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Abstract—Intravenous immunoglobulin (IVIG) is widely

used for the treatment of autoimmune and inflammatory

disorders because of its immunomodulatory effects. The

interaction of IVIG with the immune system has been

extensively investigated over the past 30 years but, since

IVIG modifies responses of many immune cells, a unifying

model is far from reach. Here we survey the in vitro studies

on the effects on IVIG on macrophages, B cells, T

regulatory cells, dendritic cells and soluble factors, as well

as the in vivo studies in murine disease models and the

preliminary in vivo findings in humans.

Keywords — intravenous immunoglobulin, autoimmune

disease, immune regulation.

I. INTRODUCTION When human immunoglobulin was introduced in the 1950s for the treatment of primary immunodeficiency diseases, it could be injected only subcutaneously or intramuscularly because severe reactions caused by molecular aggregates prevented intravenous administration. With the advent of de-aggregated Ig preparations that could be safely administered intravenously (intravenous immunoglobulin, IVIG), a number of benefits of high-dose treatment became rapidly evident. A potential for effects beyond those of mere antibody replacement was first demonstrated in 1981, when IVIG was used to treat the children with antibody deficiency that developed autoimmune thrombocytopenia. In fact, IVIG administration was rapidly followed by temporary normalization of platelet counts[1]. Since then, the use of IVIG for the treatment of a wide range of systemic inflammatory and autoimmune disorders has steadily increased. In addition to its licensed indications for Kawasaki disease, immune thrombocytopenic purpura (ITP),

Sumbmitted ad March 1 and accepted March 4th, 2014 This work was supported by the Intramural Research Programme, Sapienza

University of Rome (to M.F.). 1Department of Clinical Immunology, Sapienza University of Rome, viale dell‟Università 37, 00185 Rome, Italy.

*Correspondence to Milica Mitrevski (e-mail: mitmilica@tiscali.it).

Guillain–Barré syndrome, and chronic inflammatory demyelinating polyneuropathy (CIDP), IVIG is also used as an anti-inflammatory drug for numerous off-label applications [2]. Although in many cases positive results have been reported, well-controlled clinical studies addressing the beneficial effects of IVIG therapy are still needed for most of these diseases. Moreover, large part of our knowledge of the mechanisms of IVIG originates from studies in vitro or on animal models. Intravenous immunoglobulins are a unique immune-modulating therapy that has a wide range of effects on the immune system at multiple levels, varying from one disease treated to another. Although a variety of potential modes of action have been proposed, the exact mechanisms of the immunoregulatory effects of IVIG remain to be described, even for approved indications. Being obtained from the pooled plasma of thousands of healthy donors, IVIG contains a large repertoire of antibody specificities. IVIG preparations contain antibodies to non-self antigens (microbial antigens), antibodies to self antigens (natural autoantibodies), and antibodies that recognize other antibodies, also known as anti-idiotypic antibodies. Both F(ab′)2- and Fc-related mechanisms may be involved depending on the pathogenesis of the disease. Indeed, the variety of immunoregulatory mechanisms might explain the beneficial effects of IVIG in a wide range of pathogenetically heterogeneous disorders. Clinically, the beneficial effects of IVIG extend beyond the half-life of infused IgG, therefore, its effects cannot merely be a result of a passive clearance or competition with pathogenic autoantibodies.

.

II. IMMUNOMODULATORY MECHANISMS OF IVIG

F(ab’)2 -mediated activities

A variety of antibodies to non-self antigens present in IVIG preparations provide a cornerstone of replacement therapy by neutralizing with the F(ab′)2 portion many different pathogens,

toxins and superantigens. On the other hand, many antibodies

Immunomodulatory Effects of Intravenous Immunoglobulin

– Assembling a Jigsaw Puzzle Milica Mitrevski1, MD, Ramona Marrapodi1, ScD, Alessandro Camponeschi1, ScD, Laura Todi1, ScD,

Guido Granata1, MD, Maria Comasia Leone1, MD, and Massimo Fiorilli1, MD

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to self antigens are found in IVIG and are thought to have important role in its immunonodulatory effects. These self antigens include the variable domains of other antibodies (which are recognized by anti-idiotypic antibodies) [3] and an array of surface receptors. The latter include CD95 (FAS) [4], CD95 ligand ( FASL) [5], sialic acid-binding immunoglobulin-like lectin 9 (SIGLEC9) expressed on neutrophils [6], SIGLEC8 expressed on eosinophils [7], T cell receptor (TCR)α/β, CD5[8], blood group antigens and

gangliosides [9], stimulatory cytokines as B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) [10], and adhesion molecules involved in leukocyte trafficking [11]. It was observed that IVIG inhibited the binding of antithyroglobulin, anti-DNA and antiintrinsic factor antibodies to their autoantigens in vitro. The inhibitory effect of IVIG was dependent on interactions between the variable regions of IVIG and variable regions of the autoantibodies, supporting the concept of a functional idiotypic network regulating autoimmune responses in man [3]. Induction of immune cell apoptosis is believed to be an important mechanism of action of IVIG. Both agonistic and antagonistic antibodies directed to the death receptor CD95 are present in IVIG. However, the effects of IVIG on CD95 may be opposite depending on dosage, since at low-dose IVIG inhibits in vitro CD95-mediated apoptosis of neutrophils while at high-dose induces cell death [12]. Both F(ab′)2 and Fc fragments are involved in the modulation

of cytokine expression and function and in complement inhibition by IVIG. Large fragments of complement, such as C3b and C4b, are preferentially bound by the Fc region, while C3a and C5a anaphylatoxins are neutralized by low-affinity interaction with the constant domain of the F(ab′)2 of

antibodies contained in IVIG. It was shown that binding of IVIG to complement fractions inhibits the generation of the C5b-9 membrane attack complex and subsequent complement mediated tissue damage in dermatomyositis [13]. It has also been shown that IVIG can scavenge activated complement proteins; in fact, association of IgG F(ab′)2 fragments with C3a and C5a may result in

neutralization of their biological effects in animal models of anaphylatoxin-mediated pathology [14]. Fc-mediated activities

The Fc fragments of IgG play an important role in the immunomodulatory mechanisms of IVIG. This concept was first demonstrated in a clinical setting by the observation that treatment of ITP patients with Fc fragments of IVIG was highly effective [15]. Furthermore in many mouse autoimmune models, such as arthritis, nephrotoxic nephritis (NTN), and ITP, Fc fragments are as effective as the whole IVIG preparations [16, 17]. It is currently believed that the Fc-mediated mechanisms of action of IVIG involve

interactions with neonatal Fc receptor (FcRn), classical activating and inhibitory FcγRs, and complement. During the very first stages of life, FcRn mediates the passive transfer of IgG from mother to offspring both before and after birth. The function of FcRn in the adult is to bind serum IgG that has been endocytosed by endothelial cells or myeloid cells and recycled back to the cell surface. A possible FcRn-mediated mechanism of IVIG is to accelerate the degradation of pathogenic autoantibodies by saturation of the FcRn. It was demonstrated that the therapeutic efficacy of high-dose IVIG treatment in the pemphigus and pemphigoid mouse models is completely dependent on FcRn [18]. Recent studies, however, cast doubt on the extent of FcRn contribution to IVIG‟s anti-inflammatory effects. One study showed that the therapeutic activity of IVIG is lost if the carbohydrate moiety that is attached to the CH2 domain of all IgG subclasses is removed [17]. Although this results in a complete loss of IgG binding to FcγRs, it does not alter IgG binding to FcRn.

Another study demonstrated that the IVIG-mediated amelioration of ITP is not altered in FcRn-deficient mice [19]. The family of FcγRs consists of several activating members and one inhibitory member, FcγRIIB. The current paradigm in

FcγR biology states that cell activation is balanced by the

activating and inhibitory FcγRs. Alterations in the expression

or function of these receptors may therefore result in unbalanced immunity and inflammation [20]. IVIG can block destruction of antibody-opsonized platelets within minutes to hours after its transfer with anti-platelet IgG. In ITP patients, IVIG transiently decreases the rate of clearance of Ig-opsonized radiolabeled RBCs by directly inhibiting phagocytosis; the simplest explanation for this phenomenon is FcγR blockade by

IVIG-related aggregates and immune complexes [21]. Blocking of FcγRIIIA in a transgenic mouse model and in

human ITP patients interferes with platelet depletion [16, 22]. In contrast, blocking or deleting the high-affinity FcγRI in ITP

patients or in autoimmune mice is therapeutically ineffective, highlighting the importance of low-affinity FcγRs for

antibody-mediated effector functions [23]. Low- or medium-affinity receptors, such as human FcγRIIA/B/C and

FcγRIIIA, cannot interact with monomeric IgG but can only

bind IgG in the form of an immune complex. Therefore, monomeric IgG, which constitutes more than 97% of the IVIG preparation, cannot be responsible for a direct blockade of activating FcγRs [24]. However, different IVIG preparations

may contain varying levels of dimeric or multimeric IgG molecules. In vitro, IVIG suppresses the expression of FcγRIIA on human

dendritic cells (DCs), thereby preventing their activation by immune complexes [25]. Thus, the clinical effects of IVIG in ITP may involve the interaction of IVIG with activating FcγR

on dendritic cells [26]. IVIG is effective in autoimmune diseases in which soluble

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immune complexes are not a common feature (for example, ITP, chronic inflammatory demyelinating polyneuropathy and Guillain-Barre´ syndrome), whereas is of rather ineffective in diseases in which soluble immune complexes are usually elevated (for example, SLE and rheumatoid arthritis). It is possible that, in autoimmune states where endogenous soluble immune complexes are present, chronic stimulation of dendritic cells via FcγRs occurs and, therefore, IVIG does not

provide significant benefit [26]. The other classical FcγR that has been associated repeatedly

with the antiinflammatory activity of IVIG in vivo is the low-affinity inhibitory FcγRIIB receptor. Inhibitory FcγRIIB

may be an important checkpoint of humoral tolerance in the human immune system [27]. With the exception of T cells and NK cells, FcγRIIB is expressed on all cells of the immune

system, and it is the only classical Fc receptor regulating B cells through activating signals delivered by immune complexes retained on the FDC to the B cell receptor (BCR). In a murine model of immune thrombocytopenia the inhibitory Fc receptor, FcγRIIB, was required for protection, because

disruption either by genetic deletion or with a blocking monoclonal antibody reversed the therapeutic effect of IVIG. One study demonstrated the importance of both the activating FcγRIII and the inhibitory FcγRIIB for anti-inflammatory activity of IVIG, by showing that anti-human FcγRIII

antibodies block ITP in hFcγRIII transgenic mice while

FcγRIIB deficient mice are not protected by IVIG [16]. Other studies confirmed that FcγRIIB is required for preventive

and therapeutic effects [26, 28]. IVIG protection results from the induction of FcγRIIB on infiltrating macrophages but not

neutrophils, indicating a critical role for macrophage activation. Disease induction but not IVIG protection was observed in colony stimulating factor 1 (CSF-1)-deficient mice in K/BxN arthritis, thus defining different macrophage subsets in these processes [29]. These results suggest a two-step model for IVIG protection in which CSF-1-dependent macrophages act as innate "sensors" for the Fc fragment of IVIG, leading to the induction of FcγRIIB on CSF-1-independent "effector" macrophages thereby raising the threshold required for FcγRIII

activation and preventing autoantibody-triggered inflammation [29]. Another recent study found that untreated patients with CIDP, compared with healthy controls, showed consistently lower FcγRIIB expression levels on naive B cells, and failed to

up-regulate or to maintain up-regulation of FcγRIIB as B cells

progressed from the naive to the memory compartment. Also, FcγRIIB protein expression was up-regulated on monocytes and B cells after clinically effective IVIG therapy [30]. Sialylated IgG-mediated activities

IgG antibodies are glycoproteins that contain a carbohydrate moiety attached to each of the asparagine 297 residues in the two chains of the antibody Fc fragment. This glycan moiety is

an integral structural component of the IgG molecule, forming part of the scaffold for FcγR binding. In addition, depending on

the variable region sequences, nearly 20% of serum IgG antibodies have a F(ab′)2 fragment–attached N-linked sugar side chain[24]. Recently, several studies have shown an unexpected central role for IgG glycosylation and for novel IgG glycoform-specific FcγRs for the efficacy of IVIG therapy in

vivo. This could explain why such high quantities of IVIG have to be given to obtain therapeutic activity and how IVIG induces the upregulation of the inhibitory receptor FcγRIIB. It was first

demonstrated in 2006 that removal of the asparagine 297-linked sugar moiety from IgG in IVIG preparations resulted in the loss of the anti-inflammatory activity of IVIG in the mouse K/BxN serum-transfer model of rheumatoid arthritis [17]. This landmark study identified a sialylated component of IgG with reduced affinity for FcγRs as the anti-inflammatory moiety. There are many glycovariants for IgG, and it was observed that treatment of IVIG preparations with neuraminidase, which removes terminal sialic acid residues, abrogated IVIG activity to a level comparable with that of aglycosylated IVIG [17]. Moreover, it was shown that only the enrichment of terminal sialic acid residues of Fc, but not of F(ab′)2, fragments increased the therapeutic activity of IVIG [32]. The precise sialylation pattern that is responsible for the anti-inflammatory activity of IVIG in experimental models is the 2,6-linkage of sialic acid to galactose on the glycan found at Asn297 in the CH2 region of human IgG [33]. These SA-rich IgG glycovariants have a decreased affinity for classical FcγRs

in mice and humans, consistent with their strongly impaired activity in vivo [17][31]. This rules out the possibility that SA-rich IVIG preparations block access of autoantibody immune complexes to activating FcγRs, rather suggesting a

novel receptor on regulatory macrophages that can specifically recognize sialic acid-rich IgG. A family of molecules with the capacity to recognize terminal sialic acid residues is the sialic acid-binding immunoglobulin-like lectin (SIGLEC) receptor family, one of them being CD22, an inhibitory receptor expressed on B cells. In fact, most of these receptors have intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or ITIM-like motifs with the capacity to trigger cell inactivation. A recent study using CD22-deficient mice in models of ITP and K/BxN arthritis could not demonstrate a role for CD22 in the immediate anti-inflammatory activity of IVIG [34]. On the other hand, another murine cell-surface receptor, SIGNR1, which has significant homology with the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), was shown to be crucial for the anti-inflammatory activity of IVIG in mouse models of ITP and K/BxN arthritis [35]. SIGNR1 is expressed on marginal zone macrophages, medullary macrophages and some dendritic cell subpopulations. By extending these studies to the human

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system, it was demonstrated that the DC-SIGN can also bind to sialic acid-rich IgG glycovariants [35]. As for SIGNR1, it seems likely that it is not the sialic acid-rich sugar moiety in the Fc fragment by itself that is detected by DC-SIGN, but rather a structural change in the amino acid backbone of the Fc fragment. Upon sialylation, the affinities for Fcγ receptors are

reduced, whereas those for alternative cellular receptors, such as DC-SIGN/CD23, are increased. It was also shown that a novel TH2-type cytokine-driven pathway (Il-4 and IL-33) requiring basophils may explain how IVIG infusion upregulates FcγRIIB expression in a model of K/BxN arthritis

[37], but not involved in the model of ITP [36]. Very recently, one study investigated four independent in vivo animal model systems of autoantibody-mediated autoimmune disease to identify a common pathway explaining IVIG activity under therapeutic conditions in vivo. This study showed that, irrespective of the model system, IVIG activity is strictly dependent on the presence of terminal sialic acid residues and of the inhibitory FcγRIIB. In contrast, SIGNR1, previously demonstrated to be essential under preventative treatment conditions, showed a disease-specific impact on IVIG-mediated resolution of established autoimmune disease [38].

Effects on dendritic cells

Triggering activating FcγRs on dendritic cells in vitro and then

adoptively transferring them into mice can have IVIG-like effects and can ameliorate murine ITP; thus, investigators concluded that IVIG could be replaced by antibodies specific for activating FcγRs [26]. High-dose IVIG therapy was shown to inhibit the differentiation and maturation of normal human dendritic cells, to inhibit the up-regulation of the co-stimulatory molecules CD80 and CD86, and the ability of dendritic cells to process and present self antigens [39]. Both Fc and F(ab′)2

fragments of IgG were able to mediate the suppression of dendritic cells. In contrast, dendritic cells of patients with CVID differentiated in the presence of low-dose IVIG and presented with an up-regulated expression of CD1a and the co-stimulatory molecules CD80, CD86 and CD40 [40]. A recent report demonstrated that myeloid dendritic cells express elevated levels of CD1a and CD1b in the presence of low levels of IgG in vivo (in CVID patients). This aberrant expression pattern was normalized by IVIG therapy, suggesting that IgG can regulate group I CD1 molecules in vivo [41]. Effects on T cells

It has been demonstrated that IVIG inhibits human T-cell and dendritic cell proliferation and cytokine production, induced by mitogenic and allogeneic stimulation in vitro, as effectively as calcineurin inhibitors [42]. Even though this could be partially due to suppression of antigen presenting cells, a direct effect of IVIG was also demonstrated [43, 44] as well as the induction of lymphocyte apoptosis via Fas (CD95) and activation of caspases [4]. Furthermore, it was shown that IVIG affects T

regulatory cells by increasing their suppressive function [45] and by inducing the expansion of CD4+CD25+ regulatory T cells [46]. In 2008, novel Treg epitope peptides, called tregitopes, were identified; these epitopes are contained in the highly conserved framework regions of Fab and Fc of IVIG [47]. Tregitopes may provide one explanation for the expansion and stimulation of Treg cells following intravenous immunoglobulin therapy. In vitro studies suggest that the mechanism of action of tregitopes is to induce Tregs to produce regulatory signals. Tregs are induced directly by IVIG (tregitopes on F(ab′)2) or indirectly, by antigen presenting cells

presentation of Fc-tregitopes. This is followed by the modulation of dendritic cell phenotype, resulting in down-regulation of co-stimulatory molecules (i.e. CD80, CD86, and MHC II) and up-regulation of tolerogenic factors such as the immunoglobulin-like transcript 3 (ILT3) receptor [48]. Effects on B cells

The balance of negative and positive signals in maintaining an appropriate B cell activation threshold is critical in B lymphocyte immune tolerance and autoreactivity. In addition to the effects described above, a number of studies have revealed key regulatory effects of IVIG on B cells, such as up-regulation of FcγRIIB-mediated suppression of the expansion of auto-reactive B cells, anti-idiotype mediated inhibition of BCR, inhibition of B cell-mediated antigen presentation, neutralization of BAFF and APRIL, and differentiation of human CD40-activated B cells to IgG-secreting cells [49-54]. In vitro treatment with IVIG induces apoptosis of human B cells through a Fas- and caspase-dependent pathway [4, 55]. A recent study showed that in vitro IVIG treatment of B cells renders them refractory to BCR stimulation, suppresses the PI3K signaling pathway and induces a long-term state of tolerance, inducing a program of long-term functional silencing similar to anergy [56]. As a further similarity with B cells anergy, it was shown that in vitro binding of sialic acid-bearing IVIg to CD22 results in sustained activation of extracellular-signal-regulated kinase (ERK) and negative regulation of some B-cell functions [57]. To investigate whether IVIG modulates ERK signaling in vivo, we measured constitutive and BCR-induced ERK phosphorylation in CVID patients before and immediately after IVIG infusion. Constitutive ERK activation was significantly increased in unstimulated naive and in IgM+ memory B-cells after IVIG infusion, whereas BCR-induced activation, expressed as the fold increase respect to the constitutive ERK level, decreased in these cells (manuscript submitted). Moreover, we observed an increase in B cells expressing low levels of CD21 (CD21low B cells), a peculiar population of anergic-like B cells that is spontaneously expanded in a subset of CVID patients [58, 59], in CVID patients‟ peripheral blood mononuclear cells drawn after IVIG infusion and cultured overnight in the absence of stimuli (unpublished data). These

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findings suggest that IVIG may induce anergy-like changes in B cells in vivo. The ultimate effects of IVIG on B-cell function may not necessarily be adverse. In fact, a recent study [60] suggests that IVIG induces B-cell proliferation and IgM synthesis in CVID

patients treated with „replacement‟ IVIG dose (rather than high-dose), probably due to a natural anti-CD40 IgG that are capable of restoring B-cell proliferation and immunoglobulin secretion, compensating for possible defective T-cell signaling. Effects on glucocorticoid resistance

Glucocorticoid resistance (GCR) or insensitivity has been described in many autoimmune and inflammatory conditions. IVIG has the potential to act as a steroid-sparing agent in systemic lupus erythematosus and in autoimmune blistering diseases [61]. In one study of asthmatic patients, IVIG therapy acted synergistically with dexamethasone in suppressing lymphocyte activation and in significantly improving GCR-binding affinity after 3 and 6 months of therapy [62]. Using a steroid-resistant CEM-C1 lymphoblastoid cell line cell model, one study suggested that one of the steroid-sparing effects of IVIG is mediated through the improved GCR-binding affinity [63].

III. CONCLUSIONS The information accumulated over the past thirty years makes our knowledge of the effects of IVIG on the immune system resembling more and more a jigsaw puzzle (Fig. 1). In this

puzzle, however, the new pieces move other pieces from a central to a marginal position. Although animal experiments may provide an important tool in studying disease-specific effects of IVIG in vivo, the use of human-pooled immunoglobulin in animals remains uncertain in terms of potential loss-of-function or gain-of-function effects. A number of different factors, including batch-to-batch variations (i.e. specific antibody titers or the ratio between agonistic and antagonistic antibodies, the amount of sialylated Ig fraction, etc.), the method of preparation, the presence of IgG aggregates and many other characteristics of individual IVIG preparations may influence the ultimate effects of treatment. These differences may explain some discrepant results of clinical trials as well as of in vitro studies. Trials with tailored IVIG preparations, for example enriched for terminal sialic acid residues or with well-defined in vitro activities on key regulatory pathways, may represent a promising approach for optimizing treatments. Finally, the better understanding of the mechanisms of action of IVIG in specific diseases will help to identify patient subpopulations that are likely to benefit the most.

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Fig. 1. A jigsaw puzzle representation of the interaction of IVIG with the immune system.

Fig. 1 A jigsaw puzzle representation of the interaction of

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