Intravenous immunoglobulin and immune response

Intravenous immunoglobulin (IVIg) is a pooled preparation of polyspecific, polyreactive immunoglobulin (Ig)G molecules from several thousand healthy donors. The immunoglobulin (Ig) molecule has Fab region, which is involved in antigen-binding and the Fc portion, which is involved in effector function. As IVIg is prepared from multiple donors, it contains numerous antibodies directed against a wide range of antigens; consequently, the variable regions on the Ig Fab fragments in IVIg preparations are diverse. The variable region can bind to non-self-antigens (foreign antigens), self-antigens and anti-idiotypic antibodies. IVIg contains a broad spectrum of antibody specificities against bacterial, viral, parasitic and mycoplasma antigens, that are capable of both opsonization and neutralization of microbes and toxins.

In addition to its initial use as replacement therapy in primary and secondary immunodeficiencies, IVIg is widely indicated in a large spectrum of autoimmune and inflammatory diseases. One of the first proposed mechanisms of action of IVIg was via Fcγ receptor blockade 1. This study demonstrated that infusion of Fcγ fragments in idiopathic thrombocytopenic purpura/immune thrombocytopenia (ITP) patients increased platelet count, mediated by the blockade of Fcγ receptors 1. It has since been demonstrated in vivo that Fcγ fragments, particularly if sialylated, can exert anti-inflammatory effects 2. This suggests that the clinical benefits of IVIg may be mediated via an Fc pathway; indeed, to date there have been no studies that demonstrate the clinical benefit of Fab fragments alone.

However, non-Fc mechanisms have been proposed that provide an insight into the possible molecular mechanisms of action of IVIg, although these do not exclude the potential co-operation of Fab and Fc portion of IgG to elicit the effects of IVIg.

One mechanism of action was proposed by Sultan et al., who found that anti-idiotypic antibodies in IVIg were effective in the treatment of autoimmune haemophilia 3. This led to the study and characterization of anti-idiotypic antibodies in IVIg which neutralize pathogenic autoantibodies 4,5. IVIg was found to contain anti-idiotypes against anti-factor VIII, anti-neutrophil cytoplasmic antibody, anti-DNA, anti-thyroglobulin, anti-acetylcholine receptors and anti-neuroblastoma antigens. Furthermore, anti-idiotypic antibodies have been found to play a role in transplantation due to the anti-human leucocyte antigen antibodies.

In addition to ITP and haemophilia, IVIg has been found to be effective in several inflammatory and autoimmune diseases. Therefore, anti-inflammatory effects of IVIg were studied and were shown to be mediated in part through anti-complement effects. Dermatomyosis is a condition mediated by C5b/C9 membranolytic attack complexes (MACs) in intramuscular capillaries. The formation of MAC occurs when C3 is hydrolyzed into C3b, which leads to the activation of C5b and the formation of MACs. In a study conducted by Basta et al. 6, IVIg was found to form complexes with C3, preventing MAC formation and deposition in patients with dermatomyosis. This suggests that the clinical benefit of IVIg can be attributed to complement scavenging, demonstrating an additional distinct mechanism of action that may be mediated by F(ab′)₂ and whole IVIg, but not Fcγ fragments alone.

Anaphylatoxins are complement peptides that are produced when the complement system is activated. A study by Basta et al. implicates F(ab′)₂ in the neutralization of anaphylatoxins, such as C3a and C5a 7. IVIg is able to suppress C3a- and C5a-induced release of thromboxane B2 and histamine, which have proinflammatory properties. Moreover, circulatory collapse caused by C5a was prevented in pigs pretreated with F(ab′)₂ IVIg. The neutralization of C3a and C5a were observed in cells treated with F(ab′)₂ IVIg and whole IVIg and not Fcγ IVIg fragments, suggesting that F(ab′)₂ and not Fcγ are implicated in this process.

IVIg has also been found to be beneficial in a murine model of brain ischaemia and stroke, via a complement scavenging mechanism. Administration of IVIg, either prior to an ischaemic event or during reperfusion, led to a two- to three-fold improvement in functional outcomes in ischaemia and reperfusion. C3 levels were higher in injured compared to non-injured brain regions. Furthermore, compared with wild-type mice, C5-deficient mice were protected from ischaemia and reperfusion. IVIg decreased C3 and caspase 3 activation, suggesting that IVIg inhibits complement-mediated cell damage via scavenging of complement proteins to elicit beneficial effects 8. In addition to a role in scavenging complement in inflammatory and immune diseases, IVIg has also been shown to alter the cytokine network and mediate the balance between T helper (Th) types. Th cells can be classified into several subsets, such as Th1, Th2, Th17 and regulatory T cells, which produce distinct cytokines. Th1 cells produce cytokines such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α, Th2 cells produce IL-4, IL-5, IL-13 and IL-10, Th17 cells produce IL-17, IL-21 and IL-22, and regulatory T cells which are immunosuppressor cells produce TGF-β and IL-10. In a study conducted by Ruiz de Souza et al., peripheral blood monocytes treated with IVIg induced an up-regulation of anti-inflammatory cytokine IL-1 receptor antagonist and down-regulation of several proinflammatory cytokines 9. By the early 2000s there was an increasing focus on the role of dendritic cells and their effect on T cell polarization. Mature dendritic cells can stimulate naive T helper cells (Th0) and polarize them into distinct subsets. Our study demonstrated that both the F(ab′)₂ and Fc fragments of IVIg are capable of inhibiting the differentiation and maturation of dendritic cells, suggesting that IVIg is capable of inducing tolerogenic dendritic cell phenotypes 10.

As a consequence of IVIg-induced tolerogenic dendritic cells, regulatory T cells are up-regulated. Using a murine model of autoimmune encephalomyelitis (EAE), prophylactic IVIg was found to increase CD4⁺CD25⁺forkhead box protein 3 (FoxP3⁺) regulatory T cells 11. This proliferation of regulatory T cells has also been observed in humans following high-dose IVIg treatment in patients with autoimmune rheumatic disease 12.

We recently reported that, in EAE mice, IVIg inhibits the differentiation of CD4⁺ T cells to Th1 and Th17 cells 13. The down-regulation of Th1 and Th17 cells was observed with a concomitant up-regulation of regulatory T cells, demonstrating the reciprocal regulation mechanism of IVIg. Furthermore, the reciprocal regulation was suggested to be F(ab′)₂-dependent due to the comparable inhibition of Th1 and Th17 cells observed in mice treated with F(ab′)₂ fragments or IVIg 13.

IVIg-induced expansion of regulatory T cells may be due to several mechanisms. Mazer et al. propose that IVIg renders dendritic cells tolerogenic via its interaction with dendritic cell immunoreceptor (DCIR) 14. This leads to increased levels of FoxP3⁺ regulatory T cells which can attenuate autoimmune disease severity. Another mechanism of action for regulatory T cell expansion is provided recently by our group. Our report suggests that IVIg-induced expansion of regulatory T cells is due to the induction of cyclo-oxygenase 2-dependent prostaglandin E₂ production in dendritic cells 15. Inhibition of cyclo-oxygenase 2 enzymatic activity significantly reduced IVIg-mediated regulatory T cell expansion both in vitro and in vivo in EAE mice. This mechanism was dependent on Fab fragments of IVIg but not Fc.

Immunomodulatory mechanisms of IVIg in autoimmune conditions are not fully understood, although several mutually non-exclusive effects have been proposed. Individually, each of these mechanisms may participate to a certain extent in the overall effect of IVIg. While some of the effects may rely upon the binding of the Fc moiety of IgG to Fcγ receptors on target cells, others may be primarily dependent on the range of variable regions of IgG.

Disclosure

The authors have received research and travel grants from CSL Behring.