Immunomodulatory action of intravenous immunoglobulin (IV Ig)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1782817/

Comment; The complexity of immunology is mind-boggling! So much to sort out!  It depends on dose used (physiologic or high/therapeutic) and other factors, genetic engineering may provide new sources of valuable biologic agents.

W A C Sewell^* and S Jolles^†

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INTRODUCTION

Intravenous immunoglobulin (IV Ig) 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, IV Ig is used at a ‘replacement dose’ of 200–400 mg/kg body weight, given approximately 3-weekly. In contrast, ‘high-dose’ IV Ig (hdIV Ig), 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 hdIV Ig 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 hdIV Ig, 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 hdIV Ig.

IV Ig 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 IV Ig 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 IV IG

Anti-proliferative effects

IV Ig 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 IV Ig inhibited proliferation more than commercial multiple-donor preparations, but there was no difference between standard IV Ig preparations and cytomegalovirus hyperimmune globulin.⁴

Both antigen-dependent and antigen-independent responses are inhibited by IV Ig in a dose-dependent manner.⁵ T-cell proliferation in response to anti-CD3 or tetanus toxoid was shown to be inhibited by IV Ig 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 IV Ig 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 IV Ig was shown to enhance the suppressive activity of the patient’s lymphocytes, an effect that was reversed on cessation of IV Ig therapy.

Modulation of apoptosis and the cell cycle

IV Ig 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 IV Ig in this study. In a further in vitro study¹⁰ a dual effect by IV Ig 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 IV Ig, resulting in long-term survival. Additional studies have confirmed that IV Ig causes the arrest of cells at the G0/G1 phase of the cell cycle, and inhibits cells from entering S-phase.¹¹

IV Ig 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 IV Ig preparations.¹² In contrast hdIV Ig 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 IV Ig.¹⁴ Taken together these studies show that although IV Ig 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 IV Ig-binding antibodies in a small number of patients with autoimmune disorders has suggested that IV Ig can act in a manner analogous to a B-cell superantigen¹⁵ and that B cells using V_H3·23 and V_H3·30/3·35 are selectively activated following IV Ig therapy.¹⁶

Dimers and higher-order aggregates of IgG in IV Ig preparations have been shown to activate neutrophils via triggering of macrophages. In a rat model, administration of IV Ig containing IgG dimers (aged IV Ig) resulted in an influx of activated neutrophils into pulmonary tissue compared to those containing low dimers (fresh IV Ig).¹⁷

Intact IV Ig, and F(ab′)₂ and Fc fragments of IV Ig, 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 IV Ig.¹⁹ High-dose IV Ig can reduce serum intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) levels in patients with atopic dermatitis.²⁰ IV Ig 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 β₅.²¹Blockade 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

IV Ig 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 IV Ig²² 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

IV Ig contains anti-idiotypes

Alteration of the idiotypic network by anti-idiotypic antibodies in IV Ig 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 IV Ig. F(ab′)₂ fragments block antibody/antigen binding, and F(ab′)₂ derived from IV Ig and bound to a column will remove autoantibodies.^24,25 IV Ig itself bound to a column will also remove these autoantibodies.^26,27 IV Ig does not, however, contain antibodies against common immunoglobulin allotypes.²⁸

IV Ig 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 IV Ig is derived from human donors, anti-cytokine antibodies may also be present within IV Ig preparations. Anti-interferon-γ (IFN-γ) antibodies in IV Ig have been shown to inhibit IFN-γ production in vitro³² and such anti-IFN-γ antibodies were present at greater concentrations in IV Ig than in polyclonal immunoglobulins directed against hepatitis B or cytomegalovirus.³³

IV Ig contains antibodies against the β-chain of the T-cell receptor³⁴ and also against CD5 and CD4.^35,36Binding 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 IV Ig 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.

IV Ig 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 IV Ig, which is most pronounced for Fc fragments.^29,30 IV Ig 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 IV Ig 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 IV Ig in recurrent epilepsy,⁴² IV Ig 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 IV Ig treatment. No changes in cytokines in response to IV Ig 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 IV Ig (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 IV Ig-treated, antibody-deficient patients increase only in patients with an adverse reaction to IV Ig.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, IV Ig 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 IV Ig 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 IV Ig.

In a smaller study of patients receiving high-dose IV Ig for atopic eczema, hdIV Ig 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 IV Ig 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 IV Ig 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

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EFFECTS AS A RESULT OF FC FRAGMENT

Inhibition of phagocytosis via inhibitory FcR

The ability of the Fc portion of immunoglobulin in IV Ig 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 IV Ig in treating autoimmune cytopenias. There are four main lines of evidence for this: firstly, IV Ig reduces the clearance of autologous erythrocytes coated with anti-D.⁵¹ Secondly, IV Ig 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 IV Ig 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 IV Ig 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, IV Ig 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 IV Ig. Protection was associated with the ability of IV Ig 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.

IV Ig 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 IV Ig.

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 IV Ig was found to act synergistically with steroids in improving the clinical parameters of asthma. IV Ig acted synergistically with dexamethasone in suppressing lymphocyte activation as measured by a shift in the dexamethasone dose–response curve by 1 log-fold. IV Ig therapy was also associated with significantly improved glucocorticoid receptor binding affinity. IV Ig resulted in significant reductions in oral glucocorticoid requirements and hospitalizations in a group of patients with severe asthma, with IV Ig 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 IV Ig 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 IV Ig.

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EFFECTS AS A RESULT OF COMPLEMENT RECEPTORS

Inhibition of complement deposition

IV Ig 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 hdIV Ig is known to be effective.⁶¹ Administration of IV Ig 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 IV Ig. C1q, C3 and C4 binding were inhibited by IV Ig in a dose-dependent manner, suggesting that one mechanism of action of IV Ig is to inhibit deposition of classical pathway components. Intact IgG, and Fc but not F(ab′)2 was responsible for this effect.⁶²

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EFFECTS AS A RESULT OF SUBSTANCES OTHER THAN ANTIBODY WITHIN IV IG PREPARATIONS

IV Ig contains immunoregulatory substances

IV Ig 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 IV Ig 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 IV Ig preparations.⁶⁵ This TGF-β may have antiproliferative effects. However, IL-6, IL-10, or TNF-α have not been detected in any IV Ig product.

Conclusions

It is clear that the observed immunomodulatory effects of IV Ig 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 IV Ig 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 IV Ig. 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) IV Ig therapy, a product containing a restricted repertoire of immunoglobulins produced synthetically, may be sufficient to exert a therapeutic effect. Thirdly, non-immunoglobulin components in IV Ig 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 IV Ig, clinical research in this area is urgently needed. This may inform us not only about the mechanism of action of IV Ig but also the mechanisms involved in the pathogenesis of disease.