Oestrogen receptor specificity in oestradiol-mediated effects on B lymphopoiesis and immunoglobulin production in male mice

Abstract

Oestrogen treatment down-regulates B lymphopoiesis in the bone marrow of mice. Meanwhile it up-regulates immunoglobulin production. To understand better the oestrogen action on bone marrow male mice lacking oestrogen receptor alpha (ERalpha; ERKO mice), lacking ERbeta (BERKO mice), lacking both receptors (DERKO mice) or wild-type (wt) littermates were castrated and treated for 2.5 weeks with 30 microg/kg 17beta-oestradiol (E2) or vehicle oil as controls. The B lymphopoiesis in the bone marrow was examined by flow cytometry and mature B-cell function was studied using an ELISPOT assay enumerating the B cells in bone marrow and spleen that were actively producing immunoglobulins. In wt mice the frequency of B-lymphopoietic (B220+) cells in the bone marrow decreased from 15% to 5% upon E2 treatment. In ERKO and BERKO mice significant reduction was seen but not of the same magnitude. In DERKO mice no reduction of B lymphopoiesis was seen. In addition, our results show that E2 mediated reduction of different steps in B lymphopoiesis require only ERalpha or both receptors. In wt and BERKO mice E2 treatment resulted in significantly increased levels of B cells actively producing immunoglobulin, while in ERKO and DERKO mice no such change was seen. Similar results were found in both bone marrow and spleen. In conclusion our results clearly show that both ERalpha and ERbeta are required for complete down-regulation of B lymphopoiesis while only ERalpha is needed to up-regulate immunoglobulin production in both bone marrow and spleen.

Figure 1

Both ERα and ERβ are needed to achieve a full inhibitory effect on B lymphopoiesis after oestrogen treatment. The figure shows the percentage of B220⁺ cells in bone marrow of castrated mice of all ER genotypes treated 5 days/week for 2·5 weeks with 30 μg E₂/kg or olive oil as controls. A two-way anova reveals that both ERα and ERβ contribute to achieve full inhibitory effect. ERα: P < 0·001, ERβ: P < 0·001. Bars represent mean ± SEM. *P < 0·05, **P < 0·01 versus controls (non-parametric Mann–Whitney U-test).

Figure 2

E₂ treatment changes the phenotype pattern of B220⁺ cells in bone marrow. The figure shows representative FACS plots from bone marrow of castrated mice of all ER genotypes treated 5 days/week for 2·5 weeks with 30 μg E₂/kg or olive oil as controls. Means and SEM for the B lymphopoietic fractions 1–3 are shown in Table 2 and means and SEM for the activated immunoglobulin-switched B cells (fraction 4) in Table 3.

Figure 3

ERα but not ERβ is required to achieve an increased frequency of B cells actively producing immunoglobulins. The figure shows the frequency of B220⁺ cells actively producing immunoglobulin in bone marrow(a)and spleen (b)of castrated mice of all ER genotypes treated 5 days/week for 2·5 weeks with 30 μg E₂/kg or olive oil as controls. Data are shown as immunoglobulin SFC/10³ B220⁺ cells. A two-way anova reveals that ERα, but not ERβ, is required to achieve full effect. ERα: P < 0·001 (a); P < 0·05 (b); ERβ: not significant. Bars represent mean ± SEM for the summation of IgM, IgG and IgA. *P < 0·05, **P < 0·01 versus controls (non-parametric Mann–Whitney U-test).