Unraveling the <scp>IVIG</scp> mystique

Over the past three decades, the elucidation of the mechanism of action of therapeutic intravenous immunoglobulin (IVIG) has been a major challenge in medicine. IVIG is processed from thousands of pooled individual-donor plasmas and contains millions of IgG antibodies targeting various pathogens. Because of this diversity of the antibody population, immunoglobulin therapy was first introduced in the 1950s as an intramuscular injection for IgG replacement therapy to prevent infections in patients having primary humoral immunodeficiency disorders. IVIG became available in the 1970s and IgG replacement therapy continues to be a primary and essential use of IVIG.1, 2 However, IVIG use was to be transformed forever in the early 1980s and has now been shown to have modest to substantial efficacy in the treatment of more than 100 diseases. In 1981, Imbach and colleagues3, 4 described their seminal findings that showed that IVIG used in pediatric patients with immune thrombocytopenia (ITP) resulted in increasing the platelet (PLT) count. This report was the first evidence that IVIG had therapeutic activity in an autoimmune disease and was quickly confirmed by others in both children and adults with ITP.5 It was also Imbach4 who established the high-dosage requirement for IVIG in the treatment of ITP, 1 to 2 g/kg, which has remained the standard dose, although the effective dosage has never been systematically examined. Imbach and others subsequently suggested that IVIG may be a useful therapy in other autoimmune diseases.6, 7 Indeed, since the middle 1980s, IVIG use has risen substantially each year and has been a successful therapeutic agent for the treatment of numerous autoimmune and inflammatory disorders,8-18 and it is even used to treat autoimmune diseases in veterinary medicine.19 Although IVIG has shown efficacy for the amelioration of a number of human disorders, its mechanism of action remains elusive. Over the past 30 years, many mechanisms have been proposed with some supporting evidence; however, for most of the proposed mechanisms, there is also rebuttal evidence creating some confusion but making it likely that there is not a single unifying mechanism to explain all of its beneficial effects. Indeed, as these postulated mechanisms are nonmutually exclusive, it is likely that IVIG has multiple mechanisms of action that include many or all of those previously described. There has been a plethora of proposed mechanisms over the years: 1) functional Fcγ receptor (FcγR) blockade, 2) anti-idiotypic antibodies, 3) modulation of complement, 4) modulation of cytokines, 5) blocking of neonatal Fcγ receptor (FcγRn)-mediated clearance of pathogenic antibody, 6) up regulation of functional inhibitory Fcγ receptor (FcγRIIb), 7) sialylated Fcγ as effector, 8) immune cell modulation, 9) natural autoantibodies in IVIG as effectors, and 10) IVIG dimers and immune complex formation. Many of these postulated mechanisms of IVIG action have only equivocal supportive data and remain controversial, requiring more extensive scrutiny. Some of the most contentious proposed mechanisms include: 1) FcγR blockade, 2) involvement of FcγRIIb, 3) role of Fcγ sialylation, 4) role of FcγRn, and 5) dimerization of IVIG or formation of immune complexes. There have been claims that the mechanism of action of IVIG is due to aggregates of IgG contained in the IVIG preparations, specifically IgG dimers.20-22 However, the current manufacturing process for IVIG results in only a very small population of dimerized IgG molecules with most of the IgG in a monomer state.23 Two studies have directly addressed the role of dimers in IVIG preparations, suggesting that dimers are, indeed, effectors for the action of IVIG.21, 22 In one study,21 it was found that a single dose of IVIG (1 g/kg) having high dimer content was effective at reversing the PLT count in a mouse model of ITP. In contrast, IVIG predominately consisting of monomeric IgG failed to ameliorate the ITP. In a second study,22 dimers of IVIG antibodies interacting with IVIG autoantibodies were enriched and found to have increased autoantibody activity. The authors suggest a paradigm where only IgG dimers contained in IVIG provide efficacy. As reported in this issue of TRANSFUSION, Tremblay and colleagues24 now challenge the paradigm that dimers contained in IVIG provide its effector function. These authors describe a passive antibody mouse model of ITP and use both dimer-deficient and dimer-enriched IgG preparations to show that amelioration of ITP does not require dimerization of the IgG molecules. The investigators collected blood from 10 different donors to prepare their own native IgG, devoid of dimers, using a series of purification steps. They have used gel filtration chromatography to show that this preparation contains only IgG monomers. In addition they used fractionation of commercial IVIG to obtain monomeric IVIG devoid of dimers. They also used IVIG enriched for dimers by using storage as previously described21 to increase the dimer content to approximately 15% compared to approximately 4% in nonstored IVIG. Using all preparations, native IgG or IVIG devoid of IgG dimers or aggregates, IVIG enriched for IgG dimers, or unmanipulated IVIG, these investigators found no differences in the amelioration of experimental ITP. The evidence is convincing that only the monomer portion of IVIG is responsible for its immunomodulatory effects. These results are completely opposite to those of Teeling and colleagues.21 However, both studies prepared the immunoglobulin preparations in a similar manner and examined the effect of IVIG monomers, dimers, and unmanipulated IVIG using a "prevention" protocol where IVIG is administered before the PLT antibody. Both studies used the same PLT antibody, MWReg30, and the same dose of immunoglobulin, 1 g/kg. Perhaps the differences in results obtained in these two studies was due to the use of different mouse strains.25 In the study by Tremblay and coworkers,24 BALB/c mice were used and efficacy of the immunoglobulin preparations on PLT counts was examined 6 hours after ITP induction. In contrast, in the study by Teeling and colleagues,21 C57BL/6 mice were used and PLT numbers were evaluated after only 180 minutes had elapsed. Teeling and colleagues21 also used a "treatment" mouse model where ITP was induced before administering the IVIG and found similar results, concluding a total lack of efficacy to ameliorate ITP when using monomer IVIG. With such opposing results and the use of completely different mouse models of ITP, it will take more studies to completely understand the role of IgG dimers as the effectors in the mechanism of ITP. This question is becoming crucial, however, as manufacturers are already trying to minimize the dimer content of their IVIG products for other reasons.23 Therefore, resolution of the efficacy of dimers compared to monomers in IVIG is an important question that requires additional studies. In the same report,24 Tremblay and colleagues also examined a potential role for immune complexes as the effector mechanism of IVIG.26-29 For these studies, the authors used an in vitro approach to document the percentage of immune complexes that could form between human IVIG and mouse plasma proteins. Based on these results, the authors were able to determine the lowest level of immune complexes that would not have an effect on ITP. They then determined that their in-house, native IgG preparation formed approximately fivefold less immune complexes with mouse plasma then did IVIG, likely due to thousands of donors used to prepare IVIG compared to only 10 donors used to prepare the in-house IgG. Nonetheless, this in-house IgG still showed efficacy to ameliorate ITP though its percentage of immune complexes that were predicted to form in vivo was similar to that found in IVIG doses that were unable to ameliorate the ITP. The final conclusion from this work is that both dimers and immune complexes are dispensible to prevent experimental ITP. Although many mechanisms for the beneficial effects of IVIG have been proposed and models have been established, recently, contradictory evidence has placed some of these hypotheses in jeopardy. Indeed, evidence is equivocal for some of the most popular mechanisms. It is worthwhile to remind ourselves that hypotheses cannot be proven, only supported by evidence, but they can be disproven, by a single counterexample, as suggested by the great science philosopher, Sir Karl Popper.30 Whether this paper can be regarded as a definitive study to reject the dimer hypothesis will depend on whether the model system applies to the biology being studied. IVIG may legitimately have multiple mechanisms working in humans that may be difficult to sort out in animal model systems which may not apply to the entire human spectrum. Only careful ongoing work and time will tell the answer to this fundamental underlying issue. In the article by Tremblay and coworkers24 another proposed mechanism for IVIG action has been challenged. Based on their report, it can no longer be claimed that dimers or immune complexes are the effector mechanism for IVIG effect. Indeed, this group has clearly shown that only monomers are required for IVIG efficacy. The strength of findings from well-constructed experimental design that do not support previously postulated mechanisms for the action of IVIG is that it forces, or should force, investigators to use an unbiased approach in order to elucidate the precise mechanism or mechanisms of action of this amazing therapeutic. None.

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