IVIg increases interleukin-11 levels, which in turn contribute to increased platelets, VWF and FVIII in mice and humans.

The mechanisms of action of intravenous immunoglobulins (IVIg) in autoimmune diseases are not fully understood. The fixed duration of efficacy and noncumulative effects of IVIg in immune thrombocytopenia (ITP) and acquired von Willebrand disease (AVWD) suggest other mechanisms besides immunological ones. Additionally to the peripheral destruction of platelets in ITP, their medullary hypoproduction emerged as a new paradigm with rescue of thrombopoietin receptor agonists (TPO-RA). In an ITP mouse model, interleukin (IL)-11 blood levels increase following IVIg. IL-11 stimulates the production of platelets and other haemostasis factors; recombinant IL-11 (rIL-11) is thus used as a growth factor in post-chemotherapy thrombocytopenia. We therefore hypothesized that IVIg induces IL-11 over-production, which increases platelets, VWF and factor VIII (FVIII) levels in humans and mice. First, in an ITP mouse model, we show that IVIg or rIL-11 induces a rapid increase (72 h) in platelets, FVIII and VWF levels, whereas anti-IL-11 antibody greatly decreased this effect. Secondly, we quantify for the first time in patients with ITP, AVWD, inflammatory myopathies or Guillain-Barré syndrome the dramatic IL-11 increase following IVIg, regardless of the disease. As observed in mice, platelets, VWF and FVIII levels increased following IVIg. The late evolution (4 weeks) of post-IVIg IL-11 levels overlapped with those of VWF and platelets. These data may explain thrombotic events following IVIg and open perspectives to monitor post-IVIg IL-11/thrombopoietin ratios, and to assess rIL-11 use with or without TPO-RA as megakaryopoiesis co-stimulating factors to overcome the relative hypoproduction of platelets or VWF in corresponding autoimmune diseases, besides immunosuppressant.

Role of coagulation-associated processes on factor VIII immunogenicity in a mouse model of severe hemophilia A.

BACKGROUND: Immune responses to therapeutic factor VIII remain a major problem, affecting 30% of patients with severe hemophilia A. The primary factors that drive immune responses in these patients remain elusive. There have been conflicting reports on a role of coagulation (or thrombin) in anti-FVIII immune responses. OBJECTIVE: To assess the importance of coagulation-associated processes for the onset of the anti-FVIII immune response. METHODS: Using FVIII-deficient mice, we compared the immunogenicity of recombinant FVIII or the inactive FVIII(V) (634M) mutant. In parallel, the involvement of tissue factor (TF) activity in the anti-FVIII immune response was investigated upon injection of a neutralizing anti-TF antibody or by the use of chimeric mice that lack TF expression in myeloid cells. The development of the anti-FVIII immune response was also monitored after treatment with warfarin. RESULTS: The kinetics of the development of antibody responses to FVIII(V) (634M) were indistinguishable from those of wild-type FVIII. Inhibition of TF activity did not modulate immune responses to exogenous FVIII. Additionally, global inhibition of coagulation with warfarin failed to reduce the anti-FVIII immune response. CONCLUSIONS: Thrombin generation or coagulation-associated processes do not modulate the anti-FVIII antibody response in mouse model of severe hemophilia A.

Predictive immunogenicity of Refacto AF.

The administration of therapeutic factor VIII (FVIII) to treat or prevent haemorrhages in haemophilia A patients results, in up to 30% of the cases, in the development of inhibitory anti-FVIII antibodies. Much debate has taken place on the relevance of the nature of the FVIII product as a risk factor for inhibitor development. Thus, the plasma-derived vs. recombinant origin, the second vs. third generation of the product, or the presence of the B domain have been controversially evoked. A few years ago, Refacto AF, a third-generation recombinant B domain-deleted FVIII was marketed. The aim of this study was to compare the immunogenicity of Refacto AF to that of two recombinant full-length FVIII products: Helixate and Advate. For the three recombinant FVIII products, we compared the binding to the mannose-sensitive endocytic receptor CD206, the dose-dependent endocytosis by immature monocyte-derived dendritic cells (DCs), the activation by FVIII-loaded DCs of a FVIII-specific HLA-DRB1*0101-restricted mouse T-cell hybridoma and the induction of inhibitory anti-FVIII IgG in FVIII-deficient mice. At elevated FVIII concentrations, Refacto AF was less endocytosed than full-length recombinant products. At lower concentrations, however, Refacto AF was endocytosed by DCs and activated T cells as well as Helixate and Advate. The levels of inhibitory anti-FVIII IgG induced by Refacto AF in FVIII-deficient mice were lower or equal to that induced by Helixate and Advate respectively. The predicted immunogenicity of Refacto AF is identical to or lower than that of the two recombinant full-length FVIII products available on the French market.

Heme binds to factor VIII and inhibits its interaction with activated factor IX.

BACKGROUND: Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme-binding proteins. Free heme has been proposed to affect coagulation and has been co-purified with the factor VIII (FVIII)-von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. OBJECTIVES: To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. METHODS AND RESULTS: Heme bound to several sites on FVIII with high apparent affinity. Heme-binding inhibited FVIII procoagulant activity in a dose-dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme-mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme-binding protein known for its protective activity in hemolytic conditions. CONCLUSIONS: Our data identify FVIII as a novel heme-binding protein. Occupation of high affinity heme-binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme-mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.

Immunoprotective effect of von Willebrand factor towards therapeutic factor VIII in experimental haemophilia A.

The development of inhibitory anti-factor VIII (FVIII) antibodies in patients with haemophilia A following replacement therapy is associated with several types of risk factors. Among these, the purity of FVIII concentrates, and in particular the presence of von Willebrand factor (VWF), was controversially proposed to influence the immunogenicity of exogenous FVIII. We re-assessed in vivo and in vitro the immuno-protective effect of VWF towards FVIII. The immuno-protective effect of VWF towards FVIII was investigated in vivo, in a model of haemophilia A. We studied the endocytosis of FVIII by murine bone marrow-derived dendritic cells and evaluated the capacity of VWF to block the internalization of FVIII. We characterized the relevance of VWF for the accumulation of FVIII in the marginal zone of the spleen, a secondary lymphoid organ where the immune response to therapeutically administered FVIII initiates. Our results confirm that VWF reduces the immunogenicity of FVIII in FVIII-deficient mice. Paradoxically, VWF is important for the accumulation of FVIII in the marginal zone of the spleen. We propose that VWF exerts at least two non-mutually exclusive immunoprotective roles towards FVIII in haemophilic mice: VWF prevents the endocytosis of FVIII by professional antigen-presenting cells by blocking the interaction of FVIII with as yet unidentified endocytic receptor(s). Hypothetically, VWF, by virtue of increasing the half-life of FVIII in the circulation, may allow an increased contact time with tolerogenic marginal zone B cells in the spleen.

Bortezomib delays the onset of factor VIII inhibitors in experimental hemophilia A, but fails to eliminate established anti-factor VIII IgG-producing cells.

BACKGROUND: Replacement therapy with exogenous factor VIII to treat hemorrhages induces inhibitory anti-FVIII antibodies in up to 30% of patients with hemophilia A. Current approaches to eradicate FVIII inhibitors using high-dose FVIII injection protocols (immune tolerance induction) or anti-CD20 depleting antibodies (Rituximab) demonstrate limited efficacy; they are extremely expensive and/or require stringent compliance from the patients. OBJECTIVES: To investigate whether the proteasome inhibitor bortezomib, which depletes plasmocytes, modulates the anti-FVIII immune response in FVIII-deficient mice. METHODS AND RESULTS: Preventive 4-week treatment of naïve mice with bortezomib at the time of FVIII administration delayed the development of inhibitory anti-FVIII IgG, and depleted plasma cells as well as different lymphoid cell subsets. Conversely, curative treatment of inhibitor-positive mice for 10 weeks, along with FVIII administration, failed to eradicate FVIII inhibitors to extents that would be clinically relevant if achieved in patients. Accordingly, bortezomib did not eradicate anti-FVIII IgG-secreting plasmocytes that had homed to survival niches in the bone marrow, despite significant elimination of total plasma cells. CONCLUSIONS: The data suggest that strategies for the efficient reduction of anti-FVIII IgG titers in patients with hemophilia A should rely on competition for survival niches for plasmocytes in the bone marrow rather than the mere use of proteasome inhibitors.

Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A.

BACKGROUND: Alloimmune responses to intravenously administered protein therapeutics are the most common cause of failure of replacement therapy in patients with defective levels of endogenous proteins. Such a situation is encountered in some patients with hemophilia A, who develop inhibitory anti-factor (F)VIII alloantibodies after administration of FVIII to treat hemorrhages. OBJECTIVES: The nature of the secondary lymphoid organs involved in the initiation of immune responses to human therapeutic has not been studied. We therefore investigated this in the case of FVIII, a self-derived exogenous protein therapeutic. METHODS: The distribution of intravenously administered FVIII was followed after FVIII-deficient mice were injected with radiolabeled FVIII and using immunohistochemistry. The role of the spleen and antigen-presenting cells (APC) in the onset of the anti-FVIII immune response was analyzed upon splenectomy or treatment of the mice with APC-depleting compounds. RESULTS: FVIII preferentially accumulated in the spleen at the level of metallophilic macrophages in the marginal zone (MZ). Surgical removal of the spleen or selective in vivo depletion of macrophages and CD11c-positive CD8 alpha-negative dendritic cells resulted in a drastic reduction in anti-FVIII immune responses. CONCLUSIONS: Using FVIII-deficient mice as a model for patients with hemophilia A, and human pro-coagulant FVIII as a model for immunogenic self-derived protein therapeutics, our results highlight the importance of the spleen and MZ APCs in the initiation of immune responses to protein therapeutics. Identification of the receptors implicated in retention of protein therapeutics in the MZ may pave the way towards novel strategies aimed at reducing their immunogenicity.

Immunomodulation of autoimmune and inflammatory diseases with intravenous immunoglobulin.

Intravenous immunoglobulin (IVIg) has been used in the treatment of primary and secondary antibody deficiencies for over two decades. Since the early 1980s, the therapeutic efficacy of IVIg has been established in idiopathic thrombocytopenic purpura, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, dermatomyositis and Kawasaki syndrome, and the prevention of graft versus host disease in recipients of allogeneic bone marrow transplants. Its use has also been reported in a large number of other autoimmune and systemic inflammatory conditions. In this review, we discuss the mechanisms by which IVIg exerts immunomodulatory effects in immune pathologies.

Mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases.

Intravenous immunoglobulins (IVIg) exert a broad range of immunoregulatory functions that provide a basis for the beneficial effects of IVIg in autoimmune and systemic inflammatory disorders. This review focuses on the effects f IVIg on humoral and cellular immunity that may be of relevance for the treatment of inflammatory neurological diseases.

Mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases.

Therapeutic polyclonal intravenous immunoglobulin (IVIg) consists of normal IgG obtained from the pools of plasma of several thousand healthy blood donors. IVIg is used as substitutive treatment of primary and secondary immunodeficiences. Since the first study of Paul Imbach who demonstrated the beneficial effect in idiopathic thrombocytopenic purpura, IVIg is also used in a number of autoimmune and inflammatory diseases. The immunoregulatory effects of IVIg in autoimmune diseases depend on the interaction of Fc portion of immunoglobulins with Fc receptors and on the selection of lymphocyte repertoires of patients through variable regions of infused immunoglobulins. IVIg modulates the activation and effector functions of B and T lymphocytes, neutralizes pathogenic autoantibodies, interferes with antigen presentation and has a strong anti-inflammatory effect which depends on its interaction with the complement system, cytokines and endothelial cells. The immunomodulatory potential of IVIg in patients is thus a result of a variety of complex mechanisms that act in a synergy.

Variable sensitivity of lymphoblastoid cells to apoptosis induced by Viscum album Qu FrF, a therapeutic preparation of mistletoe lectin.

BACKGROUND: Viscum album (VA) Qu FrF preparation consists of an aqueous extract of Viscum album, the European mistletoe. It has cytotoxic and immunomodulatory properties that support its usefulness in cancer therapy. However, only little information is available on the interaction between mistletoe lectin (ML) and the humoral compartment of the immune system. METHODS: Inhibition of proliferation of cells was performed by incorporation of (3)H-thymidine, and cell viability was assessed by the uptake of propidium iodide. Detection of Bcl-2 and related molecules was performed by SDS-PAGE followed by immunoblotting. RESULTS: We demonstrated that B lymphocyte lines are insensitive to VA Qu FrF-induced apoptosis. We showed, using Fas-sensitive and Fas-resistant cell lines, that VA Qu FrF-induced apoptosis is Fas independent. In both B and T lymphocytes, treatment of the cells with VA Qu FrF was associated with decreased levels of Bcl-2 and Bcl-X. CONCLUSIONS: Our results suggest that B lymphocyte depletion is not involved in the modulation of humoral immunity by ML. These observations should be of value in appropriate designing of cancer therapy using compounds containing mistletoe extracts.