In-vitro-derived Bone Marrow Macrophages

Cultures of bone marrow stem cells, grown in the presence of L-cell-conditioned medium, were harvested on successive days to determine the expression of Ia antigens and the acquisition of Ir4 gene-regulated antigen presentation. I-A subregion antigen expression was detected by an indirect radiobinding assay (RBA) as early as day 3 and reached maximal binding at day 7, before declining with additional time in culture. Indirect immunofluorescence (IIF) demonstrated 20% Ia+ cells on day 3 of culture and peaked at 60% on day 7 before declining with continued incubation. A mixed lymphocyte reaction (MLR) across an I region difference was used to assess the kinetics of bone-marrow-derived macrophage (BMDM) Ia expression. Maximal stimulation occurred with BMDM stimulator cells obtained from 5–9-day cultures. To investigate the acquisition of Ir gene function, BMDM harvested after various days in culture were used to reconstitute the T cell proliferative response to poly Glu60Ala30Tyr10 (GAT). The ability of BMDM to present GAT was detected after day 5, increased to maximal levels on day 7, and then declined with weak proliferative responses obtained using day 12 BMDM. The presentation of GAT by BMDM was inhibited by monoclonal anti-Ia.17 antibody. Thus, the acquisition of Ir gene function by BADM was found to parallel the expression of Ia molecules. Additional experiments were performed to determine whether treatment of BMDM at day 7 with lymphokines or β-interferon could extend Ia antigen expression. Whereas treatment of day 7 BMDM cultures with Con-A-stimulated rat splenocyte supernatants extended and augmented Ia expression for an additional three days, β-interferon treatment did not result in augmentation or extension of Ia antigen expression.