The goal of the present study is to analyze the mechanisms through which signals produced by soluble mediators of the immune response at the cell surface are transduced to produce the intracellular changes that ultimately lead to B cell proliferation. This will be approached by studying a novel, 2 signal pathway for stimulating B cells. In this pathway, DNA synthesis is induced by cytochalasin in B cells activated by treatment with anti-immunoglobulin antibody (anti-Ig) or by treatment with calcium ionophore. Cytochalasin appears to circumvent the need for, or replace, a second signal for B cell proliferation such as that provided by B cell growth factor, with the implication that actin or proteins associated with it may be involved in generating or transducing growth promoting signals for B cells. These issues will be addressed by examining several aspects of cytochalasin mediated responses. 1) What are the characteristics of the cytochalasin-responsive B cell? The phenotype of the responsive cell will be defined in terms of size, sIg, and responsiveness to various anti-Ig reagents. Responses to specific antigen plus cytochalasin will be assessed. Hybridomas will be produced from B cells stimulated with anti-Ig plus cytochalasin to determine whether primary or secondary cells are affected. 2) What does anti-Ig do that activates, or prepares, B cells to respond to cytochalasin? The association between activation for responsiveness to cytochalasin, G-0 to G-1 transition, and Ia expression will be studied. The association between activation and morphological changes in actin filament structure will be determined. The calmodulin dependence of activation will be examined using calmodulin inhibitors. 3) What does cytochalasin do that delivers a stimulatory signal to B cells? Actin binding proteins will be isolated and studied by polyacrylamide gel electrophoresis. Stimulated changes in phosphorylation and rate of synthesis will be assessed with appropriate radioisotopes. Most helpful will be comparison between cytochalasin responsive B cells and cell populations that do not respond to cytochalasin (xid B cells and T cells) but do respond to phorbol ester. Elucidation of the mechanisms underlying normal B cell proliferation may increase understanding of, and suggest means of influencing, diseases associated with unregulated clonal expansion, such as autoimmune states and neoplastic dyscrasias of B cells.
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