Alan H. Lazarus

INTRAVENOUS IMMUNOGLOBULIN AMELIORATES ITP VIA ACTIVATING FC GAMMA RECEPTORS ON DENDRITIC CELLS

Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fcγ receptors (FcγR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 105 IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory FcγRIIB on the initiator cell, although FcγRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating FcγR on dendritic cells.

Of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an inhibitor of Syk

To determine whether inhibition of Syk would be useful in FcgammaR-dependent cytopenias such as immune thrombocytopenic purpura (ITP) or autoimmune hemolytic anemia, mouse models were used to evaluate efficacy of R406, an inhibitor of Syk function, in treating cytopenia. Both disease models responded favorably to treatment, with amelioration of ITP being more dramatic. Thus, phase 2 clinical trial was initiated to study the effects of Syk inhibition in humans with ITP. Sixteen adults with chronic ITP were entered into an open-label, single-arm cohort dose-escalation trial beginning with 75 mg and escalating as high as 175 mg twice daily. Doses were increased until a persistent response was seen, toxicity occurred, or 175 mg twice daily was reached. Eight patients achieved persistent responses with platelet counts greater than 50 x 10(9)/L (50 000 mm(3)) on more than 67% (actually 95%) of their study visits, including 3 who had not persistently responded to thrombopoietic agents. Four others had nonsustained responses. Mean peak platelet count exceeded 100 x 10(9)/L (100 000 mm(3)) in these 12 patients. Toxicity was primarily GI-related with diarrhea (urgency) and vomiting; 2 patients had transaminitis. In conclusion, inhibition of Syk was an efficacious means of increasing and maintaining the platelet count in half the patients with chronic refractory ITP. (ClinicalTrials.gov, no. NCT00706342).

A murine model of severe immune thrombocytopenia is induced by antibody- and CD8+ T cell-mediated responses that are differentially sensitive to therapy

Immune thrombocytopenia (ITP) is a bleeding disorder characterized by antibody-opsonized platelets being prematurely destroyed in the spleen, although some patients with ITP may have a cell-mediated form of thrombocytopenia. Although several animal models of ITP have been developed, few mimic primary chronic ITP nor have any shown cell-mediated platelet destruction. To create this type of model, splenocytes from CD61 knockout mice immunized against CD61(+) platelets were transferred into severe combined immunodeficient (SCID) (CD61(+)) mouse recipients, and their platelet counts and phenotypes were observed. As few as 5 x 10(4) splenocytes induced a significant thrombocytopenia and bleeding mortality (80%) in recipients within 3 weeks after transfer. Depletion of lymphocyte subsets before transfer showed that the splenocytes ability to induce thrombocytopenia and bleeding completely depended on CD4(+) T helper cells and that both CD19(+) B cell (antibody)- and CD8(+) T cell (cell)-mediated effector mechanisms were responsible. Treatment of the SCID mouse recipients with intravenous gamma-globulins raised platelet counts and completely prevented bleeding mortality induced by antibody-mediated effector mechanisms but did not affect cell-mediated disease. This novel model not only shows both antibody- and cell-mediated ITP and bleeding but also suggests that these 2 effector mechanisms have a differential response to therapy.

IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity.

Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody‐dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region‐reactive (anti‐idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet‐specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre‐established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti‐idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody‐sensitized red blood cells. F(ab′)2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti–idiotype interactions are not required.

Can antibodies with specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the treatment of autoimmune disease

Intravenous Ig (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP) as well as numerous other autoimmune states; however, the active antibodies within IVIg are unknown. There is some evidence that antibodies specific for a cell-associated antigen on erythrocytes are responsible, at least in part, for the therapeutic effect of IVIg in ITP. Yet whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP or other autoimmune diseases is also unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenic purpura. Mice experimentally treated with soluble OVA + anti-OVA versus mice treated with OVA conjugated to rbcs (OVA-rbcs) + anti-OVA were compared. In both situations, mice were protected from ITP. Both these experimental therapeutic regimes acted in a complement-independent fashion and both also blocked reticuloendothelial function. In contrast to OVA-rbcs + anti-OVA, soluble OVA + anti-OVA (as well as IVIg) did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcgammaRIIB (FcgammaRIIB(-/-) mice). Similarly, antibodies reactive with the endogenous soluble antigens albumin and transferrin also ameliorated ITP in an FcgammaRIIB-dependent manner. Finally, broadening the significance of these experiments was the finding that anti-albumin was protective in a K/BxN serum-induced arthritis model. We conclude that IgG antibodies directed to soluble antigens ameliorated 2 disparate IVIg-treatable autoimmune diseases.

Therapeutic Effect of IVIG on Inflammatory Arthritis in Mice Is Dependent on the Fc Portion and Independent of Sialylation or Basophils

High-dose i.v. Ig (IVIG) is used to treat various autoimmune and inflammatory diseases; however, the mechanism of action remains unclear. Based on the K/BxN serum transfer arthritis model in mice, IVIG suppression of inflammation has been attributed to a mechanism involving basophils and the binding of highly sialylated IgG Fc to DC-SIGN–expressing myeloid cells. The requirement for sialylation was examined in the collagen Ab-induced arthritis (CAbIA) and K/BxN serum transfer arthritis models in mice. High-dose IVIG (1–2 g/kg body weight) suppressed inflammatory arthritis when given prophylactically. The same doses were also effective in the CAbIA model when given subsequent to disease induction. In this therapeutic CAbIA model, the anti-inflammatory effect of IVIG was dependent on IgG Fc but not F(ab′)2 fragments. Removal of sialic acid residues by neuraminidase had no impact on the anti-inflammatory activity of IVIG or Fc fragments. Treatment of mice with basophil-depleting mAbs did not abrogate the suppression of either CAbIA or K/BxN arthritis by IVIG. Our data confirm the therapeutic benefit of IVIG and IgG Fc in Ab-induced arthritis but fail to support the significance of sialylation and basophil involvement in the mechanism of action of IVIG therapy.

The Mechanisms of Action of Intravenous Immunoglobulin and Polyclonal Anti-D Immunoglobulin in the Amelioration of Immune Thrombocytopenic Purpura: What Do We Really Know?

Intravenous immunoglobulin (IVIg) has been used for more than 25 years to treat an ever-increasing number of autoimmune diseases including immune thrombocytopenic purpura. Although the exact mechanism of action of IVIg has remained elusive, many theories have been postulated, including mononuclear phagocytic system blockade/inhibition, autoantibody neutralization by anti-idiotype antibodies, pathogenic autoantibody clearance due to competitive inhibition of the neonatal immunoglobulin Fc receptor, cytokine modulation, complement neutralization, and immune complex formation leading to dendritic cell priming. Polyclonal anti-D immunoglobulin is a polyclonal IVIg product enriched for antibodies directed to the RhD antigen on red blood cells and that has also been successfully used to treat immune thrombocytopenia in RhD(+) patients. The primary theory to explain polyclonal anti-D immunoglobulin function has classically been mononuclear phagocytic system blockade, although modulation of Fcgamma receptor expression and/or immunomodulation may also play a role. Work using a murine model of immune thrombocytopenic purpura to further our understanding of the mechanism of action of these 2 therapeutic agents is a focus of this article.

Intravenous immunoglobulin inhibits anti-glycoprotein IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia.

We have previously shown that injection of anti‐glycoprotein (GP) IIb induces murine immune thrombocytopenia (ITP) and that intravenous immunoglobulin (IVIg) ameliorates ITP. We hypothesise that murine ITP may be associated with platelet apoptosis, which is upregulated by anti‐GPIIb and downregulated by IVIg. The current study demonstrated that anti‐GPIIb injection induced three critical apoptosis manifestations in platelets: (i) mitochondrial inner transmembrane potential (ΔΨm) depolarisation; (ii) caspase‐3 activation; and (iii) phosphatidylserine (PS) exposure. IVIg administration inhibited caspase‐3 activation and PS exposure, but not ΔΨm‐depolarisation, in anti‐GPIIb‐treated platelets, demonstrating that IVIg ameliorates thrombocytopenia concomitantly with inhibiting late, but not early mechanisms of platelet apoptosis.

Mechanism of action of IVIG and anti-D in ITP

Infusion of large amounts of intravenous immunoglobulin (IVIG) or polyclonal anti-D can reverse thrombocytopenia in patients with idiopathic thrombocytopenic purpura within hours of the administration of these products. It has been suggested that the effects of IVIG appear to far outlast several half-lives of the product. Several mechanisms have been proposed to explain both the acute and long term effects of IVIG. These will be discussed in this review.

Flow cytometric analysis of platelets from children with the Wiskott-Aldrich syndrome reveals defects in platelet development, activation and structure.

The pathophysiology of platelet dysfunction in the Wiskott‐Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet‐associated IgG and reduced platelet‐dense granule content. Levels of reticulated ‘young’ platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar‐sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin‐ and ADP‐induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F‐actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.