Intravenous immunoglobulin (IVIg) is a blood product prepared from the serum of between 1000 and 15 000 donors per batch. It is the treatment of choice for patients with antibody deficiencies. In this indication, IVIg is used at a ‘replacement dose’ of 200–400 mg/kg body weight, given approximately 3-weekly. In contrast, ‘high-dose’ IVIg (hdIVIg), given most frequently at 2 g/kg/month, is used as an ‘immunomodulatory’ agent in an increasing number of immune and inflammatory disorders. Initial use of hdIVIg was for idiopathic thrombocytopenic purpura (ITP) in children.1 Despite a lack of double-blind, randomized, placebo-controlled trials, many other conditions are managed with hdIVIg, including numerous haematological, rheumatological, neurological and dermatological disorders.2 In this article, we review the current understanding and recent developments in the immunomodulatory mechanisms of action of hdIVIg.
IVIg may, for the purposes of clarity, be considered to have four separate mechanistic components: (1) actions mediated by the variable regions F(ab′)2, (2) actions of Fc on a range of Fc receptors (FcR), (3) actions mediated by complement binding within the Fc fragment, and (4) immunomodulatory substances other than antibody in the IVIg preparations (Fig. 1). It is likely that these components act concurrently, however, different mechanisms may be important in different settings. We will address the mechanisms under these broad headings although in some cases more than one mechanism is operative or our understanding does not allow accurate categorization.
F(ab′)2 Mediating binding site interactions of ivig
IVIg has been shown to have a considerable inhibitory effect on mitogen-induced T-cell proliferation in vitro.3 This effect has been shown for intact immunoglobulin G (IgG), with less evidence for a role for Fc fragment.4 Single-donor preparations of IVIg inhibited proliferation more than commercial multiple-donor preparations, but there was no difference between standard IVIg preparations and cytomegalovirus hyperimmune globulin.4
Both antigen-dependent and antigen-independent responses are inhibited by IVIg in a dose-dependent manner.5 T-cell proliferation in response to anti-CD3 or tetanus toxoid was shown to be inhibited by IVIg in a dose-dependent manner over a range of IgG concentrations (0–10 mg/ml).6 The inhibition was reversible by exogenous interleukin-2 (IL-2) and the authors concluded that the effects were a result of interference with cytokine-mediated T-cell proliferation.
Both pooled normal human immunoglobulin, and single donor immunoglobulin were shown to reduce pokeweed mitogen (PWM) -induced plaque-forming cell formation following 300 mg/kg infusions into patients with common variable immunodeficiency.7 This effect was short-lived, and sera collected >24 hr post-infusion no longer inhibited cell proliferation. The suppressive effects of IVIg when used at replacement dose (100–200 mg/kg) were demonstrated in antibody-deficient children.8 In this study, the effects of the children's sera on the immunoglobulin-producing activity of PWM-stimulated normal lymphocytes was assessed. Even this low-dose IVIg was shown to enhance the suppressive activity of the patient's lymphocytes, an effect that was reversed on cessation of IVIg therapy.
Modulation of apoptosis and the cell cycle
IVIg has been shown to suppress the proliferation of antigen-specific T cells without inducing apoptosis, the cells remaining refractory to induction of apoptosis by CD95 ligation.9 Interestingly, Bcl-2 expression was not affected by IVIg in this study. In a further in vitro study10 a dual effect by IVIg was found. The incidence of apoptosis was elevated in activated Ki-67 and CD95-positive peripheral blood mononuclear cells (PBMC), whereas it was lower in small, non-activated cells. The cells that survived exhibited a striking increase in the expression of p21/WAF-1, suggesting G1 arrest. A concomitant up-regulation of Bcl-2 was also observed following exposure to IVIg, resulting in long-term survival. Additional studies have confirmed that IVIg causes the arrest of cells at the G0/G1 phase of the cell cycle, and inhibits cells from entering S-phase.11
IVIg has been demonstrated to induce apoptosis in leukaemic lymphocytes and monocytes as well as normal tonsillar B cells, an effect mediated at least in part by anti-CD95 antibodies present within the IVIg preparations.12 In contrast hdIVIg used to treat toxic epidermal necrolysis (TEN) has been shown to block Fas inducing keratinocyte apoptosis.13 In addition in atopic dermatitis T-cell-mediated, Fas-induced keratinocyte apoptosis is inhibitied by IVIg.14 Taken together these studies show that although IVIg appears to be broadly anti-apoptotic and causes cell cycle arrest, in certain circumstances it may also be pro-apoptotic.
Activation of specific cells
Sequencing of IVIg-binding antibodies in a small number of patients with autoimmune disorders has suggested that IVIg can act in a manner analogous to a B-cell superantigen15 and that B cells using VH3·23 and VH3·30/3·35 are selectively activated following IVIg therapy.16
Dimers and higher-order aggregates of IgG in IVIg preparations have been shown to activate neutrophils via triggering of macrophages. In a rat model, administration of IVIg containing IgG dimers (aged IVIg) resulted in an influx of activated neutrophils into pulmonary tissue compared to those containing low dimers (fresh IVIg).17
Intact IVIg, and F(ab′)2 and Fc fragments of IVIg, inhibit IgE production in a dose-dependent manner by human B cells stimulated with anti-CD40 and IL-4, F(ab′)2 has a more inhibitory effect than Fc.18
Effects on cell adhesion
Adhesion of T cells to extracellular matrix following activation by phytohaemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA) has been shown to be reduced by IVIg.19 High-dose IVIg can reduce serum intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) levels in patients with atopic dermatitis.20 IVIg also contains antibodies to the Arg-Gly-Asp (RGD) motif, the attachment site for a number of adhesive extracellular matrix proteins and integrins β1, β3 and β5.21 Blockade of integrin binding may have effects on lymphocyte recirculation/migration, access of cells to sites of inflammation and the activation state of cells.
Antibodies against pathogens
IVIg contains antibody specificities to a broad range of pathogens, reflecting the antibody repertoire of the thousands of donors included in each batch of commercial preparation. Anti-staphylococcal superantigen antibodies have been demonstrated in IVIg22 and these were able to inhibit superantigen-mediated activation of T cells. It is possible that this is a mechanism of immunomodulation in disorders such as Kawasaki disease and atopic dermatitis, which may be superantigen-driven, however, it should be remembered that antibody levels against specific pathogens can vary enormously between batches.23
IVIg contains anti-idiotypes
Alteration of the idiotypic network by anti-idiotypic antibodies in IVIg is an important mode of action. This was first demonstrated by the fall in anti-factor VIII titre in a patient with anti-factor VIII autoimmune disease treated with IVIg. F(ab′)2 fragments block antibody/antigen binding, and F(ab′)2 derived from IVIg and bound to a column will remove autoantibodies.24,25 IVIg itself bound to a column will also remove these autoantibodies.26,27 IVIg does not, however, contain antibodies against common immunoglobulin allotypes.28
IVIg contains antibodies to a range of immunoregulatory molecules
Antibodies to interleukin-1α (IL-1α) and tumour necrosis factor-α (TNF-α) have been demonstrated in the sera of healthy individuals.29,30 Anti-IL-8 antibodies have been reported in connective tissue disorders and in normal volunteers.31 Since IVIg is derived from human donors, anti-cytokine antibodies may also be present within IVIg preparations. Anti-interferon-γ (IFN-γ) antibodies in IVIg have been shown to inhibit IFN-γ production in vitro32 and such anti-IFN-γ antibodies were present at greater concentrations in IVIg than in polyclonal immunoglobulins directed against hepatitis B or cytomegalovirus.33
IVIg contains antibodies against the β-chain of the T-cell receptor34 and also against CD5 and CD4.35,36 Binding of these molecules is likely to have downstream consequences in terms of cellular activation. CD5 is expressed on T cells but also on a subset of B cells known to be involved in the production of autoantibodies.
Effects on cytokine levels
The reported effects of IVIg on cytokines have been confusing because various technologies have been employed. Cytokine measurements in serum are severely hampered by sensitivity problems, and the presence of circulating inhibitors. Output of cytokines into lymphocyte culture supernatants does not indicate which lymphocyte population was producing which cytokine. Intracellular cytokine assays by flow cytometry give the most information about the potential for cytokine production by cells of a defined phenotype, yet may not reflect true cytokine output in vivo. The types of cells studied will also have a bearing on the potential mechanism by which cytokine production is altered, as different cell types (e.g. monocytes and T cells) will express different amounts of Fc receptors.
IVIg has been shown to reduce IL-137 in macrophages and IL-638 production by monocytes in vitro. No effect was noted for TNF-α,38 however, some studies have shown a dose-dependent reduction in TNF-α by IVIg, which is most pronounced for Fc fragments.29,30 IVIg selectively increases gene transcription and secretion of IL-1 receptor antagonist (IL-1Ra) and IL-8.39
Assessment of intracellular cytokine production by mitogen-stimulated PBMCs in the presence of IVIg showed no change in IL-2, IL-10, TNF-α and IFN-γ, and a reduction in IL-3, IL-4, IL-5, TNF-β and granulocyte–macrophage colony-stimulating factor producing cells.40,41
In a trial of IVIg in recurrent epilepsy,42 IVIg was given at 600 mg/kg every 4 weeks to 18 patients with intractable epilepsy. Plasma levels of IFN-γ, IL-2, IL-4 and IL-6 were measured before and 20 min after each IVIg treatment. No changes in cytokines in response to IVIg at months 1, 3, or 6 were noted. However, there was a statistically significant increase in both plasma IL-6 and IFN-γ immediately following each infusion. In a few patients, kinetic studies were performed and demonstrated peaks in IFN-γ at various times up to 3 days post-infusion, with a slower peak in IL-6 production.
Plasma levels of cytokines compared before and 1, 3, 20 and 44 hr following a single infusion of IVIg (400 mg/kg) infusion showed a rapid and significant rise in plasma TNF-α as well as IL-6 and IL-843 in hypogammaglobulinaemic patients. However, more detailed studies have shown that TNF-α levels in the plasma of IVIg-treated, antibody-deficient patients increase only in patients with an adverse reaction to IVIg.44 The TNF-α level actually increased during the infusion itself, and was not abnormally elevated prior to treatment. In human immunodeficiency virus (HIV) -infected patients with high circulating levels of TNF-α, IVIg was shown to reduce both plasma TNF-α levels (with an increase in circulating TNF-receptor levels) and to reduce the production of TNF in LPS-stimulated PBMC supernatants.45
In patients with common variable immunodeficiency (CIVD) using single-cell flow cytometric assessment of cytokine expression, it has been shown that replacement dose IVIg can significantly increase IL-2 expression by CD4+ T cells, and TNF-α expression by CD8+ CD28– cells.46 There was no effect on IFN-γ expression by replacement-dose IVIg.
In a smaller study of patients receiving high-dose IVIg for atopic eczema, hdIVIg had no statistical effect on IFN-γ expression by CD4 and CD8 T cells, however, CD69 expression was reduced post-activation in all patients (manuscript submitted for publication). High-dose IVIg has no discernible effect on natural killer cell IFN-γ expression following stimulation with PMA and ionomycin47 nor on monocyte IL-12 expression.48
Soluble cytokine receptors such as TNF-receptors increase following IVIg infusion, and there is a considerable increase (1000-fold molar excess) in IL-1Ra levels.43 This increase in IL-1Ra post-infusion was also noted in other studies.49,50
Effects as a result of fc fragment
Inhibition of phagocytosis via inhibitory FcR
The ability of the Fc portion of immunoglobulin in IVIg to bind to FcR and modulate the activity of the FcR-bearing cell is believed to be the main mechanism of action in the use of IVIg in treating autoimmune cytopenias. There are four main lines of evidence for this: firstly, IVIg reduces the clearance of autologous erythrocytes coated with anti-D.51 Secondly, IVIg treatment of patients with ITP impairs the ability of their monocytes to form rosettes with IgG-coated erythrocytes.52 Thirdly, the in vivo effects of IVIg in ITP can be stimulated by using antibodies to FcγRIII.53,54 Fourthly, anti-D immunoglobulin can increase platelet counts in patients with ITP who are RhD-positive. The beneficial effects of IVIg in immune cytopenias are likely to be a result of the saturation of FcR on splenic macrophages.
In ITP, autoantibody-coated platelets bind to FcγRIII on macrophages, the resulting FcγRIII cross-linking initiates phagocytosis of the platelet. In a murine model of ITP, IVIg or Fc [but not F(ab′)2] was shown to prevent platelet consumption triggered by a pathogenic autoantibody.55 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. Protection was associated with the ability of IVIg administration to induce surface expression of FcγRIIB on splenic macrophages. Modulation of inhibitory (FcγRIIB) signalling is thus a potent mechanism for attenuating autoantibody-triggered inflammatory diseases.
IVIg is also believed to act on antigen-dependent cellular cytotoxicity (ADCC) by saturating FcR on macrophages, preventing the binding of pathogenic antibodies.
Effects on antibody levels
Neonatal Fc receptor FcRn transfers maternal IgG across the placenta to the fetus; there also appears to be a role for FcγRIIB in this process. FcRn is structurally related to major histocompatibility complex (MHC) class I molecules and is present on endothelial cells, epithelial cells of mammary gland, liver, intestine and kidney and also on monocytes, macrophages and DCs.56 A further role for FcRn is to recycle internalized IgG to the cell surface for release, if IgG is not FcRn bound it is degraded. This maintains IgG levels systemically and locally and would inhibit the recycling and enhance catabolism of pathogenic autoantibodies following saturation with IVIg.
FcγRIIB signalling in B cells through inhibitory ITIM motifs may also reduce antibody production.57 It has been shown that when IgG levels in the plasma reach 200% of normal values the half-life of IgG is reduced from 21 to 12 days.58
Effects on glucocorticoid receptor binding affinity
In a study on asthmatic patients59 IVIg was found to act synergistically with steroids in improving the clinical parameters of asthma. IVIg acted synergistically with dexamethasone in suppressing lymphocyte activation as measured by a shift in the dexamethasone dose–response curve by 1 log-fold. IVIg therapy was also associated with significantly improved glucocorticoid receptor binding affinity. IVIg resulted in significant reductions in oral glucocorticoid requirements and hospitalizations in a group of patients with severe asthma, with IVIg being as effective in patients with steroid-insensitive asthma as in patients with steroid-sensitive asthma.
It is not clear whether this effect is mediated through Fc receptors or F(ab′)2, however, it does suggest that additional benefit may be gained when IVIg is used as adjunctive therapy rather than monotherapy. It also remains to be investigated if the effects of other conventional therapies can be enhanced in a more than additive way by IVIg.
Effects as a result of complement receptors
Inhibition of complement deposition
IVIg can act by intercepting assembly and deposition of the complement membrane attack complex (MAC) on endomysial capillaries by forming complexes between antibody and C3b, preventing the incorporation of activated C3 into the C5 convertase.60 Deposition of complement terminal attack components in intramuscular capillaries is one of the mechanisms underlying the pathogenesis of dermatomyositis, a condition for which hdIVIg is known to be effective.61 Administration of IVIg to rats in an animal model of dermatomyositis suppressed the necrotic changes (at 200–400 mg/kg) and the inflammatory changes (at 400–800 mg/kg) with a reduction in deposition of C3 and immunoglobulin by IVIg. C1q, C3 and C4 binding were inhibited by IVIg in a dose-dependent manner, suggesting that one mechanism of action of IVIg is to inhibit deposition of classical pathway components. Intact IgG, and Fc but not F(ab′)2 was responsible for this effect.62
Effects as a result of substances other than antibody within ivig preparations
IVIg contains immunoregulatory substances
IVIg itself may contain cytokines and other molecules including soluble cytokine inhibitors, soluble CD4, and MHC class II.63 Stabilizing agents, mainly various sugars, exert an effect. Both maltose and sucrose, at concentrations present in commercial IVIg preparations, can inhibit PHA-induced, and to a lesser extent PMA-induced, proliferative responses in vitro.64 Maltose, but not sucrose, was able to inhibit an anti-CD3 induced response. Significant quantities of transforming growth factor-β1 (TGF-β1) and TGF-β2 (∼10 ng/ml), but not TGF-β3, have been found in commercial IVIg preparations.65 This TGF-β may have antiproliferative effects. However, IL-6, IL-10, or TNF-α have not been detected in any IVIg product.
It is clear that the observed immunomodulatory effects of IVIg therefore depend on both the dose used (replacement or high dose) and the disease being investigated. Furthermore, in vitro studies should only cautiously be extrapolated to in vivo effects, and further in vivo studies, employing a variety of read-outs, will be required. There is convincing evidence that IVIg works via a wide variety of parallel mechanisms, the relative importance of each depending on the circumstances in which it is used. Three important features are encouraging given the current world shortage of IVIg. The first is that intact immunoglobulin is not required for all these effects, and future studies should examine the use of genetically engineered Fc fragments of chosen subclasses, to exploit FcR mediated inhibitory effects. It may also be possible to modulate FcR expression to maximize this effect. Second, certain idiotypes are obviously important and in immunomodulatory (but probably not replacement) IVIg therapy, a product containing a restricted repertoire of immunoglobulins produced synthetically, may be sufficient to exert a therapeutic effect. Thirdly, non-immunoglobulin components in IVIg preparations need to be examined more carefully for immunomodulatory effects in vivo.
All these approaches remain speculative, but given the risks, expense and shortage of supply of IVIg, clinical research in this area is urgently needed. This may inform us not only about the mechanism of action of IVIg but also the mechanisms involved in the pathogenesis of disease.
We would like to thank Frank Johnson and Joe Brock for expert help with graphics. Stephen Jolles is a Leukaemia Research Foundation Project Grant holder and has received support from the Peel Medical Research Trust.