplasm, different phenotypes based
on station eg Kuffer cells in liver,
CD14 membrane marker
Typical outgrowths , main station is skin
and lining epithelium of RT or GIT ,
Derived from monocytes or ??
Lymphocyte precursor cells
Derived from basophils, main station is
connective tissues
Contain eosinophilic granules in the
cytoplasm , main station is the blood,
levels raised in parasitic infestation and
atopy
Non Professionals
 Phagocytosis is not their main function ,
do not have specific receptors.
Lymphocytes
NK and LGL cells (Large Granular lymphocytes)
Epithelial cells
Endothelial cells
Fibroblasts

Paoletti R., Notario A. and Ricevuti G., (editors), Phagocytes: Biology,

Physiology, Pathology, and Pharmacotherapeutics, The New York
Academy of Sciences, 1997
How does it take place ?
 Entry of the pathogen

Recognition & phagocytosis by tissue

phagocytes

SOS signals

Recruitment of phagocytes & inflammation

How does a phagocyte recognize
the bugs ?
By using PRRs
 Toll like receptors
 FC receptors Surface
 Complement receptors
 Scavenger receptors
 NOD receptors
 RIG1
 MDA5 Intracellular
 OAS
Toll like receptors
Fcreceptors
 FcαR and Fc γ recognize microbes that
are coated with antibodies.
Fc receptors Location
Fc γ R
I Monocytes , Macrophages & Inf γ
stimulated Neutrophils

IIA Phagocytes & NK cells

IIB T & B lymphocytes

IIIA Monocytes & Macrophages

IIIB Neutrophils

FcαR Neutrophils, Monocytes &

Macrophages
 Complement receptors
Bind complement components deposited on the microbes

Complement receptor Special feature

CR1 Found on RBC`s & WBC`s except eosino
& basophils. Cannot trigger phagocytosis
unless the phagocyte is preactivated
CR3 Member of Integrin family of receptors,
expressed abundantly on macrophages and
poorly on neutrophils , activation
increases its levels. Also recognizes ICAM ,
Factor X and yeast polysaccharide cell wall
CR4 Member of Integrin family of receptors,
expressed poorly on neutrophils and
macrophages , monocyte – macrophage
incresases its levels , mediates phagocytosis
of iC3b opsonised particles and appears to
be important for nonopsonic , sugar ligand
dependent phagocytosis
 NOD protein structure
(Nucleotide oligomerization domain)

CARD NBD LRRs

N-term C-term NOD-1
Binds γ-D-glutamyl-meso-diaminopimelic
acid (iE-DAP)
CARD CARD NBD LRRs
N-term C-term NOD-2
Binds muramyl dipeptide (MDP)

CARD (Caspase-activating and recruitment domain)

NBD (Nucleotide binding Domain)

LRRs (Leucine-Rich Repeats)

Other IC Rceptors
 RIG 1(Retinoic acid inducible gene) and
MDA 5 (Melanoma differentiaton
associated gene) : RNA helicase with tandem
caspase activation and recruitment domains (CARDs)
that binds viral dsRNA and ultimately stimulates the
genes induced by interferon regulator factor 3 (IRF-3)
and nuclear factor B (NF-B).

 OAS: 2,5-oligoadenylate synthetase : recognises non

self dsRNA and breaks them down.
 PRR

Recognizes

Pathogen associated molecular pattern

Produce

Transcription factors

Induce

Effector molecules
Underhill and Ozinsky. Annu. Rev. Immunol. 2002
Phagocyte response to infection
 The SOS Signals
◦ N-formyl methionine
◦ Clotting system peptides
◦ Complement products

 Phagocyte response
◦ Vascular adherence
◦ Diapedesis
◦ Chemotaxis
◦ Activation
◦ Phagocytosis and killing
Clinical correlation: Leukocyte adhesion defects
How does a pseudopodia form ?

Clinical correlation : Wiskott Aldrich syndrome,neutrophil actin

dysfunction
What is the mechanism of
phagocytosis ?

 Two theories proposed to explain are

Zipper theory
Trigger theory
 Zipper theory

According to the zipper mechanism, ingestion occurs by sequential

engagement of a phagocyte's membrane against the particle surface,
and pseudopod advance proceeds no further than receptor-ligand
interactions permit.
Trigger theory

 In contrast, in the trigger mechanism particle

binding initiates an all-or-none phagocytic
response
 What is phagosome maturation?

Clinical correlation: Microbial evasion of phagocytosis

Cellular Microbiology, 1999, 1(3):195-203
 How is the microbe killed ?

Killing occurs by o2 dependent & independent mechanisms & by both

intracellular and extracellular mechanisms
Clinical correlation: Degranulation defects
What happens in the phagolysosome ?
Active
Resting
O2 O2-

p22phox gp91phox
Heme

FAD

NADPH

Rac

PKC Defective
microbial
killing
PI3K P p40phox
P p47phox

PKA P P
P67phox
MAPK
How does extracellular killing take place?

 LPS binds TLR4

 IL-1 binds IL1-R1
 TNFα binds TNF R1
and activates the iNOS
Citruline
Arginine
Reactions Between ROS and RNS

 NO + O2- NO2 + ONOO-

NONOates
S-nitrothiols
Nitrite
Nitrous Acid
 Mechanisms of Microbicidal Activity

Polyunsaturated Lipids
DNA Formation of Oxylipins disrupts membrane
Enzyme Deamination
Functionof nucleosides
Abasic sites and Nitrosylation of SH groups
Oxidization

Strand breaksof Tyrosine residues

Nitrosylation

Inactivation of metal ions at active site

Depletion of antioxidants
“the act of physically escaping from something (an opponent or a
pursuer or an unpleasant situation) by some adroit manoeuvre”

Nature 3:11, 2002 editorial

•Passive
•Active
Avoiding contact with Phagocytes
 Inhibiting chemotaxis
 Streptococcal streptolysin also kills them.
 Clostridium θ toxin.
 Mycobacterium tuberculosis.
• Covering self with substance that is
recognised as self
 T. pallidum binds fibronectin to its surface
 GAS has a hyluronic acid capsule
Inhibition of engulfment
 Polysaccharide capsules of S. pneumoniae,
Haemophilus influenzae, and Klebsiella pneumoniae
 M protein and fimbriae of Group A streptococci
 Surface slime (polysaccharide) produced as a biofilm
by Pseudomonas aeruginosa
 Vi antigen of Salmonella typhi
 Cell-bound or soluble Protein A produced by
Staphylococcus aureus. Protein A attaches to the Fc
region of IgG and blocks the cytophilic (cell-binding)
domain of the Ab.
Survival inside cells
 Inhibition of fusion of the phagocytic lysosomes with the
phagosome.
 M.Tuberculosis
 S. Typhi
 L. Pneumophila
 Leishmania
• Survival inside the phagolysosome
 Mycobacteria
 Br. Abortus
 B. Anthracis
• Escape from phagosomes
 Listeria
 Shigella
Products of bacteria that kill phagocytes

 Killing before phagocytosis

 Streptolysin O of GAS
 Leukocidins of Staphylococci
 Exotoxin A of P. Aeruginosa
• Killing after ingestion
 Mycobacteria
 Brucella
 Listeria
What are the other functions of phagocytic
system?
 Involved in apoptosis
Silent clearance is achieved by expression of phosphatidyl serine
on surface of apoptotic bodies

 Involved in immunological tolerance

Cells carrying self antigens are killed by phagocytosis

• In antigen processing and presentation

WHAT ARE THE
CONDITIONS
ASSOCIATED WITH
DEFECTIVE PHAGOCYTIC
SYSTEM?

Phagocytosis: Dr.Vinaykumar . Hallur

 Defects of degranulation
Disease Etiology Clinical consequence

Chediak Higashi Syndrome AR, impaired coalescence of Neutropenia , recurrent

lysosomal granules pyogenic infections,
oculocutaneous albinism

Specific granule deficiency AR, mutation of C/EBP Recurrent deep abscesses

which regulates specific
granule formation
 Defects of adhesion
Disease Etiology Clinical consequences

Leukocyte adhesion defect AR, absence of CD11/18 Neutrophilia, recurrent

surface adhesive bacterial infection without
glycoprotein pus

Neutrophil actin dysfunction Altered polymerization of Neutrophilia, recurrent

neutrophil actin bacterial infection without
pus
 Defects of cell motility
Disorder Etiology Clinical consequences

Increased
AR, Defective protein pyrin Recurrent fever and serositis,
Familial Mediterranean fever arthritis and amyloidisis

Decreased
Direct inhibition Ethanol , steroids Frequent infections

Immune complexes Bind Fc receptors on Recurrent pyogenic infections

neutrophils in SLE ,RA etc

Jobs Syndrome AD, variable expression of Recurrent skin and

inhibitor from mononuclear sinopulmonary infections
cells , high levels of
antistaphylococcal IgE
 Defects of microbial killing
Disorder Etiology Clinical consequence
Chronic granulomatous disease AR/XLR defective Recurret pyogenic infections
NADPH oxidase with catalase positive
organisms eg S.Aureus , B.
Cepacia ,S.
G6PD deficiency <5% activity of G6PD Marcescens.↑Fungal infections
Myeloperoxidase deficiency AR, failure to process None!!!
precursor protein due
to missense mutation
RAC2 deficiency AR, mutant protein neutrophilia , recurrent
bacterial infections
HOW TO EVALUATE A
PATIENT OF DEFECTIVE
PHAGOCYTOSIS ?

Phagocytosis: Dr.Vinaykumar . Hallur

 2
NOD 2 and Crohn`s disease
 Abnormal NOD-2 expression correlates
with defective epithelial defense

◦ NOD-2 expressed in Paneth cells of intestine

◦ Enteric bacteria induce a-defensin through NOD-2 to

kill luminal microbes.

◦ Clinical evidence: CD patients have decreased

a-defensin expression in Paneth cells
What happens when the
phagocytic system goes haywire ?
 ALI
 Septic Shock
 Renal failure
 Emphysema
Toll like receptors a boon or curse ?

Recent studies have shown mice deficient in certain TLRs to be

resistant to some infections.
Can we exploit the phagocytic
sytem for our benefits ?
 TLR4 agonist Imiquimod has been approved for the treatment of
viral warts.

 A TLR4 receptor agonist has been used recently as an adjuvant in

drugs.

 TLR7 receptor agonists have been used to treat Basal cell ca,
melanoma and other cancers.

 Drugs enhancing autophagy : Currently, a phase I/II trial of

hydroxychloroquine with radiation therapy and concomitant and
adjuvant temozolomide in patients with newly diagnosed
glioblastoma multiforme is being conducted through the New
Approaches to Brain Tumor Therapy consortium.
Summary
 Phagocytosis is a component of innate and aquired
immunity. It is the principal means of destroying
pathogenic bacteria and fungi. Phagocytosis initiates the
process of antigen presentation.

 Many phagocytic receptors recognize a diverse array of

microbial pathogens. Some pathogens (e.g., S.
pneumoniae) require opsonization by antibodies and
complement for their clearance. However, bugs fight
back.
Summary contd..
 Phagocytic leukocytes employ oxidative and non-
oxidative means of killing. The NADPH oxidase
generates reactive oxidants, such as superoxide anion
and hypochlorous acid (bleach).
 Innate immunity ushers in acquired immunity: innate
immune activation of APCs results in up-regulation of
co-stimulatory molecules and enhances the
effectiveness of antigen presentation.
 Phagocytosis is an essential component of development
and tissue remodelling. Ingestion of apoptotic bodies is
immunologically “silent” and is normally accompanied
by a suppression of inflammation. Failure of this
mechanism may result in autoimmunity.
 Science is like looking through a keyhole:
The closer you get to the keyhole, the
more you see of the room on the other
side.
-George Wald
1967 Nobel Laureate in Medicine
Thank you

Phagocytosis: Dr.Vinaykumar . Hallur