Macrophages and BCR ligation costimulate B-cell proliferation via soluble factor(s). (A) Macrophage-conditioned culture medium strongly enhances BCR-stimulated B-cell proliferation. Serial 2-fold dilutions of 7-day culture supernatants from macrophages (▪,□) or M-CSF—containing control supernatants (•,○) were cultured with B cells in either the presence (▪,•) or absence (□,○) of goat anti-human IgM F(ab′)₂ fragments for 72 hours. [³H]-thymidine incorporation was quantified as described in Figure 1A. Incubations were performed in triplicate, and results are presented as the mean ± SD of each triplicate from a representative experiment. The mean fold maximal increase in BCR-induced B-cell proliferation by macrophage-conditioned culture medium was 3.8 ± 1.4 from 4 independent experiments. (B) Enhancement of BCR-induced B-cell proliferation by macrophages does not require direct cell-cell contact. Macrophages, BAFF, or APRIL in the upper compartment were cultured with B cells in the presence of goat anti-human IgM F(ab′)₂ fragments in the lower chamber of a 24-well Transwell culture plate for 72 hours. For comparative purposes, macrophages were also cocultured in direct contact with B cells in a standard 24-well tissue culture plate under identical conditions. Cells were pulsed for the final 16 to 24 hours with [³H]-thymidine. [³H]-thymidine incorporation was quantified as described in Figure 1A. Incubations were performed in triplicate, and data are presented as the mean ± SD from 1 of 2 similar experiments. Similar results were also observed using mono-DCs. No statistical difference (P < .05) was observed between the control and Transwell groups for each of the 3 stimulated conditions (anti-IgM + BAFF, anti-IgM + APRIL, anti-IgM + macrophages).