B lymphopoiesis

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5382-5386, June 1994 Immunology

Pregnancy-related steroids are potential negative regulators of B lymphopoiesis KAY L. MEDINA AND PAUL W. KINCADE Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, OK 73104

Communicated by Max D. Cooper, January 26, 1994 (received for review November 12, 1993)

during pregnancy (11). Estrogen treatment also causes involution of the thymus, and preliminary experiments indicated that the same might be true for B-lymphocyte precursors (10, 12). The aim of the present study was to evaluate the sensitivity of B-lineage cells to elevations of pregnancyrelated steroids. Evidence is presented that some of these hormones may be effective negative regulators of B lymphopoiesis. We describe population changes among B-lineage precursors after hormone treatment that are similar to those in pregnant mice and define particular stages of differentiation that are sensitive to these hormones.

B lymphopoiesis is selectively suppressed in ABSTRACT esig that fluctuatons in systemic normal pregnant mice, hormone levels might influence local events within bone marrow. This has now been tested by sstaind experimental elevation of sex steroids by hormone-containing pellet implants. We found that while numbers of total nucleated cells declined after treatment with estrone, P-estradiol, or estriol, there was preferential suppression of B-lymphocyte lineage precursors. Progesterone pellets had no effect when used alone, but mice exposed to progesterone were sensitive to severallogarithm lower concentrations of estrogen. Changes in subpopulations of B-lymphocyte lineage cells with hormone pellets were similar to those previously recorded in pregnancy. B-lymphocyte lineage precursors in male and female mice were sensitive to these sex hormones. Acute treatment with single injections of water-soluble (-stradiol allowed temporal effects on B-lineage cells to be documented. With this protocol, total numbers of nucleated cells and myeloid progenitor cells remalned unchanged. Interleukin 7-responsive precursors dramatically declined within 1 day of injection, suggesting that estrogen influences that stage in the B-lymphocyte lineage. There was a subsequent sharp drop in small pre-B cells 4 days after this transient elevation in estrogen. These experiments demonstrate that B lymphopoiesis is sensitive to negative regulation by sex steroids. They extend findings made with pregnant animals and parallel previous studies of the thymus. Sex steroids might contribute to control of steady-state lymphopolesis, and fluctuations in their levels could have implications for human disease.

MATERIALS AND METHODS Animals. Balb/c mice of either sex were obtained from our Laboratory Animal Resource Center. They were studied at 2-4 months of age, and controls were age matched. All female

Blood cell formation is carefully controlled through a balance between the availability of stimulatory factors and the action of negative regulators. Interleukin (IL) 7 has recently been shown to be an absolute requirement for expansion of murine B-lymphocyte precursors (1). Other cytokines, such as stem cell factor, IL-li, and IL-6, may stimulate proliferation and differentiation of earlier cells in the series (2). Less information is available for inhibitors, but under certain experimental circumstances, several factors can inhibit particular events in the lineage. These include IL-1 and IL-4, as well as y interferon, transforming growth factor (3, and granulocyte/ macrophage colony stimulating factor (3-9). However, there is little evidence that any of them are important for normal steady-state regulation. We recently found that B lymphopoiesis in the bone marrow of normal mice is markedly suppressed during pregnancy (10). Aside from finding a dramatic change that is not experimentally induced, the observations were remarkable because of the selectivity for cells of this one lineage. It is possible that critical stimulatory factors decline during pregnancy or, alternatively, that production of a lineage-specific negative regulator(s) is increased. Atrophy of B-lineage lymphocytes in bone marrow parallels similar changes long known to occur in the thymus

mice were virgin and housed separately from the males. Thymectomized female mice were from Charles River Breeding Laboratories. Cell Preparation and Colony Assays. Single-cell suspensions were prepared from bone marrow, spleen, and thymus as described (10). Cells recovered from two femurs and two tibias were pooled, and the results were divided by 4 to yield total numbers of cells per bone. Colony Assays. Bone marrow (5 x 104) cells were prepared and suspended in 1 ml of assay medium as described (13). The semisolid agar cloning assay for B-lymphocyte precursors was done with 1 ng of recombinant mouse IL-7 (Upstate Biotechnology, Lake Placid, NY). The granulocyte/ macrophage progenitor assay [colony forming unit (CFU)-c] was done with 25 Al of 10 times concentrated L cellconditioned medium. Immunofluorescent Staining and Analysis. Cells were suspended in staining buffer (phosphate-buffered saline without Ca2+ and Mg2+ with 3% heat-inactivated fetal calf serum and 0.1% sodium azide). Staining was done by incubating cells with antibodies on ice for 15 min followed by washing with 10 vol of staining buffer. Unconjugated antibodies were revealed by a subsequent incubation with the appropriate fluorochrome-conjugated second antibody or, in the case of biotinylated primary antibodies, with streptavidin phycoerythrin (Biomedia, Foster City, CA) or streptavidin Peridinin (chlorophyll protein; Becton Dickinson). Total B-lineage cells in bone marrow and the proportion that represent IgM+ B cells were determined by two-color immunofluorescence with biotinylated-14.8 (CD45RA) and goat anti-mouse IgM [phycoerythrin or fluorescein isothiocyanate from Southern BioTech (Birmingham, AL)]. Subpopulations of B-lineage precursors were then resolved using a modification of the procedures described by Hardy and colleagues (14). The cells were stained with fluorescein isothiocyanate-labeled M1/69 [heat stable antigen (HSA)] (PharMingen, San Diego), biot-

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Abbreviations: DHEA, dehydroepiandrosterone; E2, (-estradiol; IL, interleukin; HSA, heat-stable antigen; CFU, colony-forming units.

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Immunology: Medina and Kincade inylated-S7 (CD43), and PE-labeled 6B2 (CD45R) (PharMingen). Similar results were obtained in some experiments where phycoerythrin-labeled 14.8 (CD45RA) (PharMingen) was used instead of 6B2 and 14.8 tended to give better resolution of dim and bright populations. Three-color analysis was then done and interpreted as follows. Pro-B and large pre-B cells are all CD43+, and very early precursors (Hardy's fraction A) were discriminated by their low expression of HSA. More mature cells (fractions B+C) display high levels of HSA. Bone marrow lymphocytes that are CD45R+ and CD43- include mature B cells and small pre-B cells (fraction D). Numbers of the latter were derived by subtraction of the percentage of B cells (determined from two-color staining as described above). Myeloid and erythroid cells were enumerated with Mac-1 (CD11b) and TER119 (from T. Kina, Kyoto University), respectively. All samples were analyzed with a FACScan flow cytometer (Becton Dickinson). Further details about these analyses are specified in the Results and in our recent publication (10). Hormone Treatment. For acute treatment, female or male mice at least 8 weeks of age were given a single 1-mg injection of water-soluble /3-estradiol or 2.5-10 mg of testosterone (Sigma) i.p., and bone marrow was examined 1-5 days later for clonable bone marrow cells. Hormone- or placebocontaining pellets were used for sustained treatments with estrone (E1), 3-estradiol (E2), estriol (E3), progesterone, combinations of estrogen (E2) and progesterone, dehydroepiandrosterone (DHEA), or dihydrotestosterone. These were 21-day time-release pellets purchased from Innovative Research of America and implanted s.c. into the backs of male or female mice by using a sterile trochar and forceps. All pellet-treated mice were examined 14 days after implantation. Radioimmunoassays. RIA kits for progesterone, estrogen, and DHEA were purchased from Diagnostic Products (Los Angeles), and all assays were done as per kit instructions. A double-antibody RIA kit was used for E2, and directly coated tubes were used for all other hormones. Serum samples were collected in glass tubes, and hormone levels were assayed for each individual mouse. Due to the strong tendency for estrogen to bind to plastic, all estrogen RIAs were done in glass tubes. Progesterone and DHEA were assayed in polypropylene plastic tubes.

RESULTS AND DISCUSSION Dose-Dependent Suppression of B Lymphopoiesis by Estrogen-Contining Pellets. We had reported (10) that B lymphopoiesis selectively declines during pregnancy and postulated that this may result from hormonal changes. In preliminary experiments, we determined that acute treatment of mice with nonphysiologic doses of estrogen suppressed numbers of IL-7-responding B-lymphocyte precursors. This has now been extended by implantation of E2-containing pellets, which produce elevated hormone levels for -21 days. A 2-week regimen was used for most experiments to be sure that an equilibrium was attained, but it should be noted that the resulting increases in estrogen are more prolonged than those that occur during pregnancy (15, 16). Pellets containing as little as 0.05 mg of E2 corresponding to the release of 2.4 ,gg per day resulted in a circulating level of E2 of 100-125 pg/ml as determined by RIA. This amount of E2 is only slightly higher than the maximum reported levels during pregnancy (=60 pg/ml) (17, 18). However, it was sufficient to preferentially deplete IL-7-responsive B-lineage precursors in bone marrow (Fig. 1). More limited studies using high-dose pellets (2.5 mg) containing estrone and estriol revealed that these forms of estrogen similarly suppressed B lymphopoiesis (data not shown). The natural hormone precursor, DHEA, has previ-

Proc. Natl. Acad. Sci. USA 91 (1994)

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FIG. 1. IL-7-responding cells in bone marrow are preferentially suppressed by sustained elevation of plasma estrogen levels. Pellets containing 0.001-2.5 mg of E2 or placebo were implanted s.c. Bone marrow suspensions were evaluated in colony assays after 2 weeks of treatment, and the results are expressed as totals per bone (mean of data from two to six mice per dose ± SEM).

ously been shown to affect the size of the thymus and other lymphoid organs (19). DHEA also suppresses regeneration of B-cell precursors in bone marrow after sublethal irradiation (20). We found that pellets containing 2.5 mg of DHEA substantially reduced numbers of IL-7-responding precursors (average of 16% of placebo control in two experiments; P = 0.001) but did not significantly change the numbers of total nucleated cells or B-lineage cells defined by flow cytometry. We also found in preliminary experiments that 2.5-mg, but not 0.5-mg, pellets of dihydrotestosterone suppressed IL-7-responsive B-cell precursors. Estrogens were the focus of all subsequent experiments. It was more difficult to flush cells from bones of estrogentreated animals, and numbers of total nucleated cells recovered were substantially less (reduced to 46% of normal with 2.5-mg pellets). This result probably reflects an increase in bone cortex versus hemopoietic marrow, as long-term treatment of mice with high doses of estrogen is known to cause osteosclerosis (21). The osteosclerotic response has been well studied and seems to result from estrogen effects on marrow cells in particular parts of the skeleton, especially the femurs (22). It should be noted that numbers of nucleated bone marrow cells do not change significantly during pregnancy (10) and did not change with single injections of soluble (3estradiol (see below). As noted above, the experimental 2-week elevation is not directly comparable to the gradual increases that occur during the mid- to late-gestation phases of pregnancy. Also there could be compensating changes involving other hormones in pregnant animals that prevent estrogen-mediated changes in marrow volume. Regardless of the cause of the osteosclerotic reaction, B-lineage precursors were depressed by estrogen much more than any other cell type. Total precursors enumerated by flow cytometry (see below) were 27% of normal, small pre-B cells (fraction D, see below) were 20%6 of normal, and IL-7-responding cells detected with a colony assay were 5% of normal for the 2.5-mg pellets (Table 1). This is in contrast to the situation for erythroid cells, detected by the TER119 antibody, or myeloid cells that we enumerated by staining

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Immunology: Medina and Kincade

Proc. Natl. Acad. Sci. USA 91

(1994)

Table 1. Preferential suppression of B-lymphocyte precursors by E2 plus progesterone-containing pellets CFU Fraction Fraction Total Nucleated CFU G/M IL-7 Fraction D B+C A precursors cells Group (n) 74 ± 17 93 ± 51 19.4 ± 5.7 3.9 ± 1.2 1.5 ± 0.4 24.0 ± 5.3 150.8 ± 34.0* Controls (15) S ± 3t 130 ± 26t 3.8 ± 1.Ot 1.7 ± 0.7t 1.2 ± 0.6 6.5 ± 1.St 68.7 ± 17.9 E2 (2.5 mg) (6) 92 ± 51 85 ± 18 23.4 ± 4.9 4.5 ± 1.0 1.5 ± 0.4 28.5 ± 5.4 117.2 ± 24.3 E2 (0.01 mg) (8) 85 ± 10 88 ± 17 20.9 ± 1.4 3.2 ± 0.4 1.5 ± 0.5 27.0 ± 4.9 150.3 ± 41.2 P (10 mg) (9) 3 ± it 110 ± 18t 3.2 ± 2.3t 2.0 ± 0.St 1.5 ± 0.4 7.8 ± 3.3t 49.1 ± 7.4 E2 (0.01 mg) + P (10 mg) (7) 62 ± 17 3.3 ± 1.4 7.1 ± 2.7t 1 ± it 9.3 ± 3.1t 1.7 ± 0.7 57.3 ± 22.7 E2 (0.001 mg) + P (10 mg) (5) Flow cytometry was used to enumerate total B-lineage precursors (CD45R+, Ig- cells) and subpopulations of these (fractions A-D). These results are given as mean percentages ± SD of the cells that fell within forward- and side-scatter gates for nucleated cells. B-lineage precursors responsive to IL-7 (CFU-IL7) and myeloid progenitors responsive to colony-stimulating factor (CFU-G/M) are shown as numbers of colonies per 105 cultured cells. All animals were examined 2 weeks after implantation of pellets containing E2, progesterone (P), or placebo. G/M,

granulocyte/macrophage. *Total numbers of nucleated cells per bone are given as means ± SD x 10-5. tData indicate statistically significant differences relative to placebo control values (P =