Blast transformation of B cells induced by an alpha-macroglobulin-associated lymphokine produced in crowded lymphoid cell cultures.

Previously we have shown that most B cells require continuous stimulation with a polyclonal B cell activator (PBA) to maintain the turnover of their surface Ig in culture (the lg-turnover assay). Based on this assay, we have demonstrated that rabbit T cells upon close contact with B cells in culture, produce an α2-macroglobulin-associated PBA. Here we show that this factor induces blast transformation of rabbit or mouse B cells and that its activity can be blocked by aprotinin, a protease inhibitor. Rabbit lymph node cells were cultured at a concentration of 106 cells/ml in a serum-free medium under crowded conditions to promote close cell contacts and under spread conditions to minimize cell contacts. The culture medium was replaced daily for 3 days and all supernatant fluids were extensively dialyzed. Whereas all crowded cell supernatants (CCSup) induced 14C-uridine incorporation in rabbit lymph node cell cultures, the spread cell supernatants (SCSup) were inactive. CCSup induced 14C-uridine incorporation in rabbit B but not T cell cultures. B cell blast transformation by the CCSup was not species specific since CCSup also induced blast transformation of nude mouse spleen cells. The CCSup was fractionated on a Bio-Gel P300 column, and the ability to induce blast transformation was recovered exclusively in fraction I, containing the macro-globulins. The material was absorbed with insolubilized goat anti-rabbit IgM (anti-µ) or anti-αM(α1 + α2). The blastogenic activity was present in the fraction absorbed with anti-µ, but not in the fraction absorbed with anti-αM, indicating that the blastogenic activity was associated with the αM. The ability to induce 14C-uridine incorporation by CCSup, fraction I, or fraction I absorbed with anti-IgM antibody was blocked by aprotinin. However, aprotinin had no effect on the blast transformation induced by LPS or DS. We concluded that the «M present in rabbit lymphoid cell cultures maintained in close contact is responsible for the polyclonal stimulation of B cells and that this stimulation is due to an αM-associated lymphokine, which is presumably a proteinase.