Biochemical separation of a human B cell mitogenic factor.
Recent studies have established the ability of human B lymphocytes to undergo G1-phase cell cycle progression and subsequent DNA synthesis upon exposure to factor(s) present in media conditioned by lectin-stimulated mononuclear cells. Procedures for the isolation of such a cytokine have been the focus of the present investigation. Conditioned medium from cells stimulated by lectin for 72 hr was fractionated by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. During the isolation procedure the proliferation-stimulating activity of the column fractions was assayed concurrently on purified human T cells, purified human B cells, and murine thymocytes. T cell and B cell stimulatory factors present in the initial conditioned medium were found to copurify during ammonium sulfate precipitation, DEAE-Sephadex chromatography, and Bio-Gel P-30 gel filtration. However, partial separation of these two activities was achieved after Bio-Gel P-100 gel filtration. Analytic polyacrylamide gel electrophoresis of radiolabeled Bio-Gel P-100 column fractions demonstrated a distinct protein band of 14,000-15,000 daltons in those column fractions predominantly supporting T cell growth and a distinct protein band of 12,000-13,000 daltons for those fractions predominantly supporting B cell growth. The fractions associated with B cell mitogenic activity induced B cell S-phase entry in a proportion of B lymphocytes in the absence of any detectable IgM secretion.
D001402:B-Lymphocytes / Q000276:immunology*; D002453:Cell Cycle; D002469:Cell Separation; D002478:Cells, Cultured; D004261:DNA Replication; D006133:Growth Substances / Q000302:isolation & purification*; D006801:Humans; D007075:Immunoglobulin M / Q000235:genetics; D015847:Interleukin-4; D007700:Kinetics; D008213:Lymphocyte Activation
D006133:Growth Substances; D007075:Immunoglobulin M; D015847:Interleukin-4