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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3349-3356
HEMATOPOIESIS
From the Laboratory of Molecular Growth Regulation, National
Institute of Child Health and Human Development; the Laboratory of
Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute;
and the Flow Cytometry Unit and Laboratory of Immunopathology, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, MD
To examine the role of retinoids in hematopoietic cell growth in
vivo, we studied female SENCAR mice made vitamin A deficient by dietary
restriction. Deficient mice exhibited a dramatic increase in myeloid
cells in bone marrow, spleen, and peripheral blood. The abnormal
expansion of myeloid cells was detected from an early stage of vitamin
A deficiency and contrasted with essentially normal profiles of T and B
lymphocytes. This abnormality was reversed on addition of retinoic acid
to the vitamin A-deficient diet, indicating that the myeloid cell
expansion is a direct result of retinoic acid deficiency. TUNEL
analysis indicated that spontaneous apoptosis, a normal process in the
life cycle of myeloid cells, was impaired in vitamin A-deficient mice,
which may play a role in the increased myeloid cell population.
Quantitative reverse transcriptase-polymerase chain reaction analysis
of purified granulocytes showed that expression of not only RAR, but
RXRs, 2 nuclear receptors that mediate biologic activities of
retinoids, was significantly reduced in cells of deficient mice. This
work shows that retinoids critically control the homeostasis of myeloid
cell population in vivo and suggests that deficiency in this signaling
pathway may contribute to various myeloproliferative disorders.
(Blood. 2000;95:3349-3356)
Retinoids are derivatives of vitamin A that control
cell growth, differentiation, and apoptosis in many types of cells.
They are also essential for embryonic development of many vertebrate species. In addition, retinoids confer resistance to various
pathogens1,2 and elicit preventive as well as
therapeutic activities against certain tumors.3-5
Two types of nuclear hormone receptors, RAR and RXR, are responsible
for mediating biologic activities of retinoids.6-8 Both RAR
and RXR bind to retinoids and their analogues through the ligand
binding domain. These receptors form RXR/RAR heterodimers, bind to the
retinoic acid (RA)-responsive element through their DNA binding domain,
and activate transcription of many retinoid responsive genes. Both RAR
and RXR have 3 subtypes, This study was designed to investigate the effects of vitamin A
deficiency on mouse hematopoietic cell growth. This work was prompted
by previous observations made with tissue culture cells of
hematopoietic origin, which showed that retinoids have a pivotal role
in their development.21-24 We found a dramatic increase in granulocytes in virtually all deficient mice, which occurred at a
relatively early stage of deficiency and persisted for the duration of
dietary restriction. Supporting a direct role of retinoids in
regulating myelopoiesis, on dietary supplementation with a physiologic
dose of RA, the number of granulocytes fell to a nearly normal level.
Our results indicate that this increase was due to impaired apoptosis
of granulocytes. Further, we show that expression of RXR Vitamin A-deficient (RA Measurement of liver retinol and retinylpalmitate levels
Cytokine gene expression by RNAse protection assay RNase protection analysis was performed using the multiprobe mouse cytokine template, mCK-2 (PharMingen, San Diego, CA), as detailed in our previous paper.27 Serum interleukin (IL)-1 (IL-1)
levels were measured by enzyme-linked immunosorbent assay using a kit
according to the manufacturer's instructions (R& D Systems,
Minneapolis, MN).
Flow cytometry analysis and histology Cells (5 × 105) suspended in RPMI 1640 were first treated with anti-FcR antibody, washed, and then incubated
with fluorescein isothiocyanate (FITC)-conjugated monoclonal antimouse
CD4, anti-IgM, or anti-Mac-1 (CD11b) antibodies in combination with
phycoerythrin (PE)-conjugated anti-CD8, anti-B220 (CD45), or
anti-Gr-1(Ly-6G) antibodies, respectively (all antibodies from
PharMingen). Stained cells were analyzed on a FACScan interfaced with
the Cellquest software (Becton Dickinson, San Jose, CA). For reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis,
granulocytes were purified from RA+ or RA
spleens by FACS sorting using FITC-labeled anti-Gr-1 antibody. Spleens
of 6 mice were pooled prior to sorting. Sorting was performed on Becton
Dickinson FACS Vantage Cell Sorter equipped with a Turbo-Sort option.
The RA sorted cells were 95% pure and RA+
sorted cells were 90% pure.
Colony-forming assay Colony-forming assays were performed using the methylcellulose-based media (Stem Cell Technology, Vancouver, Canada). Bone marrow cells (15 × 103) were cultured for 10 days in 1 mL of the media (MethoCult GF M 3534) designed to support the formation of colonies derived from the myeloid lineage for 10 days. The media contained 50 ng/mL of recombinant mouse stem cell factor, 10 ng/mL of recombinant murine IL-3 (rmIL-3) and 10 ng/mL of recombinant human IL-6, among other components. Colony-forming assays were also performed using a serum-free medium, MethoCult M3236, from the same vender.TdT-mediated dUTP biotin nick end labeling (TUNEL) assay Spleen cells (2 × 107) were incubated in 5 mL of RPMI 1640 supplemented with 10% fetal bovine serum (FBS) for 24 hours. Cells stained with PE-conjugated monoclonal antibodies to CD3, B220, or Gr-1 antibodies were fixed with 1% paraformaldehyde in PBS for 15 minutes, washed in PBS, and incubated in 1 nmol/L biotin-conjugated dUTP and 5 U terminal transferase (both from Boehringer Mannheim, IN) in 50 µL reaction mixture.28,29 Cells were washed in sodium-citrate buffer (0.1% TritonX-100 and 5% dry milk in 4 × standard sodium citrate), incubated with 2.5 µL of avidin-FITC (Boehringer Mannheim) for 30 minutes at room temperature and then washed in Hanks balanced salt solution containing 0.1% TritonX-100 and 0.5% BSA. Stained cells were analyzed on FACScan interfaced with Cellquest.Quantitative RT-PCR Total RNA was prepared from Gr-1+ cells sorted from 5 spleens or from bone marrow of individual mice in RNAsol (Tel-Test, Friendswood, TX). First strand complementary DNA (cDNA) was synthesized with Superscript II (Life Technology, Rockville, MD) according to the manufacturer's instruction. PCR reaction was performed at 94°C for 1 minute, at 55°C for 1 minute, at 72°C for 1.5 minutes for 35 cycles in the presence of 250 µmol/L dNTPs, 2.5 mmol/L MgCl2, and 500 nmol/L of 5' and 3' primers.30,31 The following pairs of PCR primers were used. RAR 5'-: CCGCATCTACAAGCCTTGC 3'-: TCCAGGGAGACTCGTTGTTC, RAR RAR1 5'-:AGCCTGGCCCAGTATGTAGG, RAR2,
5'-:ATCCCTTACCCCCCATGC, 3'-: CTTCACAGGAGCTGACCCCAT
(RAR common),12 RXR
5'-;GGTGAACTCTTCGTCCCTCAACT 3'-:
GTGAAGGAGGCCATATTTC, RAR 5'-:TAGGACCCGCGCGCTCCGGAG,
3'-ATTGAGCAGTATGCCGGTGCT, RXR 5'-:
AGCCCAGACAGCTCCTCCCCAAAT 3'-: GGACCACCTGGAGGGGGTGGA. Bax-1
5'-:GAGACACCTGAGCTGACCTT, 3'-GCACCAGTTTGCTAGCAAAG,
BclX-L5': GTGGCCTTTTTCTCCTTGG,3'-TTGAAGCGCTCCTGGCCTTT, MPO
5':TGTCAGTGAGAGGAGTTGAC, 3':ACCTCAGCTCAGGAAGTATC. Primer sets for
other genes were synthesized as described previously.32,33
PCR products were resolved on 1% agarose gel electrophoresis and
stained with ethidium bromide. Bands visualized in the UV
transilluminator were quantified in the Eagle-Eye system (Stratagene, CA).
Establishment of vitamin A deficiency in female SENCAR mice
(RA mice studied in this work were
likewise depleted of retinoids, we measured, by HPLC analysis, levels
of retinylpalmitate and retinol in liver, where much of the retinoids
are reposited.34 As seen in Figure
1, the mean levels of retinylpalmitate and
retinol both fell precipitously within 5 weeks after the initiation of dietary restriction. No retinol could be detected 2 weeks later, when
mice were 10 weeks of age. Similarly, retinylpalmitate levels reached
near zero levels starting at 8 weeks of feeding the deficient diet (11 weeks of age) and remained essentially undetectable thereafter. These
results confirm that complete retinoid deficiency in mice can be
attained by feeding a diet deficient in vitamin A, which provided a
foundation for this work. The RA mice did not show
external signs of infection and their sera were negative for a panel of
13 pathogens routinely tested in an animal facility. To look for other
signs of infection, expression of a series of cytokine genes known to
be stimulated in response to infections35-37 was examined
by RNase protection assay. Spleen samples from both RA
mice and control RA+ mice did not express detectable levels
of IL-12p40, IL-1, IL-1, IL-6, and interferon- transcripts
(not shown). In addition, serum levels of IL-1 in RA
mice were as low as control RA+ mice (17 and 18 pg/mL,
respectively).
Systemic expansion of myeloid cells in vitamin A-deficient mice Lymphoid tissues from RA mice were compared with
those of age- and sex- matched RA+ mice. Almost all mice in
the RA group exhibited marked splenomegaly by 14 weeks
of age, whereas spleens of RA+ mice were of normal size
(Figure 2A). The splenomegaly coincided with a marked increase in total cell number; spleens from
RA mice contained 50% to 70% more cells than those of
RA+ mice. Although less conspicuous, popliteal and
mesenteric lymph nodes of RA mice were also enlarged;
however, thymi appeared unaltered (not shown). Histologic examination
of RA spleens revealed extensive infiltration of
granulocytes obscuring the border between the white and red pulp
(Figure 2B). The cellular composition of bone marrow was also
drastically altered in RA mice; cells of the myeloid
lineage, both immature cells and mature granulocytes, were greatly
increased in RA bone marrow (Figure 2C). Peripheral
blood of RA mice also contained more granulocytes than
in control mice: 2900 to 3000 cells in 1 µL of RA
blood and 375 to 415 cells in 1 µL in RA+ blood (Figure
3B). Granulocytes in peripheral blood often
exhibited unusual hypersegmentation (Figure 2C).
Reversal of granulocyte increase after dietary RA repletion To test whether the observed granulocyte increase is reversible, 15-week-old mice maintained on the deficient diet were switched to the diet containing RA (3 µg/g diet). Figure 4 shows the percentage of Mac-1+/Gr-1+ cells in spleen and bone marrow after RA supplementation. As expected, mice fed continuously with the RA diet had more than twice the frequency of
Mac-1+/Gr-1+cells as those fed continuously
with the RA+ diet. However, when RA mice
were supplemented with RA for 2 weeks, the frequency of Mac-1+/Gr-1+ cells in bone marrow fell to an
essentially normal level (Figure 4), although 1 week of RA
supplementation did not have an effect (not shown). In spleen,
RA-supplemented mice showed a significant reduction in granulocytes at
1 week and further reduction in 2 weeks, although at the end of 2 weeks, their frequency was still slightly higher in RA-supplemented
mice than in control RA+ mice. Thus, vitamin A-deficient
mice can respond to RA and recover from abnormal expansion of myeloid
cells. The rapid reversibility by RA indicates that granulocyte
expansion is a direct result of vitamin A deficiency, rather than that
of an irreversible, secondary change.
Comparable colony formation by RA+ and
RA mice is attributed to alterations in the frequency of
myeloid cell progenitors. To this end, in vitro colony-forming assays were performed with bone marrow cells from RA+ and
RA mice using media designed to support the growth of
myeloid cell progenitors (see "Materials and methods"). Results
obtained from mice at 11, 13, and 15 weeks are shown in Figure
5. The number of colonies formed by
RA+ and RA bone marrow was similar in all
cases (20-40/104 cells). Differential counting of the
number of colony-forming units-granulocyte, -macrophage, and
-granulocyte/macrophage also yielded comparable results with
RA+ and RA cells. To exclude the possibility
that retinoids that might be present in the FBS in the media could have
obscured a difference in the 2 groups, similar assays were performed
with serum-free medium. Again, no significant difference was observed
between the 2 groups, ranging from 10/104 to
13/104 cells. These results indicate that the frequency of
progenitor cells capable of generating myeloid cell colonies was not
affected in RA mice.
Reduced spontaneous apoptosis of RA mice are defective in apopotosis, causing an
abnormal increase in their number. To examine this possibility, TUNEL
assay was used to detect spontaneous apoptosis of spleen cells from
RA+ and RA mice. Briefly, cells were
cultured overnight, marked for Gr-1, B220, or CD3, and then subjected
to TUNEL assay.29 Results obtained from 15-week-old mice
are shown in Figure 6. Neither B
(B220+) nor T cells (CD3+) from the 2 groups
showed a significant difference in the extent of apoptosis. In
contrast, the frequency of apoptotic cells in Gr-1+ cells
from RA mice was significantly lower than that from
RA+ mice (26% versus 53%). As might have been expected,
the frequency of apoptotic cells in total spleen cells were roughly
comparable between the RA+ and RA groups
(36%). Similar TUNEL assays were performed with 17- and 18-week-old
mice, and the percent apoptotic cells in the Gr-1+ cell
population was again lower in RA mice than that in
RA+ mice; the frequency of apoptotic cells in the
RA group ranged from 32% to 41%, whereas that in the
RA+ group ranged from 50% to 57%. These results indicate
that vitamin A deficiency leads to a reduction in spontaneous apoptosis
of the granulocyte population, although it does not completely
eliminate apoptosis.
Down-regulation of RAR and RXR expression in
RA and subtypes of the receptors. We also tested RAR because
this receptor is expressed in bone marrow Gr-1+
cells.12 To avoid a potential complication derived from
cellular heterogeneity, PCR analysis was performed with FACS-purified
granulocytes obtained from spleens. Three serially diluted RNA samples
were tested to ensure a linear range of reaction. Neither RAR nor RAR was detected in any samples tested (not shown). By contrast, expression of RAR, RXR, and RXR was clearly detectable in the samples from both groups (Figure 7A).
Importantly, expression of the 3 receptors was significantly lower in
RA cells than in RA+ cells. Levels of
hypoxanthine-guanine phosphoribosyltransferase (HPRT) RNA,
run as a control, were comparable in the 2 samples. To verify
down-regulation of RAR and RXR in RA cells, we tested
bone marrow RNA from 2 individual mice in each group. As seen in Figure
7B, levels of RAR, RXR, and RXR were again lower in
RA cells than in RA+ cells. Expression of
RAR was undetectable in bone marrow RNA, similar to the results seen
with purified granulocytes. Thus, expression of RAR and RXR is
down-regulated in myeloid cells in vitamin A-deficient mice,
indicating that expression of these receptors is regulated by the
retinoid status of the cells.
We have shown that vitamin A deficiency leads to a striking
expansion of myeloid cells in mice. The increase in Mac-1+/
Gr-1+ cells occurred at an early stage of deficiency and
persisted throughout the remaining period. The increase was first noted in bone marrow and subsequently spread to peripheral tissues, indicating that the abnormality originates in bone marrow. The onset of
the abnormality was noticeable within 1 to 2 weeks of retinoid
depletion in liver and established 5 to 7 weeks after the initiation of
the RA
We thank David Stephany and Ruth Swofford in the NIAID Flow Cytometry Unit for FACS analysis and granulocyte sorting, and Susan Kirkhoff for preparation of mice.
Submitted May 5, 1999; accepted January 28, 2000.
Reprints: Keiko Ozato, Laboratory of Molecular Growth Regulation, Bldg 6, Rm 2A01, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2753; e-mail: ozatok{at}nih.gov.
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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Abstract
Introduction
/