While IVIg’s exact mechanism of action in ameliorating autoimmune diseases such as ITP has remained poorly understood, commencing in 2001, three groups independently developed a passive murine model of immune thrombocytopenic purpura (ITP), which has greatly excelled in helping to delineate IVIg activity [1–3]. In particular, IVIg activity is now thought to likely require expression of the inhibitory Fcγ receptor (FcγR)IIB in the host animal to reverse thrombocytopenia , although the exact pathway remains unclear . Dendritic cells (DC) play a pivotal role in the priming of adaptive immune responses, yet they can also tolerize peripheral CD4+ and CD8+ T cells by inducing deletion, anergy or regulation . Thus, DC have been viewed as promising targets for immunotherapy . We have shown that the initial events in IVIg action may involve the formation of small antibody–antigen complexes , resulting in engagement of activating FcγR on DC upstream of the acute inhibition of platelet clearance . One study reported that IVIg crosslinked via a monoclonal IgG antibody to form large immune complexes was able to ameliorate murine thrombocytopenia independent of FcγRIIB expression , possibly suggesting a diminishing role for FcγRIIB when large IgG complexes are used to treat thrombocytopenia. IVIg long-term effects serve to inhibit the maturation and differentiation of human DC , and IVIg-primed DC exert immunosuppressive effects on auto- and alloreactive T cell activation and proliferation .
Summary. Intravenous immunoglobulin (IVIg) is used to treat an ever-increasing number of autoimmune diseases. While the exact mechanism of action of IVIg has remained elusive, many theories have been suggested, including mononuclear phagocytic system blockade, autoantibody neutralization by anti-idiotype antibodies, accelerated pathogenic autoantibody clearance by saturation of the neonatal Fc receptor, cytokine modulation and complement neutralization. More recently, a key role for dendritic cells (DC) in the amelioration of autoimmunity by IVIg has been suggested. Here we will focus on the role that DC may play in IVIg function using data from both mouse and human studies.
DC are a heterogeneous set of antigen-presenting cells involved in the induction of immunogenic or tolerogenic immune responses . IVIg can inhibit T-cell proliferation and T-cell cytokine production [13,14]. Bayry et al.  further expanded these results by showing that the immunosuppressive effects of IVIg on T cell activation may be mediated by DC and suggested that IVIg inhibits the maturation of DC and modulates their activation and survival, resulting in abrogation of T-cell activation and proliferation. In addition, IVIg can abrogate the production of IL-12, while increasing production of the anti-inflammatory cytokine IL-10 by the DC . IL-10 can play an important role in dampening phagocytic macrophage activation  and in fact increases in IL-10 in ITP patients undergoing IVIg therapy have been observed [16,17]. Both Fc and F(ab′)2 fragments of IVIg are able to mediate the suppression of DC, revealing that both FcγR- and non-FcγR-mediated signaling events may be involved in IVIg-mediated modulation of DC function . IVIg differentially modulates the antigen-presenting molecules on DC: the expression of MHC Class II on DC is down-regulated upon incubation with IVIg in vitro , but the expression of CD1d and activation of CD1d-restricted NK-T cells is enhanced . In experimental models, IVIg can also suppress DC function via FcγR interactions in an autoimmune giant cell myocarditis model . In addition, it has been suggested that alleviation of the incidence and severity of diabetes in NOD mice by IVIg is mediated via activating FcγRs .
DC interactions with IVIg
Previously, we speculated that IVIg might ameliorate autoimmunity via direct interactions with DC. We initially found that IVIg-primed leukocytes could, upon transfer to naïve mice, completely recapitulate the protective effects of IVIg in immune thrombocytopenia . Using this cellular therapy protocol to study IVIg function, we determined that CD11c+ DC were a specific target cell of IVIg in the amelioration of murine ITP . In particular, IVIg-primed CD11c+ DC, but not CD11c− cells, could adoptively transfer the ameliorative effects of IVIg to thrombocytopenic mice.
Similar results were also obtained by another group who showed that adoptive transfer of splenocytes from IVIg-treated mice into recipient mice had the ability to protect from fetal resorption in a murine model of spontaneous abortion . Our results have also been reproduced by Anthony et al.  who demonstrated that passive transfer of splenocytes from IVIg-treated mice could protect from experimental inflammatory arthritis.
DC and cytokines
How IVIg -primed DC (IVIg-DC) may reverse immune thrombocytopenia in the murine ITP model is currently not understood. IVIg-DC may secrete soluble mediators, such as cytokines, capable of down-regulating the macrophage response. Two groups have observed an increase in the anti-inflammatory cytokine IL-10 in ITP patients undergoing IVIg therapy [16,17]. However, using a mouse model of ITP, we found that the acute protective effect of IVIg in vivo was independent of IL-10 as well as the individual expression of the IL-1 receptor (IL-1R), IFN-γR1 (IFNGR1 subunit), IL-4, IL-12β, TNF-α, MIP-1α, or in mice deficient for the common cytokine receptor γ chain (required for signal transduction through the receptors for IL-2, 4, 7, 9, 15 and 21) . In addition, while we have confirmed an increase in mouse serum levels of the anti-inflammatory modulator IL-1R antagonist (IL-1Ra) after exposure to IVIg, a recombinant IL-1Ra did not ameliorate thrombocytopenia . A recent report has extended our observation of the apparent lack of requirement of several cytokines in the acute amelioration of murine ITP by IVIg: Aubin et al.  showed that mice exposed to IVIg exhibited no modulation of mRNA expression of 84 cytokine, chemokine or cytokine/chemokine receptor mRNAs. These results by themselves, however, do not exclude the possibility that ‘long-term’ protection afforded by IVIg-DC is dependent on these or other anti-inflammatory cytokines. For example, IVIg can, by binding FcγRIII, inhibit IFN-γ-enhanced phagocytosis in macrophages by suppression of expression of the IFNGR2 subunit of the IFN-γR . Thus the induction of an IFN-γ-refractory state may help to explain more sustained immunomodulatory effects of IVIg.
DC–NK cell interactions
Alternatively, IVIg-DC may exert a protective effect via an intermediate cell subset, such as NK cells. Several studies have observed DC–NK cell interactions . For example, DC matured in the presence of IVIg can activate NK cells, enhance NK cell degranulation, and elicit NK-cell mediated, FcγRIII-dependent lysis of the same IVIg-matured DC . Based on the observation that macrophage responses may be down-regulated following phagocytosis of apoptotic cells , it may be speculated that following adoptive transfer, IVIg-DC trigger NK cell-mediated apoptosis of target cells, leading to suppression of the macrophage response. It has recently been demonstrated that expression of CD1d and activation of CD1d-restricted NK T cells is enhanced in human monocyte-derived DC after IVIg treatment, leading the authors to speculate that these DC may activate immunoregulatory NK-T cells . In addition, there is evidence that DC, such as NK–DC  or IFN-producing killer DC [30,31] can induce apoptosis or lysis of target cells, such as T cells or tumor cells , suggesting that IVIg-DC may remove macrophages involved in platelet clearance.
Do IVIg-DC promote Treg cells?
IVIg can protect from experimental murine autoimmune encephalomyelitis (EAE) by expanding and enhancing the function of naturally occurring CD4+ CD25+ FoxP3+ T regulatory cells (Tregs) . Tregs can prevent T cell as well as B cell immune responses . Decreased levels of peripheral Tregs have been reported in patients with ITP  and recent data from Yu and colleagues suggested that functional defects in Tregs may contribute to breakdown of self-tolerance in patients with chronic ITP . Thus it is fully plausible that Tregs may play a role in the dampening of chronic ITP and IVIg-DC could stimulate the production of Tregs, downstream of the acute effects seen in mouse models of ITP. Indeed, De Groot et al.  have reported the presence of ‘Tregitopes’, Treg activating regions in the Fc portion of IgG. This finding may assist in our understanding of the long-term tolerogenic effects of IVIg in autoimmunity.
What is the molecular target of IVIg?
The actual molecular target of IVIg has remained elusive. However, in the passive murine ITP model, we have demonstrated that the protective effects of IVIg can be replaced by soluble immune complexes (sIC) . Specifically, mice treated with soluble ovalbumin (OVA) + anti-OVA, or treated with polyclonal antibodies directed to murine albumin or transferrin were successfully protected from thrombocytopenia. These results were confirmed and extended by Bazin et al.  who demonstrated amelioration of murine ITP by immune complexes formed from IVIg and a monoclonal anti-human IgG antibody. We therefore suspected that IVIg or sIC might interact with either activating or inhibitory FcγR. However, as IVIg-primed DC from FcγRIIB−/− mice ameliorated ITP in normal mice , it is very unlikely that FcγRIIB is the molecular target of IVIg.
Whether IVIg-primed DC directly act to increase FcγRIIB on macrophages or if there is an intermediary cell involved remains unclear. Samuelsson et al.  demonstrated that IVIg up-regulated FcγRIIB expression on splenic macrophages. We have shown that FcγRIIB need not be expressed on the DC which bind IVIg and need only be expressed in the recipient mouse . We suggest that IVIg directly primes DC via interaction with activating FcγR, which subsequently act directly, or indirectly, with phagocytic macrophages. This interaction then leads to anti-inflammatory effects including the up-regulation of macrophage FcγRIIB, resulting in the inhibition of platelet clearance.
Although there are numerous theories as to the mechanism of action of IVIg in autoimmune disease, research using both mouse models and human studies have highlighted a potential key role for DC in IVIg’s therapeutic effects. Whether DC directly induce an immunosuppressive effect or if there are intermediary players such as NK cells, T regulatory cells or othe DC subtypes remain to be elucidated.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.