Transcript Humoral Immune Effector Mechanisms

Humoral Immune Effector
Mechanisms
Ig of Different Isotypes
How do the functional differences impact
the immune response?
The focus will be on Ig and its interactions
with immune effector systems and receptors
Complement - an important effector system has important
functions related to immune defense
1. Lysis of cells. This is the original function identified
and causes hypotonic cell death by making hole. It is
not effective against organisms with cell walls such as fungi
and Gram positive bacteria
Complement - an important effector system has important
functions related to immune defense
2. Opsonization. Macrophage and PMNs have FcRs and
at least two different kinds of complement receptors that
aid in phagocytosis. C3b, a cleavage product formed during
activation is the major player. Antigen coated with C3b binds
to cells bearing complement receptors and if the cell is
a phagocyte the antigen will be phagocytosed.
Complement - an important effector system has important
functions related to immune defense
3. Inflammation. Peptides generated during activation
play a role in inflammation. The anaphylatoxins of which
C5a is the most potent bind receptors on mast cells and
basophils and cause degranulation with the release of
pharmacologically active mediators which induce smoothmuscle contraction and increases in vascular permeability.
C3a, C5a and C5b67 act as chemoattractants and induce
monocytes and neutrophils to adhere to vascular endothelial
cells, extravasate through the endothelial lining of the
capillaries and migrate to the site of complement activation
in the tissue.
Complement - an important effector system has important
functions related to immune defense
4. Immune clearance. Removes immune complexes from
the circulation and deposits them in the liver where they
are degraded. C3b facilitates immune complex binding to CR1
on RBCs. In the liver and spleen the complexes are stripped from
the RBC and phagocytosed. Complement also helps to solubilize
immune complexes.
Complement - an important effector system has important
functions related to immune defense
5. Enhanced immune response. CD21, part of the coreceptor on the B cell, binds cleaved C3. Recently it has
also been shown that C3 is required for optimal expansion
of T cells during a systemic viral infection.
Complement - an important effector system has important
functions related to immune defense
6. Virus neutralization. Complement mediates viral
neutralization by facilitating viral aggregation and by coating
the viral surface.
Classical pathway
Requires Ab
Mannose binding lectin pathway
Alternative pathway
IgM
YY
The first isotype made during the immune response
Exists in pentameric/hexameric forms
One consequence of polymerization is higher avidity
The higher avidity helps to compensate in part for the low
affinity of IgM made early in the immune response before
somatic mutation has taken place. Also each molecule has
ten/twelve C1q binding sites
IgG
In contrast to IgM, complement activation by IgG requires aggregation.
Monomeric IgG has an exposed C1q binding site but it is of low affinity; adjacent sites
are required in order to achieve an interaction of sufficient avidity to trigger the cascade.
IgM is a very rigid molecule
Binding of Ag by IgM leads to a conformational
change exposing the C1q binding sites.
There are multiple sites in each IgM molecule so
one IgM can bind C1q and activate the
complement cascade
C
1
q
b
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g
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C1
MBL - resembles C1q in structure except specificity is for
carbohydrate
C3a and C5a are anaphylatoxins
C3a - 77 amino acid peptide that causes smooth
muscle contraction, increases vascular
permeability and mast cell and basophil
degranulation
C5a- a77 amino acid peptide is also a
chemoattractant and activator of WBC
C3a and C5a are anaphylatoxins
Also amplify the inflammatory response by
inducing the synthesis of pro-inflammatory
cytokines.
Their receptors are present on many cell types
including leukocytes, mast cells, macrophages,
endothelial cells, astrocytes and microglial cells
C3a and C5a are anaphylatoxins
Anaphylatoxin triggered cascade of events that
contribute to the pathogenesis of a number of
disease and conditions including hypersensitivity
reactions, endotoxin shock, multiples organ
failure and respiratory distress syndrome.
Neutralization of the effects of anaphylatoxin is
therefore of substantial clinical significance
C3a and C5a are anaphylatoxins
Neutralization of the effects of anaphylatoxin is
therefore of substantial clinical significance
It has now been shown that C3a and C5a bind to
immunoglobulin molecules through the constant
region of the Fab. (Nature Medicine, published
online March 3, 2003).
Intravenous immunoglobulin (IVIG) and F(ab)’2 prepared
from it inhibit C3a and C5a induced histamine release
Binding is to F(ab)’2 and not Fc and is not inhibited
by binding to Ag
Cleavage products of C3b are opsonins
Complement receptors (CD21 = CR2) potentiate the immune response
B-cell coreceptor
C3b
Ant igen
CR2
IgM
CD19
Ig-/Ig-
Ig-/Ig- 
TAPA-1
Syk kinase
Lyn
Src kinases
Fcg receptors are important
humoral effector molecules
FcgRI (CD64)
FcgRII (CD32)
FcgRIIA: Expressed on macrophages, neutrophils, eosinophils. Ligation leads to
uptake. Not present in mouse.
FcgRIIB: These are inhibitory receptors.
On B cells crosslinking of surface IgM on resting cells in vitro induces a
proliferative response only when F(ab)’2 is used. Fc gRII provides the inhibitory
signal in this (by binding the Fc of the intact antibody).
Indeed in Fc gRII knock-out mice there was a significantly higher antibody
response to both thymus independent and thymus dependent antigens.
Therefore FcgRII acts as an inhibitory receptor on B cells. However, since loss of
FcgRII doesn’t lead to uncontrolled antibody production, there must be additional
pathways for maintaining antibody homeostasis.
FcgRII also plays an inhibitory role in mast cell degranulation through Fc RI.
FcRs function for the phagocytosis of Ab coated particles by
macrophages and killing by NK cells.
Both FcgRI and Fc gRIII require the accessory chain g or  for
function.
Phagocytosis by COS-1 cells transfected wtih FcgRIIIA-gor
PNAS 92:7381
Phagocytosis
FcgRIIIA

g

PI
+ cell, %
0
270+ 79
0
7.8 + 1.7
46 + 7
2.5 + 0.7
Phagocytic Index (PI) = (percentage of cells containing at least on particle)X(mean
number of particles per positive cell)
Fc receptors contribute to the Arthus reaction
For the Arthus reaction Ab is injected into the skin followed by
intravenous Ag
It had been thought to be complement mediated but it can be
virtually absent in g chain knock-out mice
However, there are strain differences and some strains show
more complement dependence than others
Fc receptors are required for Ab-mediated tumor protection
Mice were injected weekly with 2 µg/mg of therapeutic Ab
Inhibitory Fc receptors modulate the in vivo cytotoxicity again tumor antigens
In this experiment the mice were treated with suboptimal amounts
of antibody
Immune complexes can determine whether macrophages bias to
TH1 or TH2
Activated macrophages treated with
IFN-g and LPS are biased to IFN- g
production when stimulated by Ag
alone, but produce IL-4 when stimulated
by immune complexes. This bias is
stable and is seen when CD4+ cells
were stimulated 7 days later with OVA
alone. Intracellular cytokine expression
reflects what is seen in the secretions
(J. Immunol 168:3701)
Immune complexes facilitate DC priming of CTLs and induce DC
maturation in the absence of T cell help
J. Immunol. 168:2240-2246, 2002
There is increased binding and uptake of Ag present in ICs
J. Immunol. 168:2240-2246, 2002
FcRn plays a major role in determining serum persistence
IgG remaining (percent)
WT
2m-
Time (days)
Se le ctive de pr e s s ion of plas m a IgG conce ntration
2m-/in
mice
PNAS 93:1996
IgG
IgA
Wild-type
2200+ 100
110 + 20
Mutant
Ratio
260+ 30
8.4:1 + 0.9
110 + 20
1.0:1 + 0.2
CD89 - FcRI receptor for IgA
Found on cells of the myeloid lineage including neutrophils, eosinophils, most monocytes,
interstitial dendritic cells, and Kuppfer cells.
Can be expressed associated with FcRg.
Uptake of FITC-labeled E. coli opsonized with IgA into Kupffer cells
of transgenic mice expressing CD89
When the CD89 is occupied, it inhibits the phagocytosis of E. coli
opsonized with IgG
Buffer +
Unopsonized
E. coli
Anti-CD89 Fab
+ opsonized
E. coli
Buffer +
opsonized
E. coli
Opsonized
E. coli +
irrelevant Fab
In this case an anti-CD89 was used in place of IgA to mimic its effects
A rat mast cell line was engineered to express FcRI
Addition of IgA but not of IgG inhibited IgE mediated granule release.
However, extensive crosslinking with F(ab’)2 and rabbit anti-mouse
resulted in granule release.
Therefore a model emerges of FcaRI acting as an inhibitory receptor
at low occupany, and as a inflammatory receptor when extensively
crosslinked. Both activities required FcRg with a functional ITAM.
Protein A with Fc
Protein G with Fc
CLASSICAL PATHWAY
Antigen-antibody
C1qr2s2
(C1)
Activated C1
C2
C4
C4b
C2b
C4b2a
(C3 convertase)
C4a
C3
MBL
Activated C1-like
MASP complex
LECTIN PATHWAY
C3b
C3a
C4b2a3b
(C5 convertase)
C5
Ba
B
C3a
C3
C3b
C3bBb
(C3 convertase)
Microbial Surfaces
Factor D
ALTERNATIVE PATHWAY
MAC
C5b
C6 C7 C8 C9
C5a
C3bBb3b
(C5 convertase)
IgG1
IgG2
IgG3
IgG4