While the regulation of erythroid burst proliferation by growth promoting factors in culture media conditioned by human lymphocytes is well documented, our understanding of the mechanisms by which such factors operate is limited. We have recently shown that vesicles spontaneously shed from lymphocytes as well as purified lymphocyte plasma membranes contain significant amounts of burst promoting activity (BPA) and have provided preliminary results suggesting that this activity resides in surface components of lymphocyte membranes. The focus of this proposal is the following: (1) Purification of burst promoting activity (BPA). We propose to purify proteins or glycoproteins containing BPA from plasma membranes of human lymphocytes, or from vesicles shed spontaneously from lymphocytes in culture. We will treat lymphocyte membranes with nonionic detergents to extract integral membrane proteins as well as with high or low salt media to extract peripheral or adsorbed proteins. These extracts will be assayed in culture for BPA, and where appropriate further purified by a variety of techniques including affinity chromatography on lectin-affinity columns, sucrose gradient centrifugation and column chromatography. (2) Cross-reactivity of membrane-associated and soluble BPA. We will test the unifying hypothesis that soluble forms of BPA are derived from membrane associated BPA by shedding or proteolytic processing. Antibodies to lymphocyte plasma membranes, LCM derived vesicles or partially purified membrane BPA will be used to adsorb or neutralize soluble BPA contained in high speed LCM supernatants. The demonstration of cross reactivity would greatly advance our understanding of the source of BPA not only frm lymphocytes but possibly from other cells as well. (3) Identification of cell source and target cells of BPA. We propose to identify the cell(s) of origin of membrane-associated BPA by testing plasma membranes or LCM prepared from lymphocytes selectively enriched in particular subset(s) by a variety of cell adsorption techniques. Using similar methodology, we will attempt to identify the target cell(s) of lymphocyte membrane and vesicle-associated BPA by testing them in cultures of bone marrow and peripheral blood mononuclear cells which are selectively depleted of specific cell types.
