Srinivas V. Kaveri

Catalytic activity of antibodies against factor VIII in patients with hemophilia A.

Hemophilia A is an X chromosome-linked recessive disorder resulting in defective or deficient factor VIII (FVIII) molecules, which, in its severe form, is a life-threatening and crippling hemorrhagic disease. Infusion of homologous FVIII to patients with severe hemophilia A results, in 25% of patients, in the emergence of alloantibodies against FVIII (inhibitors)(ref. 1) that inhibit FVIII procoagulant activity by steric hindrance of the interaction of FVIII either with stabilizing molecules, with molecules essential for its activity or with activating molecules. Here, we report on the proteolysis of FVIII by alloantibodies of two patients with severe hemophilia A, demonstrating a previously unknown mechanism by which FVIII inhibitors may prevent the pro-coagulant function of FVIII. The kinetic parameters of FVIII hydrolysis indicate a functional role for the catalytic immune response in the inactivation of FVIII in vivo. The characterization of alloantibodies against FVIII as site-specific proteases may provide new approaches to the treatment of FVIII inhibitors.

V Region-Mediated Selection of Autoreactive Repertoires by Intravenous Immunoglobulin (i.v.Ig)

In addition to its use as substitute therapy for primary and secondary immunodeficiencies, intravenous immunoglobulin (i.v.Ig) is increasingly being used as an immunomodulating therapy in the treatment of patients with a variety of autoimmune and systemic inflammatory disorders (Schwartz 1990, Dietrich et al. 1992b, Dwyer 1992, Ronda et al. 1993). The use of i.v.Ig in these situations is supported by a few randomized clinical trials and a large number of uncontrolled and smaller studies. Of relevance to this chapter are that the reported beneficial effects of i.v.Ig include those in autoantibody-mediated autoimmune diseases (e.g., autoimmune cytopenias, anti-factor VIII autoimmune disease) as well as diseases in which autoaggressive T cells are primarily involved in the pathogenesis (e.g., autoimmune uveitis) (Imbach et al. 1981, Bussel et al. 1983, Sultan et al. 1984, McGuire et al. 1987, LeHoang et al. 1994). Where it is a feature of the disease, successful outcome of i.v.Ig therapy is associated with an improvement in the patients systemic inflammatory condition. Modulation of B-cell and Tcell functions and of cytokine production has further been observed in animal models of autoimmune diseases following administration of human i.v.Ig or of normal homologous IgG (Forsgren et al. 1991, Rossi et al. 1991a, Saoudi et al. 1993, Hentati et al. 1994). The design of trials to establish the efficacy and appropriate therapeutic sched-

A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes

Several therapeutic self-proteins elicit immune responses when administered to patients. Such adverse immune responses reduce drug efficacy. To induce an immune response, a protein must interact with different immune cells, including antigen-presenting cells, T cells, and B cells. Each cell type recognizes distinct immunogenic patterns on antigens. Mannose-terminating glycans have been identified as pathogen-associated molecular patterns that are essential for internalization of microbes by antigen-presenting cells, leading to presentation. Here, we have investigated the importance of exposed mannosylation on an immunogenic therapeutic self-protein, procoagulant human factor VIII (FVIII). Administration of therapeutic FVIII to hemophilia A patients induces inhibitory anti-FVIII antibodies in up to 30% of the cases. We demonstrate that entry of FVIII into human dendritic cells (DC) leading to T cell activation, is mediated by mannose-terminating glycans on FVIII. Further, we identified macrophage mannose receptor (CD206) as a candidate endocytic receptor for FVIII on DC. Saturation of mannose receptors on DC with mannan, and enzymatic removal of mannosylated glycans from FVIII lead to reduced T cell activation. The interaction between FVIII and CD206 was blocked by VWF, suggesting that, under physiological conditions, the intrinsic mannose-dependent immunogenicity of FVIII is quenched by endogenous immunochaperones. These data provide a link between the mannosylation of therapeutic self-proteins and their iatrogenic immunogenicity. Such a link would be of special relevance in the context of replacement therapy where mechanisms of central and peripheral tolerance have not been established during ontogeny because of the absence of the antigen.

Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A

Procoagulant factor VIII (FVIII) is either produced endogenously under physiologic conditions, or administered exogenously as a therapeutic hemostatic drug in patients with hemophilia A. In the circulation, FVIII interacts with a multitude of glycoproteins, and may be used for coagulation at the sites of bleeding, eliminated by scavenger cells, or processed by the immune system, either as a self-constituent or as a foreign antigen. The fate of FVIII is dictated by the immune status of the individual, the location of FVIII in the body at a given time point, and the inflammatory microenvironment. It also depends on the local concentration of FVIII and of each interacting partner, and on the affinity of the respective interactions. FVIII, by virtue of its promiscuity, thus constitutes the core of a dynamic network that links the coagulation cascade, cells of the immune system, and, presumably, the inflammatory compartment. We describe the different interactions that FVIII is prone to establish during its life cycle, with a special focus on players of the innate and adaptive immune response. Lessons can be learned from understanding the dynamics of FVIII interactions--lessons that should pave the way to the conception of long-lasting hemostatic drugs devoid of iatrogenic immunogenicity.

Stability of Natural Self-Reactive Antibody Repertoires During Aging

We have used a quantitative immunoblotting technique to analyze the repertoires of self-reactive antibodies in serum samples obtained from the same five healthy adults over a 25-year interval. The average age of the donors was 43 years at the time of the first serum sample and 69 years at the time of the second serum sample. The antibody repertoires of IgM and IgG were found to be strikingly similar among individuals in both early and late samples. Densitometric profiles of self-reactivity of serum IgM and of purified serum IgG remained unchanged over the 25-year interval. The total reactivity of serum IgG decreased significantly over the 25-year period. The observed stability of the natural self-reactive IgM and IgG antibody repertoires with aging supports the view that autoreactive B cells in the normal immune system are positively selected for reactivity with a limited set of immuno-dominant self-antigens throughout life.

Kawasaki disease: aetiopathogenesis and therapeutic utility of intravenous immunoglobulin.

Abstract Kawasaki disease (KD) is an acute febrile childhood vasculitis, associated with the development of coronary artery abnormalities in 25–30% of untreated patients. The aetiopathogenesis is not well known but it is accepted that an undefined infectious trigger in genetically predisposed individuals results in the disease. KD is characterized by an endothelial cell injury, which could be due to abnormal cytokine production and to generation of cytotoxic antibodies against the endothelial cells. Intravenous immunoglobulin IVIG is an effective treatment in preventing the occurrence of coronary artery abnormalities in KD. Several mechanisms may explain the anti-inflammatory effects of IVIG in this disease. They include modification of the cytokine balance, and alteration on both the differentiation and the function of monocytes/macrophages, neutrophils and lymphocytes.

Natural Autoantibodies as Tools to Predict the Outcome of Immune Response

Natural autoantibodies (NAbs), produced by B‐1 B‐cells, are directed against autoantigens and pathogens. NAbs can capture and present antigen to T helper cells thereby initiating adaptive immunities. It is proposed that screening for NAbs against pathogens will predict the strength of an antigen‐induced immune response and could be used as a tool for vaccine development.

Normal immunoglobulin G protects against experimental allergic encephalomyelitis by inducing transferable T cell unresponsiveness to myelin basic protein.

Normal human IgG for intravenous use (IVIg), administered intraperitoneally, protected Lewis rats against experimental allergic encephalomyelitis (EAE) induced by immunization with myelin basic protein (MBP). We demonstrate that protection was associated with an acquired unresponsiveness of lymphocytes to MBP and a decreased ability of the cells to produce IL‐2, IFN‐γ and TNF‐α and, to a lesser degree, IL‐ 4 and IL‐10, in the presence of the antigen. Lymph node (LN) cells of protected rats failed to passively transfer EAE to naive syngeneic animals. Our observations indicate that, rather than inducing selective immune deviation, IVIg induces preferential MBP unresponsiveness of Th1 cells. Whereas LN and splenic cells of IVIg‐treated rats did not proliferate nor secrete IL‐2 in the presence of the antigen, proliferation was restored by adding exogeneous recombinant IL‐2. In contrast, LN cells of IVIg‐treated rats proliferated normally and produced IL‐2 in the presence of concanavalin A, indicating the selectivity for MBP of the anergy induced by IVIg when given at the time of immunization with the antigen. Treatment with IVIg also allowed a resistance to the secondary induction of EAE, indicating that IVIg protects from EAE but does not interfere with the processes that eventually lead to resistance to re‐challenge. These data document the immunomodulatory effects of IVIg in T cell‐dependent experimental autoimmune disease and further suggest a role for normal Ig in the selection of functional T cell repertoires.

Mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases

Abstract.Intravenous immunoglobulins (IVIg) exert a broad range of immunoregularoty 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 inmmunity that may be of relevance for the treatment of inflammatory neurological diseases.

Ferrous Ions and Reactive Oxygen Species Increase Antigen-binding and Anti-inflammatory Activities of Immunoglobulin G

Polyspecific antibodies represent a first line of defense against infection and regulate inflammation, properties hypothesized to rely on their ability to interact with multiple antigens. We demonstrated that IgG exposure to pro-oxidative ferrous ions or to reactive oxygen species enhances paratope flexibility and hydrophobicity, leading to expansion of the spectrum of recognized antigens, regulation of cell proliferation, and protection in experimental sepsis. We propose that ferrous ions, released from transferrin and ferritin at sites of inflammation, synergize with reactive oxygen species to modify the immunoglobulins present in the surrounding microenvironment, thus quenching pro-inflammatory signals, while facilitating neutralization of pathogens.