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

Extensive documentation shows that macrophage efficiently present antigen t o CD 4(+) T-cells in conjunction with the MHC II molecule. Previously, a no vel fluorescent probe, FITC-BSA, was developed to analyze intracellular ant igen processing and presentation pathways within viable peritoneal murine m acrophage. The studies revealed fluorescein's accessibility to antibody bin ding when associated with peptides bound within the MHC II cleft. To determ ine if MHC II-fluoresceinated-peptide complexes on the surface of macrophag e were also sufficient to stimulate antigen-specific B-cells, nylon wool-pu rified 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 pr oduction was observed in the presence of FITC-BSA-pulsed macrophage, wherea s, macrophage incubated in the presence of unlabeled BSA were not stimulate d. 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-fluore scein 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 observ ed in the presence of antigen-pulsed macrophage from H-2(b) mice. Moreover, B-cell stimulation via this pathway was dependent upon antigen concentrati on as well as the cell to cell ratio. (C) 2000 Elsevier Science Ltd. All ri ghts reserved.