DEVELOPMENT OF A NOVEL IN-VITRO COCULTURE SYSTEM FOR STUDYING HOST RESPONSE TO NATIVE BACTERIAL-ANTIGENS

We have developed a novel co-culture system in which murine splenocyte s are cultured with live bacteria in the presence of a bacteriostatic antibiotic. Superantigens, like staphylococcal enterotoxin B (SEB) are important factors in bacterial pathogenicity. Research has shown that superantigens affect numerous immune cell types, either directly or i ndirectly, yet their involvement in pathogenic mechanisms remains poor ly defined. In these studies, we utilize the co-culture system to stud y how superantigen pretreatment affects interferon-gamma (IFN-gamma) p roduction by splenocytes co-cultured with gram-positive bacteria. Stre ptococcus mutans, S. sanguis and Bacillus subtilis were tested for sus ceptibility to a panel of antibiotics. Spectinomycin was found to main tain a bacteriostatic state of approximately 10(5) bacteria ml(-1) at optimal concentrations for each bacterial strain. Co-culturing splenoc ytes with bacteria did not affect splenocyte viability and cultured sp lenocytes responded to mitogenic stimulation as expected. Two days aft er SEE pretreatment, isolated splenocytes cultured with either Strepto coccus species produced 10-15 times more IFN-gamma than splenocytes fr om sham-injected controls; however, no differences in CD4(+) or CD8(+) T cell populations appeared in cultures with or without bacteria. Spl enocytes isolated four days after SEE treatment did not produce signif icant amounts of IFN-gamma in co-culture. Co-cultures containing live bacteria produced four times more IFN-gamma than cultures containing h eat-killed bacteria. Splenocytes depleted of natural killer (NK) cells prior to SEE treatment produced 25% less IFN-gamma after 20 h co-cult uring with S. mutans. T lymphocytes were identified to be the major pr oducer of IFN-gamma at this time point by intracellular cytokine stain ing. Apparently SEE exposure primes a response to live bacteria and th e response is evident two days after initial exposure. The in vitro co -culture system allows us to observe host responses to bacteria in the context of the multicellular interdependent immune response. With thi s assay we can more closely 'mimic' in vivo events, particularly immun e cell interactions in microfloral environments, to study how the path ogenic effects of superantigens alter this response. (C) 1998 Elsevier Science B.V.