Age and stage dependency of estrogen receptor expression by lymphocyte precursors

Abstract

Sex steroids negatively regulate B lymphopoiesis in adult mice. Paradoxically, lymphocytes arise during fetal life, when estrogen levels are high and maternal lymphopoiesis is suppressed. Here we demonstrate that embryonic B lymphopoiesis was unaffected by estrogen, but sensitive to glucocorticoids. Both fetal and adult precursors contained glucocorticoid receptor transcripts, but only adult precursors expressed estrogen receptor alpha and beta together with the androgen receptor. Fetal hematopoietic cells did not efficiently acquire functional estrogen receptors after transplantation to irradiated adult mice. Sex steroid receptors were also expressed in a stage- and developmental age-dependent fashion in human precursors. A developmental switch in responsiveness of hematopoietic cells to sex steroids may be essential for formation of the immune system.

Figure 1

B lymphopoiesis in FLOC is unaffected by estrogen. Fragments of 15-dpc fetal liver (FL) from C57BL/6J mice were cultured for 6 days in the presence of medium alone, 17β-estradiol (10⁻⁴ M), or dexamethasone (10⁻⁸ M). Flow cytometry was performed on the recovered cells, as well as suspensions of the initial fetal liver with the indicated mAbs. The regions of the contour plots containing newly formed B cells are marked with boxes (percentages are indicated above the boxes). Before culture, fetal liver lacked CD45R⁺ IgM⁺ B cells (Upper Left), but these cells emerged during 6 days of organ culture (Upper Right, 0.16 ± 0.01 × 10⁶ B cells per fragment). In three independent experiments, B cell production was unaffected by inclusion of estrogen (Lower Left, 0.18 ± 0.01 × 10⁶ B cells per fragment), whereas it was always totally suppressed by the synthetic glucocorticoid dexamethasone (Lower Right, <0.01 × 10⁶ B cells per fragment).

Figure 2

Progenitors from fetal liver, but not adult marrow, generate B lineage cells in the presence of estrogen. Bone marrow (BM) cells from 8-week-old C57BL/6J mice and fetal liver (FL) cells from 15 dpc embryos were harvested, enriched for lineage-negative cells, and sorted as Lin⁻ c-kit^Lo cells on the MoFlo. These highly enriched precursors were then cultured for 7 days under stromal cell-free, serum-free conditions in the presence of medium alone, 17β-estradiol (10⁻⁸ M) or dexamethasone (10⁻⁸ M). Recovered cells were evaluated by flow cytometry. Lymphoid progenitors from either bone marrow or fetal liver gave rise to CD19⁺ B lineage cells in stromal-cell-free, serum-free cultures (Top, 10.7 ± 0.26 × 10³ CD19⁺ cells per marrow precursor culture, and 1.84 ± 0.16 × 10³ CD19⁺ cells per fetal precursor culture). In two independent experiments, estrogen blocked B lineage differentiation from adult (Left Middle, 0.17 ± 0.03 × 10³ CD19⁺ cells per marrow precursor culture), but not fetal (Right Middle, 2.26 ± 0.14 × 10³ CD19⁺ cells per fetal precursor culture) progenitors. The synthetic glucocorticoid dexamethasone totally blocked generation of CD19⁺ lymphocytes and induced CD11b/Mac-1⁺ myeloid lineage cells regardless of the source of precursors (Bottom, < 0.07 × 10³ CD19⁺ cells recovered per culture).

Figure 3

Receptors for sex steroids are not acquired by hematopoietic cells until after birth. (A) Lin⁻ c-kit^Hi or Lin⁻ c-kit^Lo cells were sorted from 8-week-old adult mice bone marrow (BM) or 15-dpc fetal liver (FL). Transcripts corresponding to hormone receptors were then examined by RT-PCR. RNAs from uterus for ERs, kidney for AR, and 70Z/3 pre-B cells for GR were used as positive controls (PC). (B) Lin⁻ c-kit^Hi or Lin⁻ c-kit^Lo cells were sorted from bone marrow of 5-day-old (5 D), 3-week-old (3 W), or 18-month-old (18 M) mice. The expression of hormone receptors was examined by RT-PCR. (C) Freshly isolated human bone marrow (BM) and cord blood (CB) cells were sorted into CD34⁺ and CD34⁻ populations. Hormone-receptor expression was examined by RT-PCR. RNA from the MCF7 human breast cancer cell line served as a positive control.

Figure 4

Fetal hematopoietic cells do not express hormone receptors after transfer to adult mice. (A) Purified Lin⁻ c-kit^Hi Sca-1⁺ hematopoietic progenitor cells from 15-dpc fetal liver were transferred into irradiated RAG-1-deficient mice. Four weeks after transfer, bone marrow cells were harvested and sorted into fetal-liver-derived Lin⁻ c-kit^Hi Sca-1⁺ CD45.1⁺ and adult recipient Lin⁻ c-kit^Hi Sca-1⁺ CD45.1⁻ populations. The expression of hormone receptors was then examined by RT-PCR. PC, positive control. (B) We used an identical experimental design to recover spleen cell suspensions from mice transplanted 4 weeks earlier with Lin⁻ c-kit^Hi Sca-1⁺ cells isolated from adult bone marrow or fetal liver, and we examined the cells by flow cytometry. Some of the recipients were hormone-treated, and donor CD45.1⁺ cells were gated as shown for analysis with the indicated antibodies. (Upper) the engraftment and differentiation of donor Lin⁻ c-kit^Hi Sca-1⁺ CD45.1⁺ cells. B cells bearing various levels of IgM and IgD, representing various stages of maturity, were present in recipient spleens regardless of transplant source. (Lower) The remarkable estrogen resistance of fetal liver as compared with adult bone marrow-derived precursors.