Rheumatology Unit, Division of Medicine, University of Bristol, Bristol BS2 8HW, United Kingdom
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ABSTRACT |
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High-dose estrogen both stimulates new medullary bone formation and suppresses hematopoiesis in mouse long bones. To determine whether the latter response is a direct consequence of the former, we compared the time course of estrogen's effects on osteogenesis and hematopoietic bone marrow. Flow cytometry was employed to measure hematopoietic subpopulations in bone marrow from femurs of female mice killed at different times after commencing 0.5 mg estradiol/wk to each animal. Estrogen markedly reduced the number of leucocytes (CD11a positive), which had already diminished by 75% after 4 days and had virtually disappeared by 18 days. Specific populations showed a similar pattern of decline after estrogen, including B lymphocytes, monocytes, and endothelial cells. In contrast, the osteogenic precursor population showed a marked increase after estrogen treatment, as assessed by assaying alkaline phosphatase-positive colony-forming units (fibroblastic) ex vivo. However, this rise did not reach significance until 8 days after estrogen administration, suggesting that it follows rather than precedes estrogen's effects on hematopoiesis. We conclude that estrogen does not suppress hematopoiesis in mouse long bones as a direct consequence of its effects on osteogenesis.
flow cytometry; histomorphometry; B lymphocytes
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INTRODUCTION |
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IT IS WELL RECOGNIZED that estrogen exerts an important protective effect on the skeleton and that loss of this action after menopause contributes to the pathogenesis of postmenopausal osteoporosis. Although this protective action of estrogen is largely thought to be mediated by inhibition of bone resorption (3), recent evidence suggests that stimulation of osteoblast activity may also be involved, particularly at concentrations within the upper physiological range (39). To investigate the mechanisms by which estrogen stimulates osteoblast function in more detail, we utilized the mouse model. In this species, high-dose estrogen induces osteosclerosis within the shaft of long bones (9, 40) as a consequence of increased osteoblastic cellular activity (1, 32, 33). We have found that, after high-dose estrogen treatment in female mice, new sites of cancellous bone formation rapidly appear within the tibial metaphysis, presumably as a result of the generation of osteoblasts from osteoprogenitor cells within bone marrow (29).
The osteosclerosis that high-dose estrogen induces in the long bones of female mice results in a considerable reduction in the space available for hematopoiesis and is associated with the development of extramedullary hematopoiesis (21). At first sight, it would seem likely that suppression of hematopoietic marrow after high-dose estrogen is secondary to replacement of the bone marrow cavity by new bone. Alternatively, it is possible that any inhibitory effect of high-dose estrogen on hematopoietic marrow represents a primary action of this hormone, in which case the associated osteogenic response may reflect a secondary phenomenon. That osteogenesis may be a consequence rather than a cause of estrogen's suppressive effects on hematopoiesis is consistent with previous observations that other forms of hematopoietic depletion, such as that induced by gamma irradiation, are likewise associated with osteogenesis (23).
Although important functional relationships are likely to exist between medullary hematopoiesis and osteogenesis, few in vivo studies have investigated these. In light of observations that suggest that estrogen exerts major effects on medullary hematopoiesis and osteogenesis in mice, this would appear to represent a useful experimental model for examining relationships between these two processes. In the present study, we addressed this possibility by comparing the time course of estrogen's effects on hematopoiesis and osteogenesis in female mice.
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METHODS |
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Mice.
Ten-week-old CBA-1 female mice were obtained from the University of
Bristol Medical School breeding colony. Throughout the following
experiments, mice received a standard diet (Rat and Mouse Standard
Diet; B & K, Humberside, UK) and water ad libitum and were kept on a
12:12-h light-dark cycle. 17-Estradiol was dissolved in corn oil
(Sigma Chemicals, Poole, Dorset, UK) and was administered by
subcutaneous injection at 500 µg/wk to each animal. At the
termination of the experiment, animals were killed by cervical
dislocation. All experimental procedures complied with the guiding
principles in the Care and Use of Laboratory Animals.
Experiment 1.
We analyzed the temporal changes in hematopoietic subpopulations of
bone marrow from mouse femurs, after administration of 17-estradiol
as above. Animals were divided into seven groups (4/group) and were
killed immediately before or 2, 4, 8, 12, 15, or 18 days after the
first subcutaneous injection of 17
-estradiol. The left femur was
removed and cleaned of tissue, and the epiphyses were removed. Bone
marrow was collected by repeated flushing of the contents of the femur
into a tube with 1.5 ml in 0.01 M PBS, pH 7.4, using a syringe with a
25-gauge needle until the bone appeared blanched. The bone marrow was
dispersed in a cell suspension by careful use of the syringe needle
against the tube wall.
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Experiment 2. We were also keen to evaluate estrogen-induced changes in the size of the megakaryocyte population. Because these cells are rare and therefore difficult to analyze with flow cytometry, we addressed this question by histology. In preliminary studies, we sought to confirm the identity of megakaryocytes as multinucleate cell profiles that were not directly opposed to a bone surface by performing immunohistochemistry on tibial sections. Acetone-fixed serial 5-µm sections were cut from snap-frozen mouse tibiae and were incubated in 1% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. After being washed in PBS, sections were incubated with 20% normal goat serum, followed by a purified rat anti-mouse CD41 monoclonal antibody, which is specific for megakaryocytes (24; clone MWReg30; Pharmingen, San Diego, CA). Binding to CD41 was visualized using a combination of biotinylated goat anti-rat IgG antibody (Chemicon) and diaminobenzidine. Sections were counterstained with 0.25% toluidine blue.
To examine the effect of estrogen on megakaryocyte number and to relate these changes to the associated osteogenic response, mice were subsequently divided into six groups (4/group), administered 17Experiment 3.
Subsequently, we analyzed the time course of the osteogenic response to
estrogen in bone marrow of mouse femurs by assaying the osteogenic
precursor population at varying time points after estrogen treatment in
ex vivo adherent bone marrow cultures (25). Animals were
divided into five groups (4/group), administered 17-estradiol as
above, and killed before or 4, 8, 12, or 16 days after the first
subcutaneous injection. Both femurs were removed and cleaned of soft
tissue, and the epiphyses were removed.
Statistical analysis. Results were expressed as means ± SE. Differences between time points after estrogen administration for the various parameters studied were analyzed by one-way ANOVA, followed by Tukey's or Dunnett's posttest comparison in those cases where the F-value was significant (i.e., P < 0.05).
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RESULTS |
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Experiment 1.
Administration of 17-estradiol led to a gradual decline over time in
the number of cells that could be flushed from each femur, which
reached significance by day 18 (Fig.
1). In contrast, 17
-estradiol led to
rapid reductions in the number of cells that expressed CD11a, CD31, or
CD45R. The leukocyte population, as defined by expression of CD11a,
showed a striking decrease between days 2 and 4.
By day 18, the size of the population had declined further
to ~10% of baseline levels. The population of cells defined by CD31
expression, which included hematopoietic and endothelial cells (see
Table 1), displayed a minor increase in number at day 2 but
then also declined significantly by day 4; the trend continued such that the population was absent altogether at day 12. Cells of the B lymphocyte lineage expressing CD45R showed a
transient initial increase after estrogen treatment followed by a
marked decrease, mirroring the change in overall leukocyte population
as defined by the CD11a-positive fraction.
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Experiment 2.
In preliminary studies, multinucleate cells within bone marrow not
directly opposed to bone surfaces were identified (Fig. 2A), all of which showed
positive immunolabeling for the megakaryocyte-specific antibody CD41
(Fig. 2B). Control sections in which the primary antibody
was omitted are shown in Fig. 2C. In a subsequent time course study, commencement of 17-estradiol was followed by a transient increase in the number of megakaryocytes, with a peak increase of nearly 50% at 2 days (Fig. 2D). This was
succeeded by a gradual decline in numbers such that by 12 days
after starting treatment with 17
-estradiol megakaryocyte
numbers were reduced by ~75%. A similar response was observed when
megakaryocyte number was expressed against remaining bone marrow area
(results not shown). 17
-Estradiol also increased cancellous bone
volume in the tibial metaphysis, which reached significance by 12 days after starting treatment (Fig.
3).
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Experiment 3.
In bone marrow cultures established 8 days after starting
17-estradiol, a 30-fold increase in osteogenic precursors was
observed, as assessed by counting CFU-AP (Fig.
4). No increase was seen at other time
points, suggesting that this increase in the osteoprogenitor population
after estrogen is relatively transient. The total number of
fibroblastic colony-forming units showed a similar pattern of response.
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DISCUSSION |
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Our findings demonstrate that, in intact female mice, high-dose estrogen treatment is followed by remarkably rapid changes in the size of several hematopoietic subpopulations within long bone marrow. These changes were observed as early as 1 day after treatment, whereas, in contrast, the osteogenic precursor population, as reflected by CFU-AP, did not increase until after 8 days. Consistent with the latter observation, deposition of new medullary bone as assessed by histomorphometry was not observed until 12 days after estrogen treatment, which is in line with results of our previous time course study (29). On the basis of these findings, effects of high-dose estrogen on hematopoietic bone marrow would appear to precede rather than follow those on osteogenesis. Hence, changes in hematopoiesis induced by estrogen presumably represent a direct effect on this compartment rather than a secondary phenomenon as a consequence of new bone formation reducing the space available for hematopoiesis.
One limitation of this study is that, because supraphysiological doses
of estrogen were used to achieve a maximal response, it is unclear how
relevant our findings are to the action of lower estrogen levels within
the physiological range. However, doses of 17-estradiol as low as
4 µg · kg
1 · day
1
stimulate osteogenesis in mice (28), whereas
physiological doses of estrogen inhibit several hematopoietic
lineages as assessed by studies of the effect of ovariectomy on
hematopoiesis (6, 13, 18). Thus a similar
association may exist between effects of physiological estrogen levels
on hematopoiesis and osteogenesis to that found for high-dose estrogen.
It has previously been reported that high-dose estrogen reduces the number of lymphoid and granulocytic cells within bone marrow in mice 2 wk after commencement of treatment (10). To our knowledge, no previous study has examined the effects of estrogen on hematopoietic marrow at earlier time points, as reported here. In addition, as far as we are aware, no previous study has investigated the effect of estrogen on osteogenic precursors using an ex vivo approach, as outlined in this investigation. Only a proportion of CFU-AP colonies produce mature osteoblasts capable of synthesizing bone nodules in culture, which was not assessed in the present study. Therefore, further studies are required to confirm our observations that suggest that estrogen stimulates osteoprogenitor generation in vivo. However, our results are consistent with a report that estrogen stimulates the proliferation and osteogenic differentiation of osteoblast precursors in mouse bone marrow cultures in vitro (26).
Another limitation of this study is that insufficient bone marrow cells were obtained from individual mice to permit simultaneous analysis by flow cytometry and CFU-AP assay. Therefore, the suggestion from our results that estrogen's effects on hematopoiesis precede those on osteogenesis requires confirmation in further studies in which these responses are assessed concurrently. Although flow cytometry enabled the effect of estrogen on the size of hematopoietic subpopulations to be determined, this method provided limited information with respect to the associated osteogenic response; although ALP is a marker for early osteoblast differentiation (27, 37), estrogen was found to significantly reduce the ALP-positive bone marrow fraction, presumably reflecting the fact that ALP is also expressed by hematopoietic bone marrow cells such as neutrophils (8). Hence, the ALP-positive bone marrow fraction does not mirror the bone marrow osteoprogenitor population, which in contrast increased after estrogen as assessed by counting CFU-AP colonies in ex vivo bone marrow cultures. It may also be possible to analyze estrogen-induced osteogenesis by flow cytometry by employing other markers suggested to identify the osteogenic subfraction with this technique (34, 41).
A decrease in production of myeloid and lymphoid precursors may have contributed to the decline in hematopoietic populations observed after estrogen treatment. Consistent with this possibility, estrogen administration has previously been reported to reduce the number of pre-B lymphocyte colonies in mouse bone marrow (20). Any tendency of estrogen to suppress lymphocyte precursors may be mediated by a primary interaction with stromal cells, which play a major role in regulating hematopoiesis, including B lymphopoiesis (31). In support of this hypothesis, pretreatment of stromal cells with estrogen has previously been found to suppress B lymphopoiesis in vitro (18, 35). Because stromal cells are also thought to contain the osteoprogenitor population (7, 22), it is possible that estrogen's effects on hematopoietic bone marrow and osteogenesis are both mediated through an action on stromal cells. That stromal cells play a role in reciprocal regulation of hematopoiesis and osteogenesis is also suggested by studies of the senescence-accelerated mouse-P6 in which enhanced hematopoiesis and impaired osteogenesis are thought to occur as a consequence of altered stromal cell function (14).
An action on precursors may also explain our finding that estrogen expanded the erythroid population, in view of a previous report that estrogen treatment increases the number and mitotic activity of erythroid precursors in mouse spleen and bone marrow (17). On the other hand, the finding that estrogen transiently increased the number of B lymphocytes over the first 2 days may have reflected initial expansion in the population of relatively mature cells, as previously observed in mice exposed to high levels of estrogen during pregnancy (20). Estrogen's suppressive effects on leucocytes may also have involved an effect on relatively mature cell types, such as induction of apoptosis; although we saw no morphological evidence for apoptosis, further studies are required to address this question in more detail.
Our observation that estrogen transiently increases the number of megakaryocytes within bone marrow is consistent with previous findings (17). In addition, a more sustained increase in megakaryocyte number has been reported in association with elevated levels of estrogen during pregnancy in mice (19) and after estrogen replacement therapy in postmenopausal women (2). Regulation of megakaryocyte number by estrogen in this way may be mediated directly through binding with megakaryocyte estrogen receptors (2, 38). Transgenically induced megakaryocytosis in mice has previously been reported to induce osteogenesis, possibly through increased release of megakaryocyte-derived osteogenic growth factors like platelet-derived growth factor (43). The latter is known to induce profound stimulation of murine fibroblastic colony-forming units (16, 42) and is itself induced by estrogen (30). Therefore, effects of estrogen on megakaryocytes represent a further potential mechanism by which hematopoietic populations might mediate estrogen-induced osteogenesis.
In summary, we have found that high-dose estrogen leads to a rapid decline in hematopoietic populations within mouse long bone marrow. Moreover, this response appears to precede estrogen's associated stimulatory action on bone marrow osteogenic precursors. Therefore, suppression of hematopoiesis after estrogen treatment in this species does not appear to result from loss of marrow space as a consequence of estrogen's stimulatory action on osteogenesis. Further studies are required to characterize estrogen's reciprocal effects on hematopoiesis and osteogenesis in more detail, for example, by analyzing whether stromal cells play an important role in mediating these two responses.
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ACKNOWLEDGEMENTS |
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This study was funded by the Arthritis Research Campaign.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. H. Tobias, Bristol Royal Infirmary, Bristol BS2 8HW, UK (E-mail: Jon.Tobias{at}bristol.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 February 2000; accepted in final form 31 May 2000.
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