humoral immune effector mechanisms. ig of different isotypes how do the functional differences...

Humoral Immune Effector Mechanisms

Ig of Different IsotypesHow 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 isnot effective against organisms with cell walls such as fungiand Gram positive bacteria


2. Opsonization. Macrophage and PMNs have FcRs andat least two different kinds of complement receptors thataid in phagocytosis. C3b, a cleavage product formed duringactivation is the major player. Antigen coated with C3b bindsto cells bearing complement receptors and if the cell isa phagocyte the antigen will be phagocytosed.


3. Inflammation. Peptides generated during activationplay a role in inflammation. The anaphylatoxins of which C5a is the most potent bind receptors on mast cells andbasophils and cause degranulation with the release of pharmacologically active mediators which induce smooth-muscle contraction and increases in vascular permeability.C3a, C5a and C5b67 act as chemoattractants and inducemonocytes and neutrophils to adhere to vascular endothelialcells, extravasate through the endothelial lining of thecapillaries and migrate to the site of complement activationin the tissue.


4. Immune clearance. Removes immune complexes fromthe circulation and deposits them in the liver where theyare degraded. C3b facilitates immune complex binding to CR1 on RBCs. In the liver and spleen the complexes are stripped fromthe RBC and phagocytosed. Complement also helps to solubilizeimmune complexes.


5. Enhanced immune response. CD21, part of the co-receptor on the B cell, binds cleaved C3. Recently it has also been shown that C3 is required for optimal expansionof T cells during a systemic viral infection.


6. Virus neutralization. Complement mediates viral neutralization by facilitating viral aggregation and by coatingthe viral surface.

How does complement work?Is complex with over 30 components.Major components are made in liver or macrophage and are in serum in high concentration.The central event is conversion of C3 to C3b

Classical pathwayRequires Ab

Mannose binding lectin pathway

Alternative pathway

CLASSICAL PATHWAYAntigen-antibodyC1qr2s2(C1)Activated C1C4C4bC2C2bC4b2a(C3 convertase)

C3C3aC4b2a3b(C5 convertase)C5C5aC5bC6 C7 C8 C9MACLECTIN PATHWAYMBLMASPActivated C1-like complexALTERNATIVE PATHWAYC3C3aC3bBBaC3bBb(C3 convertase)C3bBb3b(C5 convertase)Microbial SurfacesC4aC3bFactor D

IgM

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 hasten/twelve C1q binding sites

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.

IgG

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

C1

C1 q binding site

MBL - resembles C1q in structure except specificity is for carbohydrate

C3a and C5a are anaphylatoxinsC3a - 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 anaphylatoxinsAlso 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 anaphylatoxinsAnaphylatoxin 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 anaphylatoxinsNeutralization 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).

C3aC5a

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

C5b C5b67 C5b678

C6C7

C9C5b678

Poly-C9

B-cell coreceptorC3b Antigen

IgM

Ig-α/ -Ig β-Ig α/ -Ig β

2CR

19CD

-1TAPA

Lyn Src kinases

Syk kinase

Complement receptors (CD21 = CR2) potentiate the immune response

Immune response in CR1/CR2 +/+ (triangles) or -/- (squares) micePNAS 93:3360PRIMARYMice immunized with SRBC and titers determined by ELISA

Fc receptors are important humoral effector molecules

FcRI ( 64CD )

Expressed only on macrophages and neutrophilsCan mediate ADCC and phagocytosis in response to X-linkingOnly receptor that binds monomeric Ig with high affinity: IgG1,3 -- Ka approximately 109 IgG4 108M-1; IgG2 doesn’t bindSite of interactions mapped to the hinge proximal regionIts cytoplasmic domain is associated with a ; chain in chain

- knock out mice there is not expression of Fc RI on the macrophages and neutrophils

Fc ( 32)RII CDFcRIIA : , , . Expressed on macrophages neutrophils eosinophils Ligation leads to

. .uptake Not present in mouse

FcRIIB : . These are inhibitory receptors

On B cells crosslinking of surface IgM on resting cells in vitro induces a ( )’2 . proliferative response only when F ab is used Fc RII provides the inhibitory

( ). signal in this by binding the Fc of the intact antibody

Indeed in Fc - RII knock out mice there was a significantly higher antibody . response to both thymus independent and thymus dependent antigens

Therefore Fc . , RII acts as an inhibitory receptor on B cells However since loss ofFc ’ , RII doesn t lead to uncontrolled antibody production there must be additional

. pathways for maintaining antibody homeostasis

Fc RII also plays an inhibitory role in mast cell degranulation through Fcε . RI

Fc ( 16)RIII CD

- IIIA ( 203); transmembrane receptor with a cytoplasmic tail Phe found on, , ( ), monocytes macrophages NK the only Fc receptor on these T cells

and ζ chains associated with Fc ; RIIIA as homo or heterodimers they are . required for its surface expression

- IIIB ( ) secured in membrane by a glycosyl phosphatidyl inositol GPI anchor that 203require Ser

FcRs function for the phagocytosis of Ab coated particles by macrophages and killing by NK cells.

P h a g o c y t o s i s b y C O S - 1 c e l l s t r a n s f e c t e d w t i h F c R I I I A - o r ζ

P N A S 9 2 : 7 3 8 1

P h a g o c y t o s i s

P I + c e l l , %

F c R I I I A

α

α +

α + ζ

2 7 0 + 7 9 7 . 8 + 1 . 7

0 0

4 6 + 7 2 . 5 + 0 . 7

Both Fc RI and Fc RIII require the accessory chain orζ for.function

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 bevirtually absent in chain knock-out mice

However, there are strain differences and some strains showmore 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 amountsof antibody

Activated macrophages treated with IFN- and LPS are biased to IFN- production when stimulated by Agalone, but produce IL-4 when stimulatedby immune complexes. This bias isstable and is seen when CD4+ cells were stimulated 7 days later with OVAalone. Intracellular cytokine expressionreflects what is seen in the secretions(J. Immunol 168:3701)

Immune complexes can determine whether macrophages bias to TH1 or TH2

Immune complexes facilitate DC priming of CTLs and induce DCmaturation 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

Selective depression of plasma IgG concentration in β2 -/- mmice

93:1996PNAS

IgG IgA

- 2200 Wild type + 100 110 + 20 260 Mutant + 30 110 + 20

8.4:1 Ratio + 0.9 1.0:1 + 0.2

CD89 - FcαRI 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 FcR.

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

Anti-CD89 Fab + opsonized E. coli

Buffer + Unopsonized 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 FcαRI

Addition of IgA but not of IgG inhibited IgE mediated granule release.However, extensive crosslinking with F(ab’)2 and rabbit anti-mouseresulted in granule release.Therefore a model emerges of FcaRI acting as an inhibitory receptor at low occupany, and as a inflammatory receptor when extensivelycrosslinked. Both activities required FcR with a functional ITAM.

IgE

IgG

Fc ε RI

Fc RII

- Co ligation of Fc ε RI and Fc RII can inhibit

mast cell degranulation

Fc receptor

Clustering

Mediator Release

Fc Receptor

Clustering

No Mediator Release

Mast

cell

Surface

Antigen

Protein A with Fc

Protein G with Fc

IgG1 IgG2 IgG3 IgG4

humoral immune effector mechanisms. ig of different isotypes how do the functional differences...

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