A novel cellular interaction involving antigen-pulsed macrophage and antigen-specific B-lymphocytes

Extensive documentation shows that macrophage efficiently present antigen to CD 4(+) T-cells in conjunction with the MHC II molecule. Previously, a novel fluorescent probe, FITC-BSA, was developed to analyze intracellular antigen processing and presentation pathways within viable peritoneal murine macrophage. The studies revealed fluorescein's accessibility to antibody binding when associated with peptides bound within the MHC II cleft. To determine if MHC II-fluoresceinated-peptide complexes on the surface of macrophage were also sufficient to stimulate antigen-specific B-cells, nylon wool-purified splenic B-cells from FITC-KLH injected BALB/c mice (H-2(d)) were co-cultured with antigen-pulsed macrophage. B-cell stimulation and antibody production was observed in the presence of FITC-BSA-pulsed macrophage, whereas, macrophage incubated in the presence of unlabeled BSA were not stimulated. Compared with control cells, similar levels of stimulation were detected following depletion of Thy 1.2(+) cells from nylon wool-based spleen cell preparations. Stimulation was inhibited upon preincubation with anti-fluorescein IgG antibodies. Stimulation was not measurable using B-cells derived from the naive mice. The interaction was inhibited upon addition of MHC II specific antibodies and leupeptin, a microbial product that inhibits MHC II-peptide complex formation. Importantly, antibody production was not observed in the presence of antigen-pulsed macrophage from H-2(b) mice. Moreover, B-cell stimulation via this pathway was dependent upon antigen concentration as well as the cell to cell ratio.