Identification, purification and characterization of a receptor for dengue virus-induced macrophage cytotoxin (CF2) from murine T cells

Abstract

Dengue type-2 virus infection in mice induces a subpopulation of T lymphocytes to produce a cytokine cytotoxic factor, which induces macrophages (Mφ) to produce a biologically active cytotoxic cytokine, the Mφ cytotoxin (CF2). Previously we have identified the presence of intermediate-affinity receptors for CF2 on mouse peritoneal Mφ. The present study was undertaken to identify the CF2-receptors (CF2-R) on murine T cells followed by their purification and characterization. Receptor binding assay and Scatchard analysis revealed single, high-affinity (1.0309 nM) receptors for CF2 on T cells (22 000 receptors per cell). The binding of [ 125I]CF2 on murine T cells was saturable and specific. Furthermore, CF2-R was purified from normal mouse T cell plasma membrane by affinity chromatography followed by reversed-phase high-pressure liquid chromatography. The presence of CF2-R was confirmed by indirect dot-blot assay and its binding with [ 125I]CF2. The purified CF2-R is a 90–95-kDa protein as characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analysis. The chemical crosslinking of [ 125I]CF2 and its receptor complex showed a product of 100–110 kDa on different subpopulations of murine T cells. The pretreatment of target cells with anti-CF2-R antisera inhibited the cytotoxic activity of CF2 in a dose-dependent manner and thus confirmed the biological significance of CF2-R. Moreover, the presence of CF2-R was also identified on normal human peripheral blood mononuclear cells and T and B cells by crosslinking with [ 125I]CF2, thus revealing the possible role of CF2 and CF2-R in the immunopathogenesis of dengue virus disease.