Effects of heme on plasma cell differentiation. (A) The expression of the Blimp-1-EGFP reporter gene determined by a FACS analysis. B220-positive cells from WT (Bach2^+/+) Blimp-1-EGFP mice and Bach2-deficient Blimp-1-EGFP (Bach2^−/−) mice were stimulated with 20 μg/mL LPS in the presence or absence of 20μM heme on day 2. Each gate shows the percentage of EGFP-positive cells. (B) The ratio of EGFP-positive cells cultured with LPS + heme or LPS alone in Bach2^+/+ (left) or Bach2^−/− (right) cells. The data are presented as the means ± SD of triplicate determinations. The statistical analyses were performed by the use of the Student t test. (C) The IgM and IgG₃ secretion from splenic B220-positive B cells was analyzed by ELISA. B220-positive B cells were cultured with 20 μg/mL LPS alone (open bars) or LPS and 20 μM heme (filled bars) for 7 days, and the secreted immunoglobulin levels were measured. The data are presented as the means ± SD of triplicate determinations. Groups of 3-5 mice were used for the statistical analysis. P values (*P < .05) were calculated by use of the Student t test. (D) The differentiation of IgM-producing plasma cells in mouse splenic B220-positive B cells stimulated with 20 μg/mL LPS in the presence or absence of 20μM heme for 2 days. IgM-producing cells were detected by ELISPOT assay. (E) The percentage of IgM-secreting cells within the B-cell population. Each bar indicates the average percentage of IgM-secreting cells in 8 × 10² B cells (open bars: stimulation with LPS alone, filled bars: stimulation with LPS and heme). The data are presented as the means ± SD of triplicate determinations. Groups of 3-5 mice were used for the statistical analysis. *P values were calculated with use of the Student t test. (F-G) The percentages of cells expressing CD138 (F) and relative expression levels of Blimp-1 mRNA (G) in splenic B cells from in vitro culture treated with LPS for 2 days. Heme and deferroxamine (μM) were added as indicated. Mean of 2 independent experiments are shown.