POTENT STIMULATION OF MURINE B-CELLS TO PROLIFERATE AND TO SECRETE IMMUNOGLOBULINS BY A LIPOTEICHOIC ACID-LIKE MOLECULE PRODUCED BY CLOSTRIDIUM-BOTULINUM-C AND CLOSTRIDIUM-BOTULINUM-D

Bacterial products have served as important immunological tools to stu dy lymphocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Sev eral Gram-positive bacteria produce exotoxins that are superantigens f or T cells. In the present study, we demonstrate that the Gram-positiv e bacteria Clostridium botulinum C and D produce a high molecular weig ht mitogen (Cb mitogen) that is a potent activator of murine B lymphoc ytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongl y stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to prolife rate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitog en demonstrated that the purified botulinum toxin was devoid of mitoge nic activity. In contrast, the fractionation of the culture supernatan t of C, botulinum C in an FPLC Superose 12 column indicated that the C b mitogen was present in the void volume of the column (MW greater tha n or equal to 300 kDa) which had no toxigenic activity. However, the f ractions containing molecules of 150 kDa were highly toxic for mice an d had no mitogenic activity. The possibility that LPS was present as a contaminant in the Cb mitogen preparations was excluded because splee n cells from the LPS nonresponder C3H/HeJ mice responded well to the C b mitogen, and the antibiotic polymyxin B, which is an inhibitor of LP S, had no effect on the Cb mitogen activity. However, an anti-lipoteic hoic acid monoclonal antibody (3-1 mAb) inhibited to a great extent th e proliferation of spleen cells induced by the Cb mitogen but had no e ffect on the LPS or concanavalin A stimulation of these cells. Moreove r, the Cb mitogen was specifically adsorbed and eluted from a protein G Sepharose column to which the anti-lipoteichoic acid 3-1 mAb had bee n conjugated. These results support the view that lipoteichoic acid is a selective B cell mitogen.