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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2059-2067
IMMUNOBIOLOGY
Estrogen influences the differentiation, proliferation, and
survival of early B-lineage precursors
Kay L. Medina,
Andreas Strasser, and
Paul W. Kincade
From the Oklahoma Medical Research Foundation, Oklahoma City, OK;
and The Walter & Eliza Hall Institute, Melbourne, Australia.
 |
Abstract |
B lymphocyte production in murine bone marrow is negatively
regulated by sex steroids and the aim of this study was to identify early hormone sensitive checkpoints. Estrogen (E2) treatment reduced cµ+ pre-B cells, a change that occurred concomitantly
with decreased Ig gene rearrangements and rag-1 transcripts.
Estrogen decreased B lineage precursors in Ig transgenic mice,
demonstrating that hormonal regulation is independent of the
recombination process. B lineage precursors in Bcl-2 transgenic mice
were resistant to estrogen treatment, suggesting that life/death
decisions are involved in hormonal regulation. A previously
uncharacterized population of CD43 cµ B
lineage precursors was identified in normal, Ig transgenic, and
RAG/ mice after estrogen treatment, revealing that
down-regulation of CD43 can occur independent of Ig heavy chain
expression. These cells expressed transcripts for both tdt and
bcl-2, characteristics of early B-cell precursors. BrdU
incorporation analysis revealed that the mitotic activity of early
B-lineage cells is reduced in hormone-treated mice. We conclude that
sex steroids modulate the production of B-lineage
cells by influencing the differentiation, proliferation, and survival
of early B-cell precursors. These findings are informative about
mechanisms of hormonal regulation, as well as the significance of some
differentiation-related events.
(Blood. 2000;95:2059-2067)
© 2000 by The American Society of Hematology.
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Introduction |
B-cell progenitors within bone marrow progress through
developmental stages characterized by changes in surface and
cytoplasmic molecules, responsiveness to growth factors, and status of
Ig gene rearrangements.1-5 Although a wealth of information
is accumulating about these events, important questions remain about
which are intrinsic to differentiating lymphocytes and which are
regulated by extrinsic factors. Gene targeting has been used to
identify transcription factors, cytokines, Ig gene products, and
survival genes that are essential for commitment of cells to, and
progression within, the B lineage.6-8 However, this
experimental approach has primarily defined molecules which act in a
positive way and the role of negative regulators in modulating numbers
of B lymphocytes produced in the marrow remains poorly resolved. A
major goal of this study was to identify critical events in B
lymphopoiesis that are sensitive to a known negative regulator, 17-
estradiol (E2).9
Steady state B lymphopoiesis is believed to be influenced by
interactions between lymphoid precursors and marrow stromal cells, as
well as by the availability of stimulatory and inhibitory regulators. Under certain experimental conditions, IL-1, IL-3, IL-4, IL-10,  -interferon,  / interferon, TGF-, and GM-CSF are capable of suppressing B lymphopoiesis.10-12 However, it has not been
established whether most of these factors are involved in normal steady
state regulation of B-cell production. The negative effects of sex
steroids on B lymphopoiesis were initially suggested by the observation that a selective reduction in pre-B cells occurred during
pregnancy.13 This was reflected by reduced numbers of IL-7
responding cells in bone marrow and diminished lymphocyte proliferation
as ascertained by BrdU incorporation. In contrast, percentages of
mature recirculating B lymphocytes, as well as myeloid and erythroid
progenitors in marrow, were not reduced. Systemic levels of estrogen
and progesterone increase during pregnancy, and we found that sustained
exposure to estrogen caused a reduction of IL-7 responsive cells and
pre-B cells in the marrow, as well as newly made B cells in the
periphery.9 Additional observations suggested that sex
steroids participate in normal, steady state control of B
lymphopoiesis. Hormone-deficient hypogonadal (HPG) or castrated mice
were found to have elevated numbers of B-cell precursors, and estrogen
replacement reduced them to within the normal range.14-16 A
recent study revealed that multiple estrogen and androgen receptors can
mediate these responses.17 Although changes in systemic
levels of estrogen can profoundly alter the numbers of new B
lymphocytes generated in the marrow, the mechanism is largely unknown.
We have now identified hormone sensitive stages in early B-cell
development by controlled exposure of normal mice, Ig rearrangement deficient mice, Ig transgenic mice, and Bcl-2 transgenic mice to
estrogen. We documented significant changes in B lineage precursors, including decreased rag-1 gene expression, frequency of Ig gene rearrangements, and numbers of cµ+ pre-B cells. However,
we also found that E2 treatment had dramatic effects on Ig transgenic
mice and immunodeficient RAG/ mice. Hormone
treatment significantly reduced the mitotic activity of B lineage
precursors, causing the accumulation of noncycling cells.
Overexpression of Bcl-2 in the B lineage abrogated hormonal regulation.
We conclude that estrogen regulates the production of B lineage
lymphocytes by influencing critical early events associated with the
differentiation, proliferation, and survival of early precursors.
| |
Material and methods |
Animals
BALB/c, RAG-2/ (purchased from
Taconic Farms, Germantown, NY), and Eµ-bcl-2 (line 36) mice
were bred and maintained in our laboratory animal resource
facility.18 Bcl-2 transgenic mice were identified by flow
cytometric analysis using the antibody to human Bcl-2, 6C8 (PharMingen,
San Diego, CA) diluted in 0.03% saponin, and revealed by FITC
conjugated goat antihamster Ig (Caltag, Burlingame, CA). Nontransgenic
littermates were used as controls. RAG-1/(S),
3-83µ transgenic, RAG-1// human µ transgenic and the appropriate controls mice have been previously
described.19,20 RAG-1/(M) mice
were purchased from Jackson Labs (Bar Harbor, ME).21
Hormone treatment in vivo
Estrogen treatment causes an osteosclerotic response in the
marrow,22 therefore all data are reported as percentages.
Implantation of pellets containing 0.05 to 0.1 mg of 17- estradiol
(E2) reduced CD45R+ B-cell precursors and IL-7 responding
cells, but allowed sufficient progenitors to remain so that populations
of early cells could be analyzed.
Time-release pellets of E2 were purchased from Innovative Research of
America (Sarasota, FL) and implanted subcutaneously with a sterile
trochar. Mice given 0.05 mg dose E2 were analyzed after 14 days,
whereas those given 0.1 mg were analyzed after 7 days.
Immunofluorescent staining and cell sorting
Cell surface staining.
Cells were harvested from femurs and tibias from individual mice and
suspended in staining buffer (phosphate-buffered saline without
Ca2+ and Mg2+ with 3% heat-inactivated fetal
bovine serum and 0.1% sodium azide), as previously
described.13 Antibodies used in these analyses were the
following: CD45R (RA3-6B2), CD45R (14.8), CD24 (M1/69 or 30F1), BP-1,
CD43 (S7), CD19 (1D3), CD25 (7D4) were purchased from PharMingen, and
SB/199 FITC (IL-7R ) was purified and labeled in our lab.
Ultra-avidin Texas Red (Leinco, Mallwin, MO) or Streptavidin Cychrome
(PharMingen) were used to reveal biotinylated reagents.
Cytoplasmic staining for µ.
After surface staining, bone marrow cells were incubated in 4%
paraformaldehyde in PBS- (pH 7.4) for 10 minutes on ice,
followed by permeabilization for 20 minutes at room temperature in PBS
containing 0.2% Tween 20. Mouse cells were then incubated with a
FITC-labeled polyclonal Goat antimouse IgM (Southern Biotechnology,
Birmingham, AL) or Rat antimouse IgM antiserum (Zymed, San Francisco,
CA). Intracellular staining with the appropriate isotype control was
used to set gates to identify positively staining cells. Human µ transgene expression was detected using affinity purified goat
antihuman IgM F(ab')2 FITC (Southern Biotechnology). Cells
were kept on ice until analyzed.
Cell sorting.
Bone marrow cells from control or E2-treated mice were harvested and
enriched for lymphoid cells by incubating cells with Gr-1, Ter119
(PharMingen), and Mac-1 (10 × concentrated culture supernatant,
hybridoma from American Type Culture Collection, Logan, UT) monoclonal
antibodies, followed by washing with staining buffer. Cells were then
incubated with goat antirat IgG coated magnetic beads (PerSeptive
Diagnostics, Framingham, MA), followed by magnetic separation to
deplete myeloid and erythroid cells. Lymphoid enriched cells were then
incubated with the appropriate combinations of antibodies, which
allowed identification of early populations.2 Cells were
sorted on the FACStarPlus cell sorter.
Ig gene rearrangement
Lymphoid enriched marrow cells were resuspended in PCR lysis buffer
(10 mmol/L Tris, pH 8.4, 50 mmol/L KCl, 2 mmol/L MgCl2, 0.5% NP-40, 0.5% Tween-20, 40 µg/mL proteinase K) and incubated overnight at 50°C. The proteinase K was inactivated by incubation at 95°C for 10 minutes and the DNA used directly for PCR. Two microliters of template was used neat, diluted 1/10, and 1/100. 50 µL
PCR reactions were performed using template, 625 ng of each primer, 5 µL of 10 × buffer, 2.0 mmol/L MgCl2, 100 µg/mL
of bovine serum albumin (BSA), 10 µmol dNTPs, and 2.5 U Taq DNA
polymerase (FisherBiotech, Pittsburgh, PA). Twenty-five cycles of
amplification were performed consisting of 1 minute at 95°C, 45 seconds at 63°, and 2 minutes at 72°C, followed by a single
incubation at 72° for 10 minutes. Ten microliters of the PCR
reaction was then electrophoresed through a 1.4% agarose gel in Tris
Borate EDTA (TBE) buffer. The gel was transferred to a
nylon membrane (MSI, Westboro, MA), baked at 80° C for 2 hours,
then cross-linked with ultraviolet (UV) light for 5 minutes. The
membranes were probed with a 32P-labeled JH34
probe23 and exposed overnight on Kodak Scientific Imaging
Film (Eastman Kodak, Rochester, NY). The primers used were alpha
actin,2 DHL(5'), and J3(3') to detect
DH-JH,24 Q52 and 7183(5'
primers used separately)3 and J3(3') to detect VH-DJH rearrangements, and Mu0(5') and
J1(3')24 to detect germline alleles.
RT-PCR analysis of gene expression
For RNA isolation, sorted cells were washed and resuspended in
Trizol Reagent (Gibco-BRL). RNA was prepared and resuspended in
DEPC-treated water. Total RNA was treated with Dnase I (Gibco-BRL) to
remove contaminating genomic DNA, and cDNA was made using oligo-dT (Gibco-BRL) and Moloney murine leukemia virus reverse transcriptase (Gibco-BRL). RNA representing 104 cell equivalents was used
neat, diluted 1:5, or 1:25 per RT-PCR reaction. The conditions for
RT-PCR were 95°C for 1 minute, 63°C for 45 seconds, 72°C
for 1.5 minutes for 35 cycles, followed by a 15 minutes incubation at
72°C. All RT-PCR reactions were performed using previously
published primer sequences.3 One-fifth of the PCR product
was electrophoresed in a 1.2% agarose gel in Tris Acetate EDTA
(TAE) buffer, transferred onto nylon membranes (MSI, Westboro, MA) and hybridized using random primed probes (Boehringer Mannheim, Germany). The blots were prehybridized for 1 to 2 hours then
hybridized overnight at 42°C using reagents purchased from 5'-3', Inc. (Boulder, CO). After washing, the blots were
exposed from 2 hours to overnight on Kodak Scientific Imaging Film.
BrdU treatment in vivo and analysis
Mice were treated with 0.1 mg pellets of E2 for 6 or 7 days.
RAG-2/ mice were treated with 0.1 mg pellets
of E2 for 10 days. BrdU at 1 mg/mL (+5% glucose to overcome taste
aversion) was given in drinking water for the last 3 days of treatment.
Bone marrow was harvested and analyzed for BrdU uptake as previously
described.13
| |
Results |
Estrogen treatment reduces early cµ+ pre-B cells
Our previous studies revealed that sustained exposure to17
estradiol selectively reduced the production of new B lymphocytes in
murine bone marrow. However, the doses of estrogen used severely depleted all B-lineage precursors, making analysis of early hormone sensitive events difficult. We found that sustained exposure to 0.05 to
0.1 mg of estrogen (2.4-4.8 µg of hormone released per day) allowed
sufficient early precursor populations to remain so that
characterization of early hormone sensitive events could be performed.
Percentages of total pro-B cells identified as
CD45R+CD43+ were similar in E2-treated mice,
compared with controls with this dose of hormone (9.0% ± 1.8% vs
8.4% ± 1.6%, respectively, and Figure
1A), whereas their progeny, the small pre-B
cells, were diminished (34.8% ± 5.5% vs 14.8% ± 3.3% in
controls vs E2 treated, respectively, Figure 1A). Recirculating
CD45RhisIgM+ cells (Mature B cells, Figure 1A)
were unaffected by this dose of E2. Similar results were obtained using
CD19, a more B-lineage restricted marker, to evaluate the effects of
this dose of hormone (summarized in Table
1).
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| Fig 1.
Early pre-B cells are depleted in estrogen-treated BALB/c
mice.
(A) B-lineage populations in bone marrow from control and E2-treated
mice were revealed by surface staining with antibodies to CD45R and
CD43. Mature B cells were identified as
CD45RhiCD43. (B) Cells within the
CD45R+CD43+ gate were then analyzed for cµ
heavy chains (solid lines) as compared with staining with a normal goat
IgG control (dotted lines). The histograms depict 50 000 events
collected within lymphocyte light scatter gates and are representative
of results obtained from 7 individual control and 9 estrogen-treated
mice.
|
|
Early cµ+ pre-B cells within the
CD45R+CD43+ compartment have been
identified.4,5,25 To determine whether the loss of pre-B cells initiated before down-regulation of CD43, early
CD45R+CD43+ B cell precursors were isolated
from marrows of control and estrogen-treated mice and analyzed for cµ
protein. We found that estrogen treatment reduced percentages of
cµ+ cells within the CD45R+CD43+
compartment (10.0% ± 3.5% vs 25.6% ± 9.4% in estrogen
treated vs controls, respectively, Figure 1B). Therefore, loss of pre-B cells in hormone-treated animals initiates before down-regulation of
CD43 in the B lineage.
Estrogen treatment reduces Ig heavy chain gene rearrangements
The reduction of early cµ+ pre-B cells in E2-treated
animals could be the result of diminished Ig gene rearrangement.
Therefore, we isolated lymphocytes from marrows of individual control
and hormone-treated animals and compared incidences of
DH-JH and VH-DJH gene
rearrangements by semiquantitative PCR. We found that
DH-JH rearrangements in hormone treated animals
were slightly, but consistently reduced compared with control mice
(Figure 2A). Next, we examined VH-DJH gene rearrangements to the Q52 and 7183 (not shown) V region families. We found a 75% decrease in
VH-DJH rearrangements, involving Q52 and 7183 V
genes (Figure 2A and data not shown). Therefore, the hormone could
regulate B-cell production by influencing the recombination process
itself, the proliferation of cells having undergone successful
recombination events, or cµ+ pre-B cells could be
particularly sensitive to E2 or a hormone-induced negative regulator.
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| Fig 2.
Estrogen treatment reduces Ig gene rearrangements in bone
marrow.
(A) DNA samples representing equivalent numbers of bone marrow
lymphocytes from control and E2-treated mice were evaluated for
DH-JH and VH-DJH gene
rearrangements by PCR and specific products were labeled with respect
to rearrangements to J1, J2, or J3. DH-JH
rearrangements were detected with a primer that recognizes 9 of 10 known D minigenes and a J3 primer.24
VH-DJH rearrangements were detected using
primers specific for the Q52 V region family and a J3
primer.3,24 Genomic DNA was serially diluted 1:1,1:10,1:100
for semiquantative analysis and 25 cycles of amplification was
performed. Alpha actin was used as a control for genome representation.
Densitometry confirmed that equal amounts of DNA were compared. These
data are representative of results obtained in 3 independent
experiments. (B) CD45R+CD43
sIg B-lineage precursors in estrogen-treated mice
are enriched for cells with germline Ig genes.
CD45R+CD43 sIg cells
were sorted from control or estrogen-treated mice. PCR was performed
with genomic DNA, using primers that amplify segments normally deleted
during Ig gene rearrangement (see "Materials and Methods"). DNA
was amplified for 30 cycles and alpha actin was used as a control for
genome representation. These data were obtained by pooling cells from 4 control and 4 estrogen-treated mice. RAG-1/
bone marrow was used as a control for germline DNA. Cells sorted from
control and estrogen-treated mice were 99% and 100% pure,
respectively, based on postsort analyses.
|
|
Expression of IL-7R has been shown to be important for
differentiation of B lineage cells and VH-DJH
rearrangements to distal VH gene families are impaired in
mice lacking this receptor. A flow cytometric analysis of IL-7R
expression on bone marrow cells from hormone-treated animals revealed
no reduction in percentages of CD45R+IL-7R+
cells (4.7% ± 1.1% vs 6.9% ± 0.8% in control vs E2 treated,
respectively). Therefore, E2 does not inhibit B-cell differentiation by
diminishing expression of the IL-7R chain.
Rag-1 transcripts are reduced in hormone-treated animals.
The above findings demonstrate that cells within, and prior to, the
pre-B compartment are hormone sensitive. Therefore, we sorted early
pro-B cells
(CD45R+CD43+CD24+BP-1)
from control and estrogen-treated mice and used RT-PCR to evaluate critical genes expressed at this stage.3 Although
rag-2 transcripts were essentially normal in cells from
estrogen-treated mice, rag-1 transcripts were
substantially reduced (Figure 3A). Estrogen
had no discernable influence on expression of mb-1,
B29,  5, tdt, or VpreB (Figure 3B and
data not shown). Taken together, this analysis suggests that particular
events in early B-lineage differentiation, and primarily those
associated with Ig gene rearrangements, are subject to hormonal
regulation.
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| Fig 3.
RAG-1 expression is preferentially suppressed in
estrogen-treated mice.
Early pro-B cells
(CD45R+CD43+CD24+BP-1
sIgM) were sorted from control and E2-treated BALB/c
mice. Total RNA representing 104 cell equivalents was
reverse transcribed into cDNA, followed by RT-PCR using previously
published primers. The data represent autoradiographs of PCR products
after membrane transfer and hybridization with appropriate
32P-labeled probes or ethidium-stained bands. Similar
results were obtained in 3 independent experiments.
|
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B cells from Ig transgenic mice are estrogen sensitive.
The above findings raised the possibility of an early, hormone
sensitive, control point that determines how many B-cell precursors are
produced. We speculated that expression of Ig heavy and light chain
transgenes, that are known to suppress endogenous
VH-DJH gene rearrangements,20 might
make B lymphopoiesis hormone resistant. Analysis of the pro-B
compartment in hormone-treated 3-83 µ Ig transgenic mice revealed
a significant decrease in percentages of B-lineage precursors starting
as early as the CD19+CD43+ stage and extending
into the pre-B stage
(CD19+CD43sIgM,
Figure 4A and summarized in Table 1).
Incidences of IL-7 responsive precursors in transgenic mice were
comparable to those in normal C57BL/6 mice and these declined an
average of 92% with estrogen treatment. Percentages of newly made
IgM+IgD B cells declined more than 80%
in estrogen-treated Ig transgenic mice (16.1% ± 2.5% in control
vs 2.5% ± 0.1% in hormone treated), whereas percentages of
recirculating IgM+IgD+ B cells did not change
appreciably (3.5% ± 0.3% in controls vs 2.9% ± 0.6% in
hormone treated). Therefore, enforced expression of Ig heavy and light
chains did not alter sensitivity of the B lineage to this hormone.
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| Fig 4.
B-lineage cells in Ig transgenic mice are sensitive to
estrogen.
Flow cytometry was used to analyze bone marrow cells from untreated
(Control) and E2-treated Ig transgenic mice. Results obtained with
murine µ heavy chain plus light chain (3-83µ) transgenic
mice are shown in Panel A. Panel B shows a similar experiment performed
with immunodeficient mice bearing a human µ transgene
(RAG-1//Hu µ). Incidences of Pro-B cells
are given in the upper right quadrants, whereas incidences of more
differentiated cells are indicated in the upper left quadrants. The
data are summarized in Table 1.
|
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Although introduction of Ig transgenes has been shown to suppress
VH-DJH Ig gene rearrangement events, endogenous
DH-JH rearrangements occur normally. The above
results demonstrate that early CD19+ CD43+
precursors are sensitive to hormonal regulation. Introduction of a
human heavy chain transgene in
rag/ mice
(RAG-1//hu µ) allows progression to the
small, CD43, pre-B cell stage.19,26
Estrogen treatment in this model caused a 70% reduction in
CD19+CD43 cells (7.4%, treated vs
30.9%, control; Figure 4B). There was a corresponding reduction in
numbers of IL-7 responding precursors from 93 to 15 colony forming
cells per 105 cultured marrow cells. From these data we
conclude that hormonal regulation of B lymphopoiesis is independent of
the recombination process.
B lymphopoiesis is resistant in Bcl-2 transgenic mice.
Diminished output of B-lineage cells in E2-treated animals might result
from decreased production and/or survival of lymphocyte progenitors.
Bcl-2 is known to be essential for the production of lymphocytes and is
expressed in early lymphocyte precursors.8,27 We found that
total CD45R+ cells were not reduced in E2 treated Bcl-2 transgenic mice
(59.0% ± 1.5% versus 55.6% ± 1.5% in E2 versus control
mice, respectively, and Figure 5), nor was
the distribution of cells in the B-lineage based on differential
expression of sIgM (data not shown). In addition, percentages of
CD19+ B-cell precursors were not affected by hormone
treatment (data not shown). Early cµ+ pre-B cells are
substantially altered by estrogen in normal mice (Figure 1B and see
Figure 7 below). However, percentages of both CD43+ and
CD43 pre-B cells remained unchanged in Bcl-2
transgenic mice after exposure to hormone (19.1% ± 4.9% vs 25.7% ± 8.9% CD43+ pre-B cells and 87.0% ± 2.0% vs
89.4% ± 2.1% CD43 pre-B cells in control and
estrogen treated, respectively). Therefore, constitutive expression of
Bcl-2 in the B lineage abrogated hormonal control of B lymphopoiesis.
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| Fig 5.
B-cell precursors in Bcl-2 transgenic mice are resistant
to hormone treatment.
Bcl-2 transgenic mice were treated with E2 for 14 days before bone
marrow harvest and analysis by flow cytometry.
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Estrogen treatment reduces the mitotic activity of B-cell
precursors.
Bcl-2 has an influence on cell cycle progression, in addition to its
function as a survival protein.28,29 B-cell precursors normally undergo proliferative expansion and cell cycle-related genes
could represent hormone sensitive targets. To evaluate this possibility, BALB/c mice were given BrdU in drinking water for the last
3 days of an E2 treatment course. In control animals, an average of
84.8% ± 0.8% of the B-lineage lymphocytes incorporated BrdU,
whereas only 53.6% ± 3.0% had undergone division during this
interval in E2-treated mice (Figure 6,
upper contour plots). Essentially all the unlabeled cells in both
groups were small as assessed by low-angle light scatter. A 3-fold
increase in small noncycling lymphocytes was documented in
hormone-treated bone marrow (13.4% ± 0.7% in control vs 39.9% ± 2.4% in estrogen-treated animals). Furthermore, there was a
conspicuous population of very heavily labeled small lymphocytes in
control, but not estrogen-treated animals. These cells presumably
represent the normal product of multiple cell divisions occurring
during the 3-day interval. Supporting results were obtained when
CD45R+CD43+ and
CD45RdullCD43 B-lineage precursors were
sorted from control and E2-treated animals, fixed, and evaluated with
respect to cell cycle status. Percentages of cells in the S+
G2/M stages of the cell cycle were reduced approximately
50% after estrogen exposure (data not shown).
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| Fig 6.
Estrogen reduces mitotic activity of B cell precursors.
BALB/c and RAG-2/ mice were given hormone
implants and BrdU (1 mg/mL +5% glucose) was administered in the
drinking water for the last 3 days of treatment. CD45R+
cells were then analyzed with respect to size (forward angle
light scatter) versus BrdU uptake. The data are representative of
results obtained with 5 control and 8 estrogen-treated BALB/c and 3 control and 3 estrogen-treated RAG-2/ mice.
|
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Similar experiments conducted with RAG/ mice
revealed that the mitotic activity of B-cell precursors is hormone
sensitive from an early stage of differentiation. Although 3 days of
BrdU exposure labeled 90.3% ± 1.2% of the pro-B cells in control
RAG/ mice, this value was reduced to 70.5% ± 2.9% with estrogen treatment (Figure 6). Particularly noteworthy
was the 7-fold increase in small BrdU cells that
resulted from hormone elevation. These represent cells that remained
out of cycle throughout the 3-day interval. Expansion of early B-cell
precursors is required to maintain normal lymphopoiesis, and we now
show that estrogen treatment reduces proliferation of B-lineage
precursors before and independent of Ig gene rearrangement.
A unique population of B-lineage cells accumulates in
estrogen-treated mice.
Our analysis of estrogen-treated animals revealed a greater degree of
change in cµ+ pre-B cells than in total B-lineage
precursors. As previously reported, almost all (93% ± 2%) of the
CD45RdullCD43 precursors in normal mice
are small, pre-B cells. In contrast, only 60% ± 6% of the cells
with these surface characteristics were cµ+ in
estrogen-treated BALB/c mice (Figure 7A).
The decrease in cµ+ pre-B cells is independent of Ig gene
rearrangement, as similar results were obtained with a hormone-treated
RAG-1//hu µ Ig transgenic mouse (Figure
7A). Loss of CD43 is generally held to represent a late event in B
lymphopoiesis and roughly corresponds to the transition from large to
small pre-B cell stages.2,30 Therefore, it initially seemed
likely that the conspicuous CD43
cµ cells in estrogen-treated animals were
defective pre-B cells that had somehow traversed this critical
checkpoint. Uniform expression of CD19, small size, and high levels of
BP-1 antigen would also be consistent with that interpretation (see
Figure 9 below).
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| Fig 7.
A normally rare population of B-cell precursors
accumulates in estrogen-treated mice.
CD45R+CD43 sIgM cells
from control or E2-treated BALB/c mice were sorted, fixed and made
permeable as described in the "Materials and Methods." The sorted
cells were then incubated with an antimouse IgM to detect cµ
expression (Panel A). In this example, 40% of the CD43
cells in hormone-treated mice were cµ
compared with 7% in the control animals. Panel B shows a typical
RT-PCR analysis of tdt and bcl-2 gene expression in
CD45R+CD43 sIgM cells
sorted from control (bone marrow pooled from 3 mice) or E2-treated
animals (bone marrow pooled from 5 mice).
|
|
More detailed analysis revealed that the
CD45R+CD43 cµ cells
lacked CD25, a marker normally acquired in conjunction with successful Ig gene rearrangement (Figure 9, below).4,31
Down-regulation of tdt represents another hallmark in B-lineage
progression.1,3,32 Tdt is normally found in cells
before Ig gene rearrangement and expression ceases with appearance of µ heavy chains. RT-PCR analyses of sorted
CD45RdullCD43 sIgM
lymphocytes revealed persisting high levels of tdt transcripts in cells from E2-treated animals (Figure 7B). Additional evidence of
the early stage of differentiation represented by these cells was
expression of transcripts for bcl-2 (Figure 7B).
Ig heavy chain gene rearrangement status represents a definitive
characteristic of late stage precursors.3,5,33 Therefore, we sorted B-lineage precursors from control and E2-treated animals and
assessed germline IgH gene segments (Figure 2B). Nonrearranged IgH
segments were increased approximately 2-fold in CD43
lymphocytes from hormone-treated animals. This is consistent with the
appearance of µ cells, persisting tdt
mRNA, and the decreased abundance of IgH gene rearrangement products in
these lymphocytes (Figure 2B). Thus, many of the
CD43 µ precursors had not
initiated Ig heavy chain gene rearrangement.
The identification of CD45R+CD43
µ cells in normal mice treated with estrogen led
us to study mice that cannot initiate Ig gene rearrangement. In
contrast to what we observed with normal or Ig transgenic mice,
sustained exposure to estrogen did not reduce percentages of total
CD19+ B-lineage precursors in
RAG/ mice (Table 1). In addition, markedly
reduced CD43 expression was observed in 3 different lines of
RAG/ mice after hormone treatment. This
population became especially conspicuous because small
CD43 cells are normally rare in these animals
(Figure
8).19,26 The CD43 lymphocytes were small in size, expressed
CD19 as well as BP-1, and lacked both CD25 and cytoplasmic µ
chains (Figure 9 and data not shown). The
CD45R+BP-1+CD43 cells were
isolated from estrogen-treated RAG-2/ mice
and compared with CD43 cells from untreated normal
mice by RT-PCR. Transcripts corresponding to tdt and
bcl-2 were abundant (Figure 7B and data not shown). Therefore,
CD43 precursors that accumulated in hormone-treated
RAG/ and BALB/c mice were similar in many
respects.
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| Fig 8.
Normally rare B-lineage cells accumulate in
hormone-treated RAG/ mice.
Mice were treated with E2 for 14 days before bone marrow harvest and
analysis by flow cytometry. The boxed regions contain B-cell precursors
that accumulate in hormone-treated animals.
|
|
View larger version (21K):
[in this window]
[in a new window]
| Fig 9.
Characterization of
CD45R+CD43 cells in
RAG-1/(M) mice.
Bone marrow cells from control (solid lines) or E2-treated (hatched
lines) RAG-1/(M) mice were harvested and
compared by flow cytometry with respect to size and expression of
B-lineage surface markers.
|
|
In the course of these studies, an interesting observation was made
with 1 line of RAG-1 targeted mice
(RAG-1/(M)).21 These animals had
a conspicuous population of CD43 low to negative cells without exposure
to hormone (Figure 8). They differed from CD43 cells
found in estrogen-treated mice in that more were large in size and BP-1
antigen expression tended to be low (Figure 9). Estrogen treatment of
RAG-1/(M) mice resulted in the appearance of
small
CD45R+CD43CD19+CD25BP-1+
lymphocytes (Figures 8 and 9). The 2 lines of RAG-1 gene targeted animals are both on a C57BL/6 background, but were produced with different gene targeting strategies.19,21
We conclude that the accumulation of CD43
µ cells in response to estrogen does not require
Ig gene rearrangement. These unusual cells probably derive from a
relatively early stage of differentiation because they express
bcl-2. Expression of CD25 and down-regulation of tdt,
represent events linked with successful IgH gene rearrangement and do
not occur in these normally rare progenitors. There is acquisition of
BP-1 and loss of CD43, although these changes were previously thought
to be exclusively associated with B-lineage progression.
| |
Discussion |
The production of new B lymphocytes in murine bone marrow is
suppressed during pregnancy or after sustained exposure to estrogen, and augmented in mice deficient in sex steroids.9,13-15,17
These data suggest that sex steroids are involved in normal steady
state regulation of B lymphopoiesis. We now show that experimental
manipulation of estrogen-influenced numbers of undifferentiated
precursors, Ig gene rearrangements, and proliferative activity.
E2-induced population changes were blocked by expression of a Bcl-2
transgene. Observations made with Ig transgenic and RAG deficient mice
demonstrated that estrogen influenced some events that precede and are
independent of Ig gene rearrangement. A normally rare population of
early lymphocytes accumulated in estrogen-treated mice. These findings are informative about potential mechanisms for hormonal control of B
lymphopoiesis, as well as the sequence and significance of some differentiation related changes.
B-lineage precursors in the bone marrow have been developmentally
ordered by changes in display of cell surface and cytoplasmic markers,
changes in patterns of gene expression, status of Ig gene
rearrangements, and dependence on stromal cells for proliferation and
survival.1-5 Any of these events could represent potential targets of hormone-mediated regulation.
CD19+CD43+ pro-B cells were depleted in most
animal models studied, revealing that hormonal regulation initiates
early in the B lineage (summarized in Table 1). Therefore, we examined
the consequences of estrogen treatment on critical events in
B-cell development, including those associated with the
differentiation, survival, and proliferation of early B-lineage precursors.
Changes at the early pro-B stage could reflect decreased expression of
genes required for recombination events and expression of the pre-B
receptor. Therefore, we characterized pro-B cells from control and
hormone-treated animals with respect to expression of critical early
genes, Ig gene rearrangements, and cµ protein. Numbers of
cµ+ cells were reduced in the CD45R+
CD43+ compartment and we found a concomitant reduction in
rag-1 transcripts and VH-DJH Ig gene
rearrangements (Figures 1 to 3). In contrast, transcripts corresponding
to other antigen receptor components, including Ig-, Ig-,
5, and VpreB, were unaffected (Figure 3 and data not
shown). Experiments with 2 types of Ig transgenic mice demonstrated
that events that precede, or are in addition to, Ig gene rearrangement
are hormone sensitive. Rearranged µ transgene products suppress
endogenous Ig gene rearrangements in normal mice and alter pro-B
population sizes.34 Regardless, estrogen treatment
dramatically reduced numbers of B-lineage cells in such animals (Table
1 and Figure 4). Furthermore, similar changes were observed in
RAG-1//hu µ mice that are incapable of
endogenous Ig gene rearrangements and where a µ transgene is known to
be expressed from a very early stage.35 Finally, none of
the changes we observed resulted from down-regulation of the IL-7
receptor on B lineage precursors. Taken together, these results
demonstrate that early differentiation related events in B
lymphopoiesis are subject to hormonal regulation.
Recent studies suggest that estrogen can influence levels of Bcl-2 in
nonlymphoid tissues such as brain neurons and breast cancer
cells.36-38 Up-regulation of Bcl-2 by estrogen may account for the resistance of some breast cancers to antitumor
drugs.39 Overexpression of Bcl-2 in the B lineage protects
precursors from apoptosis induced by growth factor deprivation,
ionizing radiation, and the glucocorticoid hormone
dexamethasone.40 We found that the same transgene provided
remarkable protection from estrogen for both pro-B and pre-B cells, as
well as IL-7 responding precursors (Figure 5 and data not shown). In
addition, we found that the CD43 cµ-
cells in hormone-treated mice expressed Bcl-2 transcripts, possibly explaining their persistance with estrogen treatment. These data suggest that hormonal regulation involves life/death decisions in early
B lineage cells.
Proliferative expansion at discrete stages must be sustained to
maintain steady state production of B cells.1,41-44
Interestingly, a Bcl-2 transgene is known to affect the mitotic
activity of early B-lineage cells.28 BrdU incorporation and
cell cycle analysis revealed that estrogen treatment dramatically
reduced the mitotic activity of B-lineage precursors in normal and
RAG/ mice (Figure 6). Early
CD43+CD45R+ cells have long-term proliferation
capacity on stromal cells, and upon differentiation, give rise to
precursors that can respond to IL-7 alone. Diminished mitotic activity
of these early precursors in estrogen-treated animals would provide
fewer cells undergoing Ig gene rearrangement, less IL-7 responding
cells, cµ+ pre-B cells, and immature B lymphocytes. We
documented all of these changes in hormone-treated animals and
previously in stromal cell cocultures in which numbers of recovered
lymphocytes were greatly reduced in the presence of
estrogen.45 In addition, diminished mitotic activity would
also result in less Ig transgene expressing precursors, as we observed
(Figure 4). BrdU uptake was not evaluated in Ig transgenic mice because
early µ expression is known to alter the kinetics of B-lymphocyte
development.34 Thus, estrogen also regulates B
lymphopoiesis via its influence on cell proliferation. The effect is
lineage specific and involves precursors from an early stage.
Overall percentages of phenotypically defined "pro-B cells" were
not reduced in hormone-treated BALB/c mice, largely because of an
accumulation of
CD45R+CD19+BP-1+CD25
cµ B-cell precursors. Most of their properties,
such as continued expression of bcl-2 and tdt, along
with the presence of these cells in RAG/
mice, are consistent with a relatively early stage of B-lineage development.1,32 Our studies also indicate that these cells are noncycling and have at least some IgH genes in germline
configuration. This normally rare population of cells may be positioned
near a hormone-regulated control point in B-lineage differentiation.
Expression of a rearranged µ heavy chain as part of a pre-B receptor
complex and signaling via associated Ig-/Ig- molecules are
necessary and sufficient for progression of cells in the B lineage.19,26,46 Subsequent events include a short period of rapid proliferation, exit from the cell cycle, reduction in cell
size, loss of CD43, gain of CD25, and initiation of Ig light chain gene
rearrangement.30,47 Virtually all of the
CD45R+CD43 lymphocytes in normal bone
marrow contain µ heavy chains of immunoglobulin (Figure 7A). For this
reason, loss of CD43 is widely used as a hallmark for transition from
the pro-B/large pre-B to small pre-B cell stages. However, we provide
evidence that down-regulation of CD43 is not dependent on µ chain
expression. Hormone treatment induced the accumulation of small,
non-cycling cells and a conspicuous population of
CD43 cells in the absence of Ig gene rearrangement.
Our results show that down-regulation of CD43 is not coupled to
expression of CD25, and CD25 to be a more valid indication of
successful IgH gene rearrangement at the pro-B/pre-B
transition.4,31 A tendency for down-regulation of CD43 in
the absence of Ig gene rearrangement was found in 1 of 2 lines of RAG-1
targeted mice (Figure 8). Unlike the CD43
cµ cells that accumulated in normal or
RAG/ mice after estrogen treatment, the
CD43 cells in RAG-1/(M)
were large in size and expressed low densities of BP-1. This finding is
of unknown significance and may reflect differences in gene targeting
strategies. However, it again brings into question the validity of CD43
as a "differentiation" marker in all circumstances.
These findings also provided insight into another
"differentiation" related change. BP-1 acquisition has been used
to discriminate late pro-B cells from earlier precursors, a transition
that corresponds to completed IgH rearrangements (either successful or
abortive).3,5 However, the CD43 cells
that accumulated in estrogen-treated animals expressed high levels of
BP-1, although they were immature in all other respects (see above).
Other studies suggest that BP-1 expression may merely reflect the
period of exposure of precursors to IL-7.48
Our observations raise many issues concerning regulation of normal B
lymphopoiesis. An early, hormone-sensitive, control point appears to
exist in murine bone marrow. Ongoing studies should reveal which cells
in murine marrow possess relevant hormone receptors and address
questions about transcription factors and cell cycle regulators known
to be estrogen regulated in other cell types.45,49-52 It
will be particularly important to learn whether parallels exist for
humans, given the widespread therapeutic use of sex steroids, their
agonists, and antagonists. Furthermore, a mechanistic connection might
be sought between our findings and the well-known influences of
estrogen on autoimmune diseases.53-55
| |
Acknowledgments |
We are grateful to Drs Chris Roman and David Baltimore for
RAG-1/(s) mice, Dr David Nemazee for
providing the 3-83 µ Ig transgenic mice, Dr. Richard Hardy for
providing RAG-1/and
RAG-1//Hu µ Ig transgenic mice, and Drs
Alan Harris and Suzanne Cory for providing the Bcl-2 transgenic mice.
We acknowledge the expert technical assistance provided by Karla
Garrett and expert flow cytometry assistance provided by Jim Henthorn
and Viji Dandapani. Finally, we appreciate the secretarial assistance
provided by Shelli Wasson.
| |
Footnotes |
Submitted August 9, 1999; accepted November 24, 1999.
Supported by grant AI 20069 from the National Institutes of Health.
Reprints: Paul W. Kincade, Immunobiology and Cancer
Program, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma
City, OK 73104; e-mail: paul-kincade{at}omrf.ouhsc.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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A. L. Zoller and G. J. Kersh
Estrogen Induces Thymic Atrophy by Eliminating Early Thymic Progenitors and Inhibiting Proliferation of beta-Selected Thymocytes.
J. Immunol.,
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C. M. Grimaldi, V. Jeganathan, and B. Diamond
Hormonal Regulation of B Cell Development: 17beta-Estradiol Impairs Negative Selection of High-Affinity DNA-Reactive B Cells at More Than One Developmental Checkpoint.
J. Immunol.,
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M. Jayachandran, A. Sanzo, W. G. Owen, and V. M. Miller
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A. Bouman, M. J. Heineman, and M. M. Faas
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R. Pelayo, J. Hirose, J. Huang, K. P. Garrett, A. Delogu, M. Busslinger, and P. W. Kincade
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Blood,
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H. Igarashi, K. L. Medina, T. Yokota, M. I. D. Rossi, N. Sakaguchi, P. C. Comp, and P. W. Kincade
Early lymphoid progenitors in mouse and man are highly sensitive to glucocorticoids
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S. M. Caucheteux, M.-C. Gendron, and C. Kanellopoulos-Langevin
Pregnancy-induced alterations of B cell maturation and survival are differentially affected by Fas and Bcl-2, independently of BcR expression
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H. Yurino, S. Ishikawa, T. Sato, K. Akadegawa, T. Ito, S. Ueha, H. Inadera, and K. Matsushima
Endocrine Disruptors (Environmental Estrogens) Enhance Autoantibody Production by B1 Cells
Toxicol. Sci.,
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G.-J. Shim, M. Warner, H.-J. Kim, S. Andersson, L. Liu, J. Ekman, O. Imamov, M. E. Jones, E. R. Simpson, and J.-A. Gustafsson
Aromatase-deficient mice spontaneously develop a lymphoproliferative autoimmune disease resembling Sjogren's syndrome
PNAS,
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K. C. Lambert, E. M. Curran, B. M. Judy, D. B. Lubahn, and D. M. Estes
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V. Paharkova-Vatchkova, R. Maldonado, and S. Kovats
Estrogen Preferentially Promotes the Differentiation of CD11c+ CD11bintermediate Dendritic Cells from Bone Marrow Precursors
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K. A. Latham, A. Zamora, H. Drought, S. Subramanian, A. Matejuk, H. Offner, and E. F. Rosloniec
Estradiol Treatment Redirects the Isotype of the Autoantibody Response and Prevents the Development of Autoimmune Arthritis
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S. L. Glaser, C. A. Clarke, R. A. Nugent, C. B. Stearns, and R. F. Dorfman
Reproductive Factors in Hodgkin's Disease in Women
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K. Oritani, S. Hirota, T. Nakagawa, I. Takahashi, S.-i. Kawamoto, M. Yamada, N. Ishida, T. Kadoya, Y. Tomiyama, P. W. Kincade, et al.
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Blood,
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T. Kouro, V. Kumar, and P. W. Kincade
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H. Igarashi, T. Kouro, T. Yokota, P. C. Comp, and P. W. Kincade
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G. H. Kline, T. A. Hayden, and P. Riegert
The Initiation of B Cell Clonal Expansion Occurs Independently of Pre-B Cell Receptor Formation
J. Immunol.,
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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
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D. Ait-Azzouzene, S. Caucheteux, F. Tchang, J. Wantyghem, R. Moutier, A. Langkopf, M.-C. Gendron, and C. Kanellopoulos-Langevin
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Biol Reprod,
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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,
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R. A. Nelson, A. M. Levine, and L. Bernstein
Reproductive Factors and Risk of Intermediate- or High-Grade B-Cell Non-Hodgkin's Lymphoma in Women
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T. J. Yun, M. D. Tallquist, A. Aicher, K. L. Rafferty, A. J. Marshall, J. J. Moon, M. K. Ewings, M. Mohaupt, S. W. Herring, and E. A. Clark
Osteoprotegerin, a Crucial Regulator of Bone Metabolism, Also Regulates B Cell Development and Function
J. Immunol.,
February 1, 2001;
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[Abstract]
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Top
Abstract
Introduction