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Identification, purification and characterization of a receptor for dengue virus-induced macrophage cytotoxin (CF2)
Pranay Deep Khare1, Meenakshi Khare, Ritu Tandon
1Postgraduate Department of Microbiology, K.G. Medical College, 226-003 Lucknow, India. khare.pranay@mayo.edu
Abstract:
Dengue type-2 virus infection in mice induces a subpopulation of T lymphocytes to produce a cytokine cytotoxic factor, which induces macrophages (Mphi) to produce a biologically active cytotoxic cytokine, the Mphi cytotoxin (CF2). Previously we have identified the presence of intermediate-affinity receptors for CF2 on mouse peritoneal Mphi. 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 (22000 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.
Insights
Researchers identified high-affinity receptors for macrophage cytotoxin (CF2) on T cells, crucial for dengue virus infection. These CF2 receptors (CF2-R) were purified and characterized, showing their role in T cell cytotoxicity and potential involvement in dengue pathogenesis.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Dengue virus type 2 infection stimulates T cells to produce a cytotoxic factor.
- This factor induces macrophages to produce macrophage cytotoxin (CF2).
- Previous studies identified intermediate-affinity CF2 receptors on macrophages.
Purpose of the Study:
- To identify and characterize CF2 receptors (CF2-R) on murine T cells.
- To purify and analyze the biochemical properties of T cell CF2-R.
- To investigate the biological significance and cross-species presence of CF2-R.
Main Methods:
- Receptor binding assays and Scatchard analysis to determine receptor affinity and density.
- Affinity chromatography and reversed-phase high-pressure liquid chromatography for CF2-R purification.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and chemical crosslinking for protein characterization.
- Inhibition assays using anti-CF2-R antisera to confirm biological relevance.
Main Results:
- High-affinity CF2 receptors (1.0309 nM) were identified on T cells, with approximately 22,000 receptors per cell.
- CF2-R was purified and characterized as a 90-95 kDa protein.
- A 100-110 kDa complex was observed upon chemical crosslinking of CF2 and its receptor.
- Inhibition of cytotoxic activity by anti-CF2-R antisera confirmed CF2-R's biological significance.
- CF2-R was also detected on human peripheral blood mononuclear cells, T cells, and B cells.
Conclusions:
- Murine T cells possess high-affinity receptors for macrophage cytotoxin (CF2).
- The purified CF2-R is a 90-95 kDa protein, crucial for CF2-mediated cytotoxicity.
- The presence of CF2-R on human cells suggests its role in the immunopathogenesis of dengue virus disease.
