ABSTRACT
Factor VIII (FVIII) inhibitor antibodies are produced in a proportion of hemophilia A patients. Development of anti-FVIII inhibitor antibodies is a T cell-dependent response, mediated by FVIII specific CD4(+) T cells. This study was performed to investigate the contribution of T helper (Th) cell-mediated cytokine response in inhibitor production. Peripheral blood mononuclear cells (PBMCs) were obtained from hemophilia A patients with (n = 14) or without inhibitor (n = 14) and from normal individuals (n = 14). Following stimulation of PBMCs with rFVIII and phytohemagglutinin (PHA) mitogen, the secreted cytokines, interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and transforming growth factor-beta1 (TGF-beta1), in culture supernatant and the proliferative response were assessed using sandwich ELISA and (3)H-thymidine incorporation, respectively. No significant proliferative response to FVIII was observed, whereas PHA induced a strong response in all groups. No cytokine secretion was observed in response to FVIII stimulation. Although PHA induced IL-10, TGF-beta1 and IFN-gamma secretion in all groups, the level of IFN-gamma was significantly lower in hemophilia A patients than in normal individuals (p < 0.0001). The levels of TGF-beta1 and IL-10 were similarly higher in patients compared with normal subjects, but the difference was not statistically significant. Lack of FVIII-induced proliferative response and cytokine production together with reduced secretion of PHA-induced IFN-gamma in both groups of patients suggest involvement of nonspecific immunosuppression possibly due to hepatitis C virus (HCV) infection observed in the majority of patients.
Subject(s)
Cytokines/biosynthesis , Factor VIII/immunology , Hemophilia A/metabolism , Isoantibodies/biosynthesis , Isoantibodies/blood , Adolescent , Adult , Animals , Cells, Cultured , Child , Cytokines/metabolism , Factor VIII/antagonists & inhibitors , Factor VIII/physiology , Female , Hemophilia A/blood , Hemophilia A/immunology , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Male , Mice , Middle Aged , Mitogens/pharmacology , Phytohemagglutinins/pharmacologyABSTRACT
Factor VIII (FVIII) inhibitor antibodies are produced against functional epitopes of FVIII in about 30% of severe hemophilia A patients leading to inhibition of its procoagulant activity. The Bethesda assay, the most commonly used method to measure FVIII inhibitors, based on inhibition of coagulant activity of FVIII, is neither able to detect noninhibitory antibodies nor their isotype. In this study we employed an indirect enzyme-linked immunosorbent assay (ELISA) to measure dif ferent isotypes and IgG subclasses of anti-FVIII anti body in the plasma of hemophiliacs (with and without inhibitor) and normal individuals using recombinant FVIII-coated microtiter plates. The results showed a predominance of IgG and IgG4, though IgA was slightly elevated in a few inhibitor-positive patients and IgM was hardly detectable. A highly significant correlation was found between the Bethesda titer and the optical density values of total Ig, IgG and IgG4 anti-FVIII antibodies obtained by ELISA (p<0.0001). These findings suggest a restricted specificity of anti-FVIII response in hemophiliacs towards functional epitopes of the molecule. Furthermore, high specificity and reasonable sensitivity of the ELISA, together with other technical advantages, suggest this method as a suitable supplementary technique for rapid large-scale screening of inhibitor-positive samples, though ELISA-negative samples need to be rechecked by the Bethesda assay to identify patients with a low inhibitor titer.
