A novel immunostimulating factor (ISTF) of Actinobacillus actinomycetemcomitans ATCC 29522 was isolated and characterized as inducing proliferation of mouse B cells and human peripheral blood mononuclear cells. This factor was isolated from the bacterial culture medium and purified by size exclusion chromatography, dye-ligand affinity chromatography, immunoaffinity chromatography using monoclonal antibodies, and preparative electrophoresis. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified ISTF migrated as a single band corresponding to a molecular mass of 13 kDa. ISTF was a proteinaceous material distinct from lipopolysaccharide; it directly induced the proliferation of B lymphocytes but had no effect on the proliferation of T lymphocytes, even in the presence of antigen-presenting cells. A B-lymphocyte-mitogenic activity of ISTF was also shown by flow cytometric analysis of responding cell subpopulations. Immunoblot analysis revealed that ISTF was a component of the outer membranes of bacteria, could exist as a soluble form, and was released by growing and/or lysed bacteria. These results suggest that ISTF produced by A. actinomycetemcomitans may play an important role in immunopathologic changes associated with A. actinomycetemcomitans infections.Actinobacillus actinomycetemcomitans, a nonmotile, gramnegative coccobacillus, is associated with several human diseases including endocarditis, meningitis, osteomyelitis, subcutaneous abscesses, and periodontal diseases (2,8,20,24,30). Although the pathogenic mechanism of A. actinomycetemcomitans is not known, it has been proposed that the impairment of the host immune mechanism by the bacteria-producing virulence factors might contribute to the disease process. Many other studies have described several virulence factors of A. actinomycetemcomitans, which exhibited alterations in immune regulation. Immunosuppressive factor (ISF; 60 kDa) inhibited mitogen-induced T-cell proliferation and immunoglobulin (Ig) production (26,27). Suppressive factor 1 (SF1; 14 kDa) downregulated T-cell proliferation and cytokine production (13). Leukotoxin (115 kDa) inhibited the responsiveness of human peripheral blood mononuclear cells (PBMC) to mitogens and antigens by subverting monocytes (23). Lipopolysaccharide (LPS) suppressed antigen-and mitogen-induced human T-cell proliferation by inducing the release of prostaglandin E2 from activated macrophages (5). As all these factors contributed to the suppression of lymphocyte activation, it has been suggested that products of A. actinomycetemcomitans might possess a lymphocyte-activating substance like a superantigen (1,16,17,32). During studies in our laboratory focusing on an understanding of the immunomodulatory activity of the products from A. actinomycetemcomitans, we became intrigued by an indication that immunosuppressing and immunostimulating activities were present simultaneously in the bacterial preparations. A consistent pattern was observed: A. actinomycetemcomitans homogenates ...