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*
Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; and
Departments of Biochemistry and Child Health, University of Missouri, Columbia, MO 65211
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) was evaluated in
gene-targeted mice, and B cell precursors were found to be within the
normal range. Our previous studies indicated that hormone receptors in
stromal cells may be important for estrogen-mediated suppression of B
lymphopoiesis. We now show that estrogen-mediated inhibition of B cell
precursor expansion in culture was blocked by a specific estrogen
receptor antagonist (ICI 182,780). Stromal cells derived from
ER-targeted bone marrow were fully estrogen responsive. RT-PCR
analyses of these stromal cells revealed splice-variant transcripts of
ER, as well as message for a recently discovered estrogen-binding
receptor, ERß. Thus, androgens may normally inhibit B lymphopoiesis
through the androgen receptor, whereas estrogens might utilize one or
more receptors to achieve the same physiologic response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Conversely, reduced levels of circulating sex steroids resulted in up-regulation of B lymphopoiesis in the bone marrow. A mutation in the gonadotrophin-releasing hormone gene prevents appreciable production of sex steroids in hypogonadal (hpg) mice (6, 7, 8). These animals had substantially higher numbers of B cell precursors than littermate controls. A less dramatic increase in B cell precursor production was seen in male castrates (3). Female castration did not always result in an elevation in B cell precursors and may be both time and strain dependent (3, 9).
Estrogen appears to suppress B lymphopoiesis by altering cells in the
marrow microenvironment. Direct treatment of B cell precursors with
estrogen had no consequence on their survival, proliferation, or
differentiation. However, the hormone reduced their expansion when
stromal cells were also present in the cultures (2). Stromal cells and
lymphocytes did not have to be in direct contact to observe the
influence of estrogen. Moreover, estrogen-pulsed stromal cells produced
some suppressive substance(s) (Ref. 2, and our unpublished
observations). One estrogen receptor (ER) was shown to be expressed
by these stromal cells, and others have demonstrated functional
receptors for both androgens and estrogens in marrow stromal cells
(2, 10, 11).
Estrogen and testosterone commonly mediate their effects through well characterized nuclear receptors. When bound by ligand, the nuclear steroid receptors interact with hormone response elements in the DNA and trans-activate specific genes (12). The androgen receptor (AR) is encoded by a single gene on the X chromosome (13). Testicular feminization mice (Tfm) have a single point mutation in the AR gene causing a frame shift that results in unstable mRNA (14). However, low levels of a smaller, androgen binding AR protein that lacks the N-terminal domain have been detected in Tfm mice (14, 15). This domain of the AR is thought to be important for transcriptional activity (14). Thus, although Tfm mice retain residual androgen binding, they are androgen insensitive. The Tfm mutation is unique to male mice. Female Tfm homozygotes cannot be obtained by normal mating since male mice with the defective AR are sterile (14, 15, 16, 17). Tfm are indistinguishable from normal female mice except for small internal testes (17). The lymphopoietic status of these mice has not been well characterized, but they have somewhat larger thymuses and spleens than littermate controls (18).
Estrogen can interact with at least two distinct nuclear
receptors designated estrogen receptor (ER) and estrogen
receptor ß (ERß). ER was cloned from mouse uterus and is
required for most estrogen induced uterine functions (19, 20). ERß
was originally cloned from the rat prostate (21). The human (22) and
mouse (23) ERß homologues have recently been cloned. The ERß is
highly homologous to ER with respect to the DNA and ligand binding
domains in all three species. Interestingly, a variety of different
size transcripts for ERß are detected in mouse ovary; whereas only a
single transcript for ER is detected in that site (23). Like ER,
ERß is a high affinity receptor that binds estrogen responsive
elements in vitro and can induce transcription of estrogen regulated
genes. A biologic function has not yet been attributed to ERß, but it
is hypothesized to mediate functions distinct from ER based on its
differential expression (24).
Recently, estrogen receptor knock out mice (ERKO) have been generated
by inserting a neomycin gene into the second exon of the ER gene
(20, 25). These mutants have no detectable wild-type ER mRNA.
However, there is some residual estrogen binding in the uteruses of
these animals that reacts with the ER-specific Ab H222. Very low
levels of mRNA for two unique splice variants of ER were detected in
ERKO mice. One of these splice variants, potentially, could produce a
protein that retains a high affinity for estrogen and could be
responsible for residual estrogen binding in the uterus. However, in
vitro studies suggest that this splice variant had a reduced
transcriptional capacity, consistent with the relative unresponsiveness
of the ERKO uterus to estrogen.
We have now exploited these deficient mice to determine the relative
importance of sex steroids and their receptors for regulation of B
lymphocyte formation. Loss of the androgen receptor resulted in an
increase in B lineage cells, particularly those not yet expressing sIg.
In vitro treatment with the antiestrogen drug ICI 182,780 suggested
that estrogen suppressed B lymphopoiesis through an ER-specific
mechanism. ER-/- mice did not have abnormal with
respect to B cell precursors. While this might be explained in part by
redundancy with respect to other sex steroids, it is also possible that
events in bone marrow are regulated by multiple estrogen receptors.
Indeed, we show that stromal cells from ER gene-targeted mice were
normally responsive to estrogen and that they express the recently
described ERß.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Tfm and wild-type control (Ta6J) mice (17), ranging in age from 5 to 17 wk, were purchased from The Jackson Laboratory (Bar Harbor, ME). BALB/c mice (female 4–6 wk) were obtained either from the Oklahoma Medical Research Foundation Laboratory Animal Resources Center, or The Jackson Laboratory. ERKO mice have been previously described (25). ERKO mice and wild-type controls ranged in age from 14 to 18 wk.
Flow cytometric analyses
Cells were isolated from the bone marrow and spleen of Tfm and ERKO mice and their respective controls, resuspended in staining buffer (PBS without Ca2+ and Mg2+ and with 3% heat-inactivated FCS) and stained as previously described (3). B lineage cells in the bone marrow and spleens were identified as follows: total B lineage cells are CD45R+, B cell precursors are CD45R+ IgM- (26). Mature B cells are CD45R+, IgM+, IgD+ (27, 28). The Abs used to identify these antigens were PE rat anti-mouse CD45R (Mab 14.8; PharMingen, San Diego, CA) and FITC goat anti-mouse IgM and PE goat anti-mouse IgD (both from Southern Biotechnology Associates, Birmingham, AL). Biotinylated Abs were visualized with either streptavidin-Peridinin CP (Becton Dickinson, Mountain View, CA) or Cy-Chrome-streptavidin (PharMingen). Appropriate isotype control Abs were used to evaluate positive staining. All samples were analyzed with a FACScan flow cytometer (Becton Dickinson).
Cell sorting
Early pro B cells were isolated from bone marrow by first depleting myeloid, erythroid, and mature B cells. To do this BALB/c bone marrow cells were stained with purified rat anti-mouse Ly6G (recognizes the myeloid differentiation marker GR-1) and rat anti-mouse TER119 Ab specific for erythroid lineage cells, both purchased from PharMingen. The stained cells were incubated with goat anti-rat Ig and goat anti-mouse IgM magnetic beads (PerSeptive Diagnostics, Cambridge, MA) to deplete the unwanted cells. Enriched B cell precursors were then stained with a modification of a scheme described by Hardy et al. (28). The following reagents were used: BP-1 Ab (a kind gift from Dr. Max Cooper, Birmingham, GA) followed with goat anti-mouse IgG2a-FITC (Baxter, Mundelein, IL), PE rat anti-mouse CD45R, and biotinlylated rat anti-mouse CD24, followed by streptavidin-Perdinin CP or Cy-Chrome streptavidin. Early pro B cells were identified as BP-1-, CD45+, CD24low cells and were gated as previously shown (1). Cells were sorted using the FACStarplus cell sorter.
Colony assays
Colony assays were done as previously described (26). Briefly, bone marrow cells were isolated and resuspended in assay medium containing 0.3% Bacto-agar (Difco Laboratories, Detroit, MI). For detecting IL-7-responding cells, 2 to 5 ng/ml of recombinant mouse IL-7 (R&D Systems, Minneapolis, MN) was used. LPS-responding B cells and myeloid precursors were detected in colony assays containing 250 ng/ml of endotoxin (Difco) or 25 µl/ml of 10x concentrated L cell or WEHI-3 conditioned medium, respectively. All cloning assays were performed in 35-mm dishes (Corning Glass, Corning, NY) and incubated at 37°C in 5% CO2. Colonies were counted on day 6.
Isolation of stromal cell clones
Stromal cells were isolated from the bone marrow of male ERKO and from wild-type mice. Single cell suspensions of bone marrow cells were plated at 5 x 105 cells/ml in stromal cell cloning medium containing either DMEM or Opti-MEM I (Life Technologies, Gaithersburg, MD) with 20% FCS, 2 mM glutamine, 100 U penicillin/ml, 100 µg of streptomycin/ml, 5 x 10-5 M 2-ME and 10% BMS2 cell-conditioned medium (2, 29). ERKO stromal cells and their wild-type controls were cloned by limiting dilution. After cultures were confluent, the cells were detached with trypsin/EDTA (Life Technologies), washed, and resuspended in stromal cell cloning medium supplemented with 75 µg/ml endothelial cell growth supplement, 50 ng/ml fibroblast growth factor (both from Collaborative Biomedical Products, Bedford, MA), and 20 ng/ml insulin-like growth factor-I (IGF-I) (HyClone). Plates were fed weekly and supplemented with cytokines.
OP42 stromal cells were derived from the spleen of a B6C3fe-a/e osteopetrotic (op/op) mouse as previously described (2). OP42 stromal cells do not produce CSF-1 (30) but can support the growth of B cell precursors in the presence of exogenous IL-7 (2). OP42 stromal cells, ERKO stromal cell clones, and the corresponding wild-type clones were maintained in estrogen-low tissue culture medium (phenol-red free RPMI 1640, 5% charcoal dextran FCS (HyClone), 2 mM glutamine, 100 U penicillin/ml, 100 µg of streptomycin/ml, and 5 x 10-5 M 2-ME).
Cocultures
Early pro B cells (BP-1-, CD45+, CD24low) were cocultured with the indicated stromal cell lines. Stromal cells were plated at 80,000 cells/well in a 24-well plate with 1 ml of estrogen-low tissue culture medium for 24 to 48 h before the lymphocytes were added. B cell precursors and 2 ng/ml IL-7 (R & D Systems) were added to the wells and the cells cocultured for four days. Estrogen (Sigma, St. Louis, MO) and/or ICI 182,780 (a kind gift from Alan E. Wakeling and ICI Pharmaceuticals, Macclesfield, U.K.) were added to some cultures before addition of lymphocytes. B cell progenitor expansion was assessed as previously described (2). Briefly, each well was treated with 2 mM EDTA to remove all the cells. The detached cells were washed and resuspended in staining buffer and counted. Stromal cells were not included in cell counts. The cells were then stained with CD45R-PE, and the percentages of B lineage cells (CD45R+) were assessed by flow cytometry.
Evaluation of ER status
The expression of ER and ERß transcripts in stromal cell
clones derived from male ERKO or wild-type control mice was compared
using RT-PCR. The FastTrack 2.0 Kit from InVitrogen (San Diego, CA) was
used to isolate mRNA. The RT reaction was done using 10 µl
MgCl2, 5 µl of each 10 mM dNTP, 2.5 µl random
hexamers, 2.5 µl (50 U) RNase inhibitor, 2.5 µl (250 U) murine
leukemia virus (MuLV) reverse transcriptase, and 0.5 µg poly(A) RNA
(all reagents from Perkin-Elmer, Foster City, CA) in a total volume of
50 µl. The RT reactions were incubated at 24°C for 12 min, 42°C
for 30 min, and 95°C for 5 min, and then held at 4°C in a
Perkin-Elmer GeneAmp 9600. The PCR reactions were then conducted in the
following mix per 25-µl reaction: 5.0 µl 5x InVitrogen buffer
(Buffer J for ER and ERß and Buffer I for ß actin), 50 pM of
each primer, 2.0 µl (10 mM) dNTP mix (InVitrogen), 0.25 µl (1.25 U)
AmpliTaq, and 5.0 µl of RT reaction. The PCR reactions were cycled 35
times at 95°C for 1 min, 58°C for 45 s, and 72°C for 30
s, and then extended at 72°C for 7 min and held at 4°C. The
ER, actin, and ERß primers have been previously described
(20, 21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used Tfm mice to assess the importance of the
AR for normal B cell production in the bone marrow. These animals have
a point mutation in the X-linked AR gene and are insensitive to
androgens (14, 15, 31). Flow cytometric analyses comparing bone marrow
from wild-type and Tfm mice showed markedly increased
percentages of B cell precursors (CD45R+,
IgM-) in the mutant animals (illustrated in Fig. 1
and summarized in Fig. 2). A similar increase was seen in their
absolute numbers (wild-type 2.1 ± 0.5 x 106 vs
Tfm 4.7 ± 1.9 x 106).
IL-7-responsive cells, which give rise to pre-B cells, were enumerated
with a semisolid agar-cloning procedure (28) and also were found to be
increased (Fig. 3). In comparison,
LPS-responsive B cells and myeloid progenitors assessed in similar
semisolid agar assays were not affected by the Tfm mutation
(Fig. 3). The number of nucleated cells in Tfm bone marrow
was slightly increased as compared with controls (Tfm
21.9 ± 7.3 x 106 vs wild-type 17.0 ±
5.3 x 106/bone) but did not reach significance
(p = 0.08). The incidence of MAC-1+
cells in the bone marrow, which are primarily myeloid cells, was
significantly decreased (Fig. 2). We also examined the production of
erythroid cells because sex steroids (32, 33) negatively regulate
GATA-1, an essential transcription factor for erythroid production.
Despite the lack of androgen responsiveness, the percentage of
TER119+ cells was reduced in Tfm bone marrow
(Fig. 2). Numbers of MAC-1+ (CD116) and TER119+
cells were not significantly decreased in the AR-deficient animals,
perhaps reflecting the slight increase in total nucleated cells.
Together, these data show a clear increase in B cell precursors in
Tfm bone marrow as assessed by flow cytometry and in a
functional assay, indicating the AR may be important in determining the
size of the B cell precursor compartment.
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) and periphery. Tfm mice had significantly more
splenic IgM+ B cells than wild-type controls (54.5 ±
10.8 x 106 wild-type vs 89.4 ± 17.5 x
106 for Tfm; p = 0.001), which
was reflected in a 1.5-fold greater number of nucleated spleen cells
(p = 0.001). This suggests that increased
production of B cells within bone marrow is not compensated for by a
decreased rate of survival in peripheral tissues. Thus, these findings
document augmented and selective expansion of B cell production in the
bone marrow of Tfm mice and a corresponding increase in the
level of B cells in the peripheral lymphoid organs such as spleen. The increase of B cell precursors in Tfm mice could result from down-regulated estrogen levels, since the AR regulates the enzymes required for estrogen synthesis in male mice (34, 35, 36). However, DHT, a nonmetabolizable form of testosterone, was able to alter stromal cell-dependent B cell precursor growth in vitro. Early pro-B cells (CD24low, CD45R+, BP-1-) from normal mice (BALB/c) were cocultured with OP42 stromal cells (2) in the presence of DHT (10-6 M). Under these defined conditions, the androgen inhibited precursor expansion by 51 ± 14% (n = 6; p = 0.002). In the same group of experiments, expansion of B cell precursors was decreased 49 ± 10% (n = 5; p = 0.004) by estradiol (10-6 M). Furthermore, like estrogen (2), DHT at this concentration had no direct effect on IL-7-responding cells in semisolid agar-cloning assays. In an average of four independent experiments, the number of colonies in cultures treated with 10-6 M DHT was 105 ± 30% of diluent treated controls. Therefore, while androgens and their receptors might be functionally linked to other endocrine networks in vivo, testosterone alone was sufficient to negatively affect B lymphopoiesis in stromal cell-containing cultures.
The pattern of selectively increased B lymphopoiesis shown here for Tfm mice is very similar to that previously observed in castrated male or hpg mice (3). Moreover, the same populations of B cell progenitors were depleted after estrogen treatment and during pregnancy of normal animals (1, 4). Together, these findings suggest that both androgens and estrogens contribute to steady state regulation of B lymphocyte formation.
ERKO mice have normal levels of B cell precursors
In contrast to the results obtained with AR-deficient, castrated,
and hpg mice, no significant increases in B cell precursors
were found in ERKO (25) mice. In fact, the percentages of
CD45R+, sIgM- cells were slightly lower, and
CD45R+, sIgM+ cells (Fig. 4) and mature B cells (sIgM+,
sIgD+; wild-type 8.1 ± 2.1% vs 5.5 ± 2.0%;
p = 0.04 (26)) were significantly lower than wild-type
animals. The number of IL-7-responding cells in the bone marrow of
these animals was no different from controls (p
= 0.15). Female wild-type and ERKO mice had similar numbers of bone
marrow cells (13.9 x 106 in wild-type mice and
12.0 x 106 in ERKO mice). In male mice, numbers of
CD45+, IgM- cells, and IL-7-responding cells
were unchanged (n = 4, p = 0.7,
p = 0.06). As noted above, these populations of B
lineage cells are the most sensitive to changes in hormone levels (1, 3, 4). As in the bone marrow, B lineage cells tended to be slightly
reduced in spleens of female mice and at control levels in male ERKO
mice. Thus, despite clear evidence that estrogen negatively regulates B
lymphopoiesis (1, 2, 3, 4), loss of a functional ER was without major
consequence.
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An alternative possibility is that hormonal regulation of lymphopoiesis
is a nonreceptor-mediated effect. Therefore, we performed experiments
with ICI 182,780, a pure estrogen antagonist (38). ICI 182,780
alleviated estrogen-mediated suppression of B cell expansion in
cultures containing sorted B cell precursors and the cloned stromal
cell line OP42. In three separate experiments, 10-7 M ICI
182,780 increased recoverable B cell precursor numbers in
estrogen-treated cultures 1.6 ± 0.3-fold (Fig. 5). These data demonstrate that estrogen
suppressed B lymphopoiesis through an estrogen receptor.
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Estrogen levels are elevated 10-fold in female ERKO mice
(20), and, although the uteri of these animals are virtually
unresponsive to this hormone, the bone marrow might retain functional
estrogen receptors. To test this possibility, we isolated stromal cells
from the bone marrow of ERKO and wild-type mice and compared their
ability to support the growth of a subset of normal B cell precursors
in the presence of estrogen. Previous studies indicated that estrogen
may induce stromal cell production of a factor(s) that reduced the
expansion of cells with undifferentiated characteristics
(CD24low, CD45R+, BP-1-)
(Ref. 2; and our unpublished observations). Estrogen suppressed B cell
precursor expansion in cocultures containing ERKO-derived stromal cells
to a level comparable with cultures with either wild-type-derived
stromal cells or the OP42 stromal cell clone (2) (Fig. 6). These results were reproducible in
four experiments done with both cloned and bulk cultured stromal cells,
demonstrating this was not a phenomenon associated with a single ERKO
stromal cell line. Thus, the loss of ER in ERKO stromal cells did
not prevent them from responding to estrogen and depressing B cell
precursor expansion.
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Estrogen might mediate its effects on B lymphopoiesis in ERKO mice
through a receptor other than the classical ER. The ability of ERKO
stromal cells to respond to estrogen and inhibit B lymphopoiesis
demonstrates they have a functional estrogen response mechanism. The
ER is encoded by a single mRNA in normal mice (19), but not in
gene-targeted animals. Two smaller splice variants of ER (ERKO-E1
and ERKO-E2) are present in these animals. ERKO-E2 does not code for a
functional protein. The ERKO-E1 splice variant can produce a protein
with similar affinity for estradiol as the full length ER, but with
decreased transcriptional activity (20). A primer set that binds
sequences 5' and 3' of the disrupted portion of the ER gene
amplified a single 685-bp product from wild-type ovary tissue and a
wild-type stromal cell clone (Fig. 7). A
514-bp product (corresponding to ERKO-E1) and a 354-bp product
(corresponding to ERKO-E2) were found in ERKO ovary tissue and in a
cloned stromal cell line derived from ERKO bone marrow. Thus, ERKO
stromal cells have detectable levels of ERKO-E1 and ERKO-E2
mRNA.
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in both ligand
and DNA binding sites, a physiologic function has not previously been
demonstrated for this receptor. Thus, estrogen could regulate
lymphocyte precursor expansion in ERKO mice by interacting with either
ERß or the functionally compromised ERKO-E1 variant. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-targeted mice indicates this receptor is
dispensable for some responses to estrogen and points to a possible
role for the recently discovered ERß within bone marrow. Marrow
stromal cells regulate both hemopoiesis and bone morphogenesis and are
potentially influenced by both androgens and estrogens (40, 41, 42, 43). Thus,
male and female sex steroid levels may have consequences on bone
structure and, as shown here, events that take place within bone
marrow.
A missense mutation in the only known AR renders Tfm mice
unable to respond to androgens (14, 15, 31). One previous study noted
that Tfm mice have increased splenic weight and cellularity
as well as alterations in the thymus (18). Another group suggested that
B lymphocyte formation might be augmented by this mutation (44), and
our observations are consistent with this hypothesis. B lymphocyte
lineage cells were selectively expanded in marrow of Tfm
mice (Fig. 2). This included cells responsive to IL-7 (Fig. 3) and ones
defined by the absence of sIgM and presence of CD45R (Figs. 1 and 2).
It is interesting that IL-7-responsive B cell precursors are depressed
to the greatest degree by pregnancy or estrogen treatment (1, 4).
Furthermore, these precursor subsets were expanded in sex
steroid-deficient hpg or castrated male mice (3). We
conclude that the availability of these hormones, as well as the
ability to respond to them, is a critical parameter for determining how
many B lymphocytes are formed.
Numbers of splenic B cells are substantially elevated in three circumstances of reduced sex steroid influence: the inability to make all sex steroids (hpg), deficiency of androgens (castrated male), or hormone unresponsiveness (Tfm mice). This interesting correlation may indicate that the average lifespan of mature lymphocytes in the periphery cannot decline sufficiently to compensate for the increased production of cells in bone marrow. While it is also possible that sex steroids have a direct influence on mature B cells (45), we observed no deficiency either during pregnancy or following estrogen treatment (1, 4). The only splenic B cells to be affected in those circumstances had characteristics of newly formed lymphocytes (1, 4). Animals treated with insulin-like growth factor-I (IGF-I) also have abnormally high numbers of splenic lymphocytes, and a case has recently been made for a role of thyroxine in promoting B lymphocyte formation (46, 47). It will be important to learn if these hormones counterbalance the negative regulatory role of sex steroids and if the target cells are similar.
It has long been known that the development of humoral immunity in birds is sensitive to androgens, and the epithelium of the bursa of Fabricius represents the most likely target (48, 49). Our previous studies suggest that one mechanism estrogen might utilize to suppress B lymphopoiesis is by inducing stromal cell production of a negative regulator(s) (Ref. 2; and our unpublished observations). Stromal cell lines have been shown to express receptors for androgens as well as estrogens (42, 43), and we now report that DHT suppresses the expansion of B lineage precursors when added to short term cocultures of lymphocytes and stromal cells. This finding demonstrates that androgens can directly influence lymphopoiesis and do not have to work by altering estrogen levels.
Tfm mice have intact estrogen receptors, but there are
at least two possible reasons why estrogen does not compensate for the
loss of AR and maintain normal levels of B cell production. The initial
step of estrogen production in males involves the conversion of
pregnenolone to either progesterone or 17-hydroxypregnenolone,
catalyzed by the steroidogenic enzyme 17-hydroxylase. This activity
is confined to Leydig cells in male rodents, and synthesis of the
enzyme appears to be regulated by the AR (34, 35, 36). Since Tfm
mice lack an AR, estrogen production may be reduced and levels of the
hormone too low to compensate for decreased androgen responsiveness. A
second possibility is that, while androgens play the dominant role in
males, estrogens are the primary sex steroid regulating B lymphopoiesis
in females.
ERKO mice provide an important model to identify estrogen-mediated
responses in which the ER is important (50) and where, potentially,
other receptors may play a predominant role (51). Male mice targeted
for ER (ERKO mice) had no perturbations of B lymphocyte formation,
and this might be taken as evidence that androgens were sufficient for
normal control of lymphopoiesis. However, we predicted that B
lymphopoiesis would be abnormally increased in female ERKO mice. This
hypothesis derived in part from the finding that estrogen replacement
of hpg mice was sufficient to bring numbers of B cell
precursors into the normal range (3). Therefore, it was surprising to
find that precursor numbers were normal or reduced in female
ER-targeted animals. This might result from the expression of an
alternative estrogen receptor(s) in bone marrow stromal cells and/or
from greatly elevated estrogen levels in the bloodstream.
Alternatively, as in males, normal or lower levels of precursors in
female mice might be due to high circulating levels of androgens.
Stromal cells isolated from ERKO mice responded to estrogen and
suppressed the expansion of B cell precursors in culture. Furthermore,
a cloned stromal cell line derived from ERKO bone marrow possessed mRNA
for ER variants unique to ERKO mice (ERKO-E1 and ERKO-E2 (20)). The
ERKO-E1 splice variant of ER can produce a protein that has
decreased transcriptional activity in vitro (20). However, no protein
of this kind was detectable in the uteri of ERKO mice by Western blot.
Transcription of genes for the progesterone receptor, lactoferrin, and
glucose-6-phosphate dehydrogenase were all up-regulated in response to
estrogen treatment in the uteri of wild-type, but not ERKO mice (20).
Although the ERKO-E1 transcript was detected in stromal cells isolated
from ERKO mice, it would be difficult to account for normal
lymphopoiesis in females in terms of this functionally compromised
receptor.
We also detected transcripts for a second type of estrogen receptor in
stromal cells derived from ERKO mice. This receptor was originally
cloned from a cDNA library isolated from rat prostate and is designated
ERß (21). Recently, clones for this receptor have been isolated in
humans (22) and mice (23). Protein for this receptor has not been
identified in normal tissues. However, ERß synthesized in vitro had
an affinity for estrogen similar to ER. Transient transfection of
Chinese hamster ovary (CHO) cells with ERß cDNA and a reporter
plasmid containing an estrogen response element suggested that ERß is
transcriptionally active (21). Furthermore, the action of ERß can be
blocked by ICI 164,384 (ICI 182,780 is a more potent derivative of this
drug (38)) and numerous other anti-estrogen drugs (22, 24). In the
rat, RT-PCR experiments revealed differential expression of ERß and
ER (24). Recent studies support the idea that ligand-mediated
activation of these receptors can have different consequences (52). Our
observations raise the interesting possibility that ERß mediates some
responses of bone marrow cells to estrogen.
Systemic estrogen levels are normally limited in females by a
pituitary feedback that inhibits further release of gonadotrophs (53).
Targeting of the ER may disrupt this pathway because female ERKO
mice have 10 times the normal level of estrogen (20). This fact,
together with the presence of one or more active estrogen receptors in
bone marrow, might account for the slightly reduced B lymphopoiesis in
female gene-targeted mice. In addition, female ERKO mice had somewhat
elevated levels of testosterone, which could potentially diminish
lymphocyte formation via the AR.
Studies of sex steroids in relationship to bone density provide precedent for our findings involving lymphocyte formation and indicate that similar biologic responses can be achieved by different classes of hormones. There is a delicate balance between bone formation by osteoblastic stromal cells and bone resorption by hemopoietically derived osteoclasts. IL-6 is important in the recruitment and activation of osteoclasts, and it has been proposed that sex steroids suppress IL-6 induction in osteoblasts. Furthermore, functional AR and ER have been detected in osteoblast-like cells (10, 42, 43, 54). If circulating levels of either estrogen or testosterone are reduced, bone loss occurs (43, 55). Reduction of estrogen or androgen levels in IL-6-deficient mice does not reduce bone mass (43, 55). Thus, androgens and estrogens may have overlapping effects on two functions of bone marrow cells, bone remodeling and lymphopoiesis.
These studies extend our understanding of the complex
interrelationships between sex steroids, their receptors, and B cell
production. Functional ARs are required for normal numbers of B cells
to be made in male mice. One potential mechanism involves ligation of
ARs in marrow stroma and subsequent alteration of their ability to
support lymphocyte growth. The situation is potentially more complex in
the case of estrogen, which similarly inhibits precursor expansion in
culture and can be blocked by ER-specific drugs. It remains to be seen
if ER and ERß are redundant with respect to responses of bone
marrow cells to this hormone, or if their differential expression
corresponds to tissue specific differences. ER gene-targeted mice and
stromal cells derived from them should provide important tools for
further investigation of these issues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul W. Kincade, Immunobiology Program, Oklahoma Medical Research Foundation, 825 N. E. 13th Street, Oklahoma City, OK 73104. E-mail address:
3 Abbreviations used in this paper: DHT, dihydrotestosterone; hpg, hypogonadal; AR, androgen receptor; Tfm, testicular feminization; ER, estrogen receptor; ERKO, estrogen receptor knock out; sIg, surface Ig; PE, phycoerythrin.
Received for publication May 8, 1997. Accepted for publication February 24, 1998.
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  J. A. Dudakov, G. L. Goldberg, J. J. Reiseger, A. P. Chidgey, and R. L. Boyd Withdrawal of Sex Steroids Reverses Age- and Chemotherapy-Related Defects in Bone Marrow Lymphopoiesis J. Immunol., May 15, 2009; 182(10): 6247 - 6260. [Abstract] [Full Text] [PDF]
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 S. Altuwaijri, K.-H. Chuang, K.-P. Lai, J.-J. Lai, H.-Y. Lin, F. M. Young, A. Bottaro, M.-Y. Tsai, W.-P. Zeng, H.-C. Chang, et al. Susceptibility to Autoimmunity and B Cell Resistance to Apoptosis in Mice Lacking Androgen Receptor in B Cells Mol. Endocrinol., April 1, 2009; 23(4): 444 - 453. [Abstract] [Full Text] [PDF]
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T. Yokota, K. Oritani, K. P. Garrett, T. Kouro, M. Nishida, I. Takahashi, M. Ichii, Y. Satoh, P. W. Kincade, and Y. Kanakura Soluble Frizzled-Related Protein 1 Is Estrogen Inducible in Bone Marrow Stromal Cells and Suppresses the Earliest Events in Lymphopoiesis J. Immunol., November 1, 2008; 181(9): 6061 - 6072. [Abstract] [Full Text] [PDF]
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 I. Mascanfroni, M. d. M. Montesinos, S. Susperreguy, L. Cervi, J. M. Ilarregui, V. D. Ramseyer, A. M. Masini-Repiso, H. M. Targovnik, G. A. Rabinovich, and C. G. Pellizas Control of dendritic cell maturation and function by triiodothyronine FASEB J, April 1, 2008; 22(4): 1032 - 1042. [Abstract] [Full Text] [PDF]
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 J.-M. Renoir, C. Bouclier, A. Seguin, V. Marsaud, and B. Sola Antioestrogen-mediated cell cycle arrest and apoptosis induction in breast cancer and multiple myeloma cells J. Mol. Endocrinol., March 1, 2008; 40(3): 101 - 112. [Abstract] [Full Text] [PDF]
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E. Carreras, S. Turner, V. Paharkova-Vatchkova, A. Mao, C. Dascher, and S. Kovats Estradiol Acts Directly on Bone Marrow Myeloid Progenitors to Differentially Regulate GM-CSF or Flt3 Ligand-Mediated Dendritic Cell Differentiation J. Immunol., January 15, 2008; 180(2): 727 - 738. [Abstract] [Full Text] [PDF]
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 R. H. Straub The Complex Role of Estrogens in Inflammation Endocr. Rev., August 1, 2007; 28(5): 521 - 574. [Abstract] [Full Text] [PDF]
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 T. Takao, C. Kumagai, N. Hisakawa, R. Matsumoto, and K. Hashimoto Effect of 17{beta}-estradiol on tumor necrosis factor-{alpha}-induced cytotoxicity in the human peripheral T lymphocytes J. Endocrinol., January 1, 2005; 184(1): 191 - 197. [Abstract] [Full Text] [PDF]
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A. C. Roden, M. T. Moser, S. D. Tri, M. Mercader, S. M. Kuntz, H. Dong, A. A. Hurwitz, D. J. McKean, E. Celis, B. C. Leibovich, et al. Augmentation of T Cell Levels and Responses Induced by Androgen Deprivation J. Immunol., November 15, 2004; 173(10): 6098 - 6108. [Abstract] [Full Text] [PDF]
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 M. J. Osborn, P. L. Ryan, N. Kirchhof, A. Panoskaltsis-Mortari, F. Mortari, and K.-S. R. S. Tudor Overexpression of murine TSLP impairs lymphopoiesis and myelopoiesis Blood, February 1, 2004; 103(3): 843 - 851. [Abstract] [Full Text] [PDF]
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 M. T. Tilli, M. S. Frech, M. E. Steed, K. S. Hruska, M. D. Johnson, J. A. Flaws, and P. A. Furth Introduction of Estrogen Receptor-{alpha} into the tTA/TAg Conditional Mouse Model Precipitates the Development of Estrogen-Responsive Mammary Adenocarcinoma Am. J. Pathol., November 1, 2003; 163(5): 1713 - 1719. [Abstract] [Full Text] [PDF]
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A. Sapino, P. Cassoni, E. Ferrero, M. Bongiovanni, L. Righi, N. Fortunati, P. Crafa, R. Chiarle, and G. Bussolati Estrogen Receptor {alpha} Is a Novel Marker Expressed by Follicular Dendritic Cells in Lymph Nodes and Tumor-Associated Lymphoid Infiltrates Am. J. Pathol., October 1, 2003; 163(4): 1313 - 1320. [Abstract] [Full Text] [PDF]
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 F. X. Lu, Z. Ma, S. Moser, T. G. Evans, and C. J. Miller Effects of Ovarian Steroids on Immunoglobulin-Secreting Cell Function in Healthy Women Clin. Vaccine Immunol., September 1, 2003; 10(5): 944 - 949. [Abstract] [Full Text] [PDF]
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 G.-J. Shim, L. Wang, S. Andersson, N. Nagy, L. L. Kis, Q. Zhang, S. Makela, M. Warner, and J.-A. Gustafsson Disruption of the estrogen receptor {beta} gene in mice causes myeloproliferative disease resembling chronic myeloid leukemia with lymphoid blast crisis PNAS, May 27, 2003; 100(11): 6694 - 6699. [Abstract] [Full Text] [PDF]
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H. Igarashi, T. Kouro, T. Yokota, P. C. Comp, and P. W. Kincade Age and stage dependency of estrogen receptor expression by lymphocyte precursors PNAS, December 18, 2001; 98(26): 15131 - 15136. [Abstract] [Full Text] [PDF]
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C. M. Grimaldi, D. J. Michael, and B. Diamond Cutting Edge: Expansion and Activation of A Population of Autoreactive Marginal Zone B Cells in a Model of Estrogen-Induced Lupus J. Immunol., August 15, 2001; 167(4): 1886 - 1890. [Abstract] [Full Text] [PDF]
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T. Kouro, K. L. Medina, K. Oritani, and P. W. Kincade Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators Blood, May 1, 2001; 97(9): 2708 - 2715. [Abstract] [Full Text] [PDF]
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 T. M. Ellis, M. T. Moser, P. T. Le, R. C. Flanigan, and E. D. Kwon Alterations in peripheral B cells and B cell progenitors following androgen ablation in mice Int. Immunol., April 1, 2001; 13(4): 553 - 558. [Abstract] [Full Text] [PDF]
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B. F. Bebo Jr., A. Fyfe-Johnson, K. Adlard, A. G. Beam, A. A. Vandenbark, and H. Offner Low-Dose Estrogen Therapy Ameliorates Experimental Autoimmune Encephalomyelitis in Two Different Inbred Mouse Strains J. Immunol., February 1, 2001; 166(3): 2080 - 2089. [Abstract] [Full Text] [PDF]
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 T. S. Thurmond, F. G. Murante, J. E. Staples, A. E. Silverstone, K. S. Korach, and T. A. Gasiewicz Role of Estrogen Receptor {alpha} in Hematopoietic Stem Cell Development and B Lymphocyte Maturation in the Male Mouse Endocrinology, July 1, 2000; 141(7): 2309 - 2318. [Abstract] [Full Text] [PDF]
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 K. Mori, T. Ushiyama, K. Inoue, and S. Hukuda Polymorphic CAG repeats of the androgen receptor gene in Japanese male patients with ankylosing spondylitis Rheumatology, May 1, 2000; 39(5): 530 - 532. [Abstract] [Full Text] [PDF]
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K. L. Medina, A. Strasser, and P. W. Kincade Estrogen influences the differentiation, proliferation, and survival of early B-lineage precursors Blood, March 15, 2000; 95(6): 2059 - 2067. [Abstract] [Full Text] [PDF]
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 P. Morales, M. V. Carretero, H. Geronimo, S. G. Copín, M. L. Gaspar, M. A. R. Marcos, and J. Martín-Pérez Influence of Prolactin on the Differentiation of Mouse B-Lymphoid Precursors Cell Growth Differ., August 1, 1999; 10(8): 583 - 590. [Abstract] [Full Text]
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J. F. Couse and K. S. Korach Estrogen Receptor Null Mice: What Have We Learned and Where Will They Lead Us? Endocr. Rev., June 1, 1999; 20(3): 358 - 417. [Abstract] [Full Text]
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Y. Ishimi, C. Miyaura, M. Ohmura, Y. Onoe, T. Sato, Y. Uchiyama, M. Ito, X. Wang, T. Suda, and S. Ikegami Selective Effects of Genistein, a Soybean Isoflavone, on B-Lymphopoiesis and Bone Loss Caused by Estrogen Deficiency Endocrinology, April 1, 1999; 140(4): 1893 - 1900. [Abstract] [Full Text]
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 P. K. Coyle Hormonal Effects on The Immune System Multiple Sclerosis, December 1, 1998; 4(6): 509 - 510. [PDF]
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