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An in vitro murine model of a penicillin specific IgE anamnestic response
M C Smith1, C Heusser, K Wagner
1Department of Pathology, State University of New York Health Science Center, Brooklyn 11203.
A new murine model system was developed to study hapten specific IgE anamnestic antibody forming cell (AFC) responses induced in vitro. BALB/c mice injected intraperitoneally with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on day 0 and 21 were killed on day 42. Spleen cells (4 X 10(7] were cultured for 0-8 days in the presence of BPO-KLH (0.25-2500 ng/ml), after which the numbers of BPO specific IgE AFC were enumerated in an ELISPOT assay at 37 degrees C using BPO-BSA coated plates. Peak BPO specific IgE anamnestic AFC responses (approximately 40 AFC/10(7) cultured cells) occurred when cells were cultured with 100 ng/ml BPO-KLH for 5 days; peak BPO specific IgG1 and IgA responses (approximately 3000 and approximately 280 AFC/10(7) cultured cells) were detected one day earlier with the same antigen concentration. No ELISPOTs of any isotype were detected if: cells were cultured in the absence of specific antigen; cells from alum treated or unsensitized mice were used; assay wells were coated with BSA alone; isotype specific antibodies were omitted; or assays were performed in the presence of cycloheximide or at 4 degrees C. Cellular interactions in IgE anamnestic AFC responses were studied using the newly developed model system. We found that these responses were partially T cell independent, and that spleens from sensitized and unsensitized adult mice, as well as neonatal mice, contained cells which suppressed these responses.
A new murine model system was developed to study hapten specific IgE anamnestic antibody forming cell (AFC) responses induced in vitro. BALB/c mice injected intraperitoneally with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on day 0 and 21 were killed on day 42. Spleen cells (4 X 10(7] were cultured for 0-8 days in the presence of BPO-KLH (0.25-2500 ng/ml), after which the numbers of BPO specific IgE AFC were enumerated in an ELISPOT assay at 37 degrees C using BPO-BSA coated plates. Peak BPO specific IgE anamnestic AFC responses (approximately 40 AFC/10(7) cultured cells) occurred when cells were cultured with 100 ng/ml BPO-KLH for 5 days; peak BPO specific IgG1 and IgA responses (approximately 3000 and approximately 280 AFC/10(7) cultured cells) were detected one day earlier with the same antigen concentration. No ELISPOTs of any isotype were detected if: cells were cultured in the absence of specific antigen; cells from alum treated or unsensitized mice were used; assay wells were coated with BSA alone; isotype specific antibodies were omitted; or assays were performed in the presence of cycloheximide or at 4 degrees C. Cellular interactions in IgE anamnestic AFC responses were studied using the newly developed model system. We found that these responses were partially T cell independent, and that spleens from sensitized and unsensitized adult mice, as well as neonatal mice, contained cells which suppressed these responses.