Identification of a Lymphocyte Mitogenic Factor Produced by Actinobacillus actinomycetemcomitans

ABSTRACT

Actinobacillus actinomycetemcomitans, a gram-negative, capnophiTic bacterium, is associated with several human diseases including periodontal disease. Products of A. actinomycetemcomitans exert immunomodulatory effects on various lymphoid populations, some of which may be implicated in the pathogenesis of periodontitis. It has been recently suggested that some of periodontopathic bacterial products might possess superantigenic (SAg) activities. In order to examine SAg activity of A. actinomycetemcomitans, we tried to purify immunomodulating factor (IMF) which can induce proliferation of mouse splenocytes and human PBMC. IMF fraction was obtained from the culture supernatant of A. actinomycetemcomitans by alcohol precipitation, ultrafiltration, size exclusion chromatography, and dye ligand affinity chromatography which has been widely used for the puri5cation of known SAgs. SDS-PAGE analysis showed that the factor migrated to a molecular mass of 40 kDa. The concentration of IMF which elicited maximal proliferative response of mouse splenocytes was ranged 1-10 ug/ml of protein on day 3 in culture. Human PBMC gave a similar response profile to IMF, but their maximal response was obtained by lower concentraion of IMF on day 2 in culture. This activity of IMF was heat and proteinase K sensitive and was not blocked by co-incubation with polymyxin B, a ligand for the lipid A region of lipopolysaccharide. T cell-enriched fraction of mouse splenocytes obtained by nylon wool column lost the response to IMF. Even though mitomycin C-treated antigen presenting cells were added to T cell-enriched fraction, the response to IMF was feeble as compared to unfractionated cells. Splenocytes depleted of T cells by anti-Thy 1.2 and complement also did not respond to IMF. These findings demonstrated that T cells are responsible for a minor proportion of the observed proliferation induced by IMF and the help of these cells are essential to the most of the proliferating cells which may be B cells. This observation was confirmed by flow cytometric analysis of responding lymphocyte subpopulations. These results indicate that IMF of A. actinomycetemcomitans does not act in a manner consistent with known SAgs but is more relevant to the explanation of pathologic findings of periodontal lesions.