Regulation of B-lymphocyte Clonal Proliferation by Stimulatory and Inhibitory

The macrophage has been implicated in the elaboration of a spectrum of molecules which alter or modulate the functions of hemopoietic and lymphoid cells. These include colony-stimulating factor (CSF),' required for the renewal and clonal proliferation of granulocyte-macrophage stem cells in semisolid culture (1), lymphocyte-activating factor which potentiates the mitogenic response of T lymphocytes to lectin and histocompatibility antigen (2-4), as well as factors which increase the helper function of T lymphocytes (5, 6) and the promotion of soluble mediator production by lymphocytes (7-9). The role of the macrophage in the regulation of humoral immune responses is however, less well understood. In contrast to the reported potentiation by cell-free macrophage supernates of B-cell differentiation into antibody-secreting cells in response to sheep erythrocytes (SRBC) and hapten-protein conjugates (10, 11), mitogeninduced B-lymphocyte proliferative responses are suppressed in the presence of macrophages (12-15). We have elected to investigate the immunoregulatory role of the macrophage in B-cell activation by utilizing a two-layer semisolid culture system which prevents macrophage-lymphocyte, as well as lymphocyte-lymphocyte contact, and in which only diffusible interactions may occur. In this system, the capacity of a B lymphocyte to undergo focal proliferation and generate a clone or colony is taken as the immunological end point of B-cell activation, a process dependent upon 2-mercaptoethanol (16) and mitogens native to laboratory-grade agar (17). As previously reported, this functional population of clonable B lymphocytes, the majority of which bear Fc receptors, IgM, and la antigens on their surface (18, 19), are heterogeneous with respect to size and density and are widely distributed in lymphoid tissues (20, 21). We report here that B-cell activation under these conditions is dependent upon diffusible factor(s) elaborated by macrophages. In addition, the ability of