Glucocorticoid Effects on Mitogen-Stimulated Isolated Murine B-Lymphocytes*

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Abstract
Glucocorticoid effects on lipopolysaccharide (LPS)-stimulated isolated B cells were compared to effects on B cells in mixed spleen cell cultures. Our previous studies had indicated that LPS-stimulated B cells in mixed spleen cell cultures that were differentiating to plaque-forming cells were less sensitive than stimulated blast-transforming B cell populations in general. In this study, the sensitivity of isolated B cells undergoing proliferation, as measured by [3H]thymidine ([3H] TdR) incorporation, was comparable to that observed for B cells in mixed spleen cell cultures. The effect of 10-7 M triamcinolone acetonide (TA), added with LPS, on cells differentiating to immunoglobulin secretion was markedly different in isolated B cells (80% inhibition) when compared to B cells in the presence of other spleen cells (45% inhibition). Viability of the different cultured populations in the presence of TA was comparable at all time points measured; thus, differential viability cannot readily explain the differences observed. Depletion experiments in which either macrophages and/or T cells were removed from the spleen cells indicated that the removal of macrophages markedly increased the sensitivity of the precursor plaque-forming cells to the glucocorticoid. Glucocorticoid effects on proliferating cells were also studied with time. The addition of 10-7 M TA was most effective in blocking [3H]TdR incorporation in LPS-stimulated cells when added at the initiation of mitogen stimulation. With addition of TA at later times, there was a progressive decrease in steroid sensitivity. Proliferating cells appear to become more resistant to glucocorticoids as early as 6–12 h after LPS stimulation. With TA treatment at the onset of LPS stimulation, [3H]TdR incorporation was inhibited 80% at 36 h, a time when the rate of thymidine incorporation in untreated cells was nearly maximal. However, TA addition at 36 h caused only a 35% reduction in thymidine incorporation during the subsequent 36-h period. Thus, with proliferation and differentiation, B cells become less sensitive to the effects of glucocorticoids.