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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2821-2828
HEMATOPOIESIS
Retinoic acid is a negative regulator for the differentiation of
cord blood-derived human mast cell progenitors
Tatsuya Kinoshita,
Kenichi Koike,
Hadija
Hemed Mwamtemi,
Susumu Ito,
Shuichi Ishida,
Yozo Nakazawa,
Yumi Kurokawa,
Kazuo Sakashita,
Tsukasa Higuchi,
Kouichi Takeuchi,
Nobukuni Sawai,
Masaaki Shiohara,
Takehiko Kamijo,
Shigeyuki Kawa,
Tetsuji Yamashita, and
Atsushi Komiyama
From the Department of Pediatrics, Second Department of Internal
Medicine, Shinshu University School of Medicine, Matsumoto; Blood
Transfusion Service, Shinshu University Hospital, Matsumoto; Research
and Development, Mitsubishi Kagaku Bio-Clinical Laboratories, Inc,
Tokyo, Japan.
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Abstract |
We examined the effects of retinoids on the human mast cell
development using a serum-deprived culture system. When 10-week cultured mast cells derived from CD34+ cord blood cells
were used as target cells, both all-trans retinoic acid (ATRA)
and 9-cis RA inhibited the progeny generation under stimulation
with stem cell factor (SCF) in a dose-dependent manner (the number of
progeny grown by SCF plus RA at 10 7 mol/L was one tenth
of the value obtained by SCF alone). The early steps in mast cell
development appear to be less sensitive to RA according to the single
CD34+c-kit+ cord blood cell culture study.
The optimal concentration of RAs also reduced the histamine
concentration in the cultured mast cells (3.00 ± 0.47 pg per cell
in SCF alone, 1.44 ± 0.18 pg per cell in SCF+ATRA, and
1.41 ± 0.10 pg per cell in SCF+9-cis RA). RT-PCR
analyses showed the expression of RAR , RAR , RXR, and RXR
messenger ribonucleic acid (mRNA) in 10-week cultured mast cells. The
addition of an RAR-selective agonist at 1010 mol/L to
107 mol/L decreased the number of mast cells grown in
SCF, whereas an RXR-selective agonist at up to 108 mol/L
was inactive. Among RAR subtype selective retinoids used at
109 mol/L to 107 mol/L, only the RAR
agonist was equivalent to ATRA at 107 mol/L in its
ability to inhibit mast cell growth. Conversely, the addition of excess
concentrations of a RAR antagonist profoundly counteracted the
retinoid-mediated suppressive effects. These results suggest that RA
inhibits SCF-dependent differentiation of human mast cell progenitors
through a specific receptor.
(Blood. 2000;95:2821-2828)
© 2000 by The American Society of Hematology.
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Introduction |
Mast cells play a pivotal role as major effector cells
in allergic disorders such as asthma, atopic dermatitis, and allergic rhinitis. Antigen-specific IgE-mediated degranulation of mast cells
leads to the subsequent release of chemical mediators and multiple
cytokines. Numerous investigators have developed antiallergic drugs to
interfere with this process. Potent inhibitors of mast cell growth may
provide a new strategy for prophylactic treatment of allergic
disorders. In this regard, interferon gamma-1b is a candidate because
this cytokine suppresses mast cell production.1
Retinoids are a group of natural and synthetic vitamin A analogues, and
exert important effects on the growth and differentiation of various
cell types, including hematopoietic progenitors.2-5 The
action of retinoids is thought to be mediated by 2 types of nuclear
retinoid receptors, the RARs and RXRs, which are members of the
steroid/thyroid hormone receptor superfamily.6-8 Each class
of the receptors comprises 3 subtypes designated  , , and  .
All-trans retinoic acid (ATRA) and
9-cis RA are high affinity ligands for RARs, and 9-cis RA additionally binds
RXRs.6-8 These receptors form RAR/RXR heterodimers and
RXR/RXR homodimers, respectively, to function as ligand-activated
transcription factors.
Stem cell factor (SCF) has been demonstrated to act as a major growth
and differentiation factor for human mast cell lineage.9-13 However, the purity of cultured mast cells grown by SCF alone has
ranged from approximately 40% to 85%.9,11,12 We have recently reported the selective growth of a large number of mast cells
from CD34+ human cord blood cells under stimulation with
SCF in long-term serum-deprived cultures.14 In this study,
we examined the effects of retinoids on the human mast cell development
using this culture system.
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Materials and methods |
Cytokines, reagents, and antibodies
Human recombinant SCF was generously provided by Kirin Brewery Co
Ltd (Takasaki, Japan). Human recombinant interleukin (IL)-4 was
purchased from R & D Systems (Minneapolis, MN). Human recombinant IL-6
was a gift from Ajinomoto Co (Kawasaki, Japan).
ATRA was obtained from Sigma (St Louis, MO), and 9-cis-RA from
Wako (Osaka, Japan). Ro 13-7410, Ro 25-7386, Ro 40-6055, Ro 19-0645, Ro
44-4753, and Ro 41-5253 were kindly provided by F. Hoffman-La Roche,
Ltd (Basel, Switzerland). Ro 13-7410 has been shown to selectively bind
and activate RARs but not RXRs.15 It does not discriminate
among RAR, RAR, and RAR. Ro 25-7386 is indicated to be a
specific RXR agonist and does not bind RARs.16 Three
synthetic retinoids (Ro 40-6055, Ro 19-0645, and Ro 44-4753) are
described to be selective for RAR, RAR, and RAR,
respectively, when used at
nanomolar concentrations.15,17,18 Ro 41-5253 acts as an
RAR antagonist.16,18 All the retinoids were dissolved in
ethanol at a concentration of 102 mol/L, and stored
in light-protected vials at  80°C. Before their use in each
experiment, they were further diluted with -medium (Flow
Laboratories, Rockville, MD) containing 1% deionized bovine serum
albumin (BSA) (Sigma Chemical). All experiments were performed in
subdued light, and the tubes containing retinoids were covered with
aluminum foil. Preliminary experiments showed that controls containing
an equivalent amount of ethanol were not different from medium controls
in the development of the cultured mast cells under stimulation with SCF.
The polyclonal rabbit antihuman IL-6 antibody (Ab, 1.2 mg/mL)14 was a gift from Ajinomoto Co. One microgram of
this Ab neutralized the activity of 3 ng IL-6, as determined with
the use of the cell line, SKW6-CL4. A polyclonal sheep antihuman IL-4
Ab was purchased from Genzyme Co (Cambridge, MA). The Ab at 0.1 to 1 µg/mL neutralized the bioactivity of a 0.25 ng/mL solution of IL-4.
The mouse monoclonal antibody (MoAb) against human
granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased
from Oncogene Science Inc (Uniondale, NY). This azide-free antibody at
2 µg/mL reduced the growth of granulocyte-macrophage colonies
supported by 10 ng/mL of GM-CSF to 37%.19 The neutralizing
antihuman transforming growth factor (TGF)-1 antibody was obtained
from R & D Systems. The ND50 of the antibody was determined
to be 0.2 to 0.6 µg/mL in the presence of 0.25 ng/mL of TGF-1,
using TGF- responsive HT-2 cells.
For immunocytochemical staining, purified MoAbs for tryptase (MAB1222)
and chymase (3D5) were purchased from Chemicon International Inc
(Temecula, CA) and Biogenesis Inc (Sandown, NH), respectively.
For the flow cytometric analysis, the MoAbs for CD34 (HPCA-2,
fluorescein isothiocyanate [FITC]) and c-kit (95C3, phycoerythrin [PE]) were purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA) and Immunotech SA (Marseilles, France), respectively.
Cell preparation
Cord blood samples were aspirated in heparinized plastic syringes
from the umbilical vein at normal delivery. Fully informed consent was
obtained from the mothers of all neonates before harvesting the
specimens. Mononuclear cells (MNCs) were separated by density centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ), washed twice, and suspended in Ca++- and
Mg2+-free phosphate-buffered saline (PBS) containing 1 mmol/L EDTA-2Na and 2.5% fetal bovine serum (Hyclone, Logan, UT).
After treatment with Silica (Immuno-Biological Laboratories, Fujioka,
Japan) for 30 minutes at 37°C, CD34-positive cells were enriched
using a Dynal CD34 Progenitor Cell Selection System (Dynal AS, Oslo,
Norway). Briefly, 2 to 4 × 107 cells were mixed
with the same number of polystyrene beads coated with an MoAb specific
for CD34 (Dynabeads M-450 CD34), and incubated for 30 minutes at
4°C. Bead-rosetted cells were separated by a magnet. For the
detachment of the beads from the cells, affinity-purified polyclonal
antibodies against the Fab portion of anti-CD34 Ab (Detach-a-Bead CD34)
were added, and incubation was carried out for 45 minutes at room
temperature. The detached beads were removed by the magnet, and the
cells were collected as CD34+ cells. More than 90% of the
isolated cells were CD34-positive, as determined by FACScan flow
cytometry (Becton Dickinson).
Suspension cultures
Serum-deprived liquid cultures were carried out in 24-well culture
plates (#3047; Becton Dickinson) using a modification of the technique
described previously.14,20 Ten thousand CD34+
cells or 5000 CD34+c-kit+ cells were cultured
in each well containing 2 mL of -medium supplemented with 1% BSA,
300 µg/mL fully iron-saturated human transferrin (approximately 98%
pure, Sigma), 16 µg/mL soybean lecithin (Sigma), 9.6 µg/mL
cholesterol (Nakalai Chemicals, Tokyo, Japan), and 100 ng/mL SCF with
or without different concentrations of ATRA. To examine the effects of
retinoids, IL-6, and IL-4 on the SCF-dependent development of mast
cells, we used the cultured cells grown in 10 ng/mL of SCF from
CD34+ cord blood cells as target cells.14
One × 104 or 5 × 104 10-week
cells were incubated for 2 weeks in 24-well culture plates containing
100 ng/mL of SCF, different concentrations of retinoids, 50 ng/mL of
IL-6 or 20 ng/mL of IL-4, alone or in combination. The plates were
incubated at 37°C in a humidified atmosphere flushed with a mixture
of 5% CO2, 5% O2, and 90% N2.
Half of the culture medium was replaced weekly with fresh medium
containing the factor(s). The number of viable cells was determined by
a trypan-blue exclusion test using a hemocytometer. We presented the
actual counts of progeny in the results.
The DNA distribution was examined by flow cytometry after the cells had
been stained with propidium iodide (PI), as described previously.21
Serum-deprived single-cell culture
Single-cell sorting was performed by 2-step sorting, as described
previously.14,21-23 Cord blood MNCs
(2 × 106) were stained with 20 µL of
FITC-conjugated anti-CD34 MoAb and 20 µL of PE-conjugated anti-c-kit
MoAb. After 2 washes, CD34+c-kit+ cord blood
cells were collected in 5-mL tubes and were resorted into the
individual wells of a 96-well U-bottomed tissue culture plate (#3077;
Becton Dickinson) containing 100 µL of -medium supplemented with
1% BSA, 300 µg/mL of fully iron-saturated human transferrin, 16 µg/mL of soybean lecithin, 9.6 µg/mL of cholesterol, 100 ng/mL of
SCF, and different concentrations of ATRA, using the
FACStarplus flow cytometer equipped with an automatic cell
deposition unit (Becton Dickinson). Ninety-nine percent of the wells
contained a single cell on the first day of culture. The plates were
incubated at 37°C in a humidified atmosphere flushed with a mixture
of 5% CO2, 5% O2, and 90% N2.
The number of cells in each well was serially counted until 4 weeks under direct microscopic visualization. Aggregates consisting of
30 or more cells were scored as colonies. Then, the colonies were
individually lifted with a 3-µL Eppendorf micropipette, spread on
glass slides using a Cytospin II (Shandon Southern, Sewickly, PA), and
the constituent cells were stained with antitryptase MoAb.
Clonal cell cultures
The mast cell colony assay was carried out in 35-mm Lux suspension
culture dishes (#171099; Nunc, Naperville, IL) using a modification of
the technique described previously.24 The culture consisted
of 10-week cultured cells (5000 cells/mL) grown in 10 ng/mL of SCF,
-medium, 0.9% methylcellulose (Shinetsu Chemical, Tokyo, Japan),
1% BSA, 300 µg/mL of fully iron-saturated human transferrin, 16 µg/mL of soybean lecithin, 9.6 µg/mL of cholesterol, and 100 ng/mL
of SCF with or without different concentrations of ATRA. Dishes were
incubated at 37°C in a humidified atmosphere flushed with a
mixture of 5% CO2, 5% O2, and 90%
N2. On day 14, aggregates consisting of 30 or
more cells were scored as mast cell colonies, and those
consisting of 10 to 29 cells as mast cell clusters. To confirm the in
situ identification of mast cells, 60 individual colonies and clusters
were lifted and stained with the antitryptase MoAb or mouse
IgG1 using the alkaline phosphatase-antialkaline phosphatase
(APAAP) technique. Almost all the constituent cells were positive
for tryptase.
Immunocytochemical staining
The cultured cells were spread on glass slides using a Cytospin II.
Reactions with mouse MoAbs against tryptase and chymase were detected
using the APAAP method (Dako APAAP Kit System, Dako Corp,
Carpinteria, CA), as described previously.25 The isotype mouse MoAb was also used as a control. Briefly,
cytocentrifuged samples were fixed with Carnoy's fluid,
washed with PBS, and preincubated with normal rabbit serum to
saturate the Fc receptors on the cell surface. After being washed with
PBS 3 times, the samples were reacted with a mouse MoAb for 30 minutes
at room temperature in a humidified chamber. After 3 more washes
with PBS, the samples were incubated with rabbit antimouse IgG
antibody, washed 3 times, and successively reacted with the calf
intestinal alkaline phosphatase-mouse monoclonal antialkaline
phosphatase complex. Finally, alkaline phosphatase activity
was detected with naphthol AS-MX phosphate, Fast Red TR, and levamisole
to inhibit nonspecific alkaline phosphatase activity. The specimens
were counterstained with hematoxylin.
The diameter of the mast cells was measured by calculating the average
of 2 perpendicular diameters of tryptase+ cells on glass
slides, using a microscope equipped with an ocular micrometer.
Reverse transcription-polymerase chain reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was
performed according to a modification of the procedure described previously.20 Total rubonucleic acid (RNA) was individually isolated from the cells, using Isogen (Wako). Next, 1 µg of RNA was
reverse transcribed in 200 U of SuperScript II (Life Technologies, Gaithersburg, MD), 107 mol/L oligo dT primer (Takara
Shuzo, Ohtsu, Japan), and 10 U RNase inhibitor (Boehringer Mannheim,
Mannheim, Germany) in 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, and 3 mmol/L MgCl2. The prepared solution was incubated at
37°C for 1 hour. The PCR reaction was performed in a volume of 10 µL containing 1 µL of RT-PCR product, 1 µL of 10 × PCR
buffer (100 mmol/L Tris-HCl, pH 8.3, 500 mmol/L KCl), 1.5 mmol/L
MgCl2, 0.5 µmol/L of each primer, 1 µL of 0.5 mmol/L
dNTP mix, and 0.5 U of Taq DNA polymerase (Takara Shuzo). The primers
for amplification were designed on the basis of previous reports
(RAR,26 the other receptors27):
5'-CATTGAGACCCAGAGCAGC -3' (nt 282-300) and
5'-CCGTCTCCGCATCATCCATC -3' (nt 1062-1081) for RAR;
5'-CACTGGCTTGACCATCGCAGACC -3' (nt 1047-1069) and
5'-GAGAGGTGGCATTGATCCAGG -3' (nt 1507-1527) for RAR;
5'-GGCCTGGGCCAGCCTGACCTC -3' (nt 288-308) and
5'-CAGCCCCAGATCCAGCTGCACG-3' (nt 803-824) for RAR; 5'-CTCCTCAGGCAAGCACTATG-3' (nt 498-517) and
5'-AGAGCTTAGCGAACCTTCCC-3' (nt 1311-1330) for RXR;
5'-TCAGGCAAACACTACGGGGT-3' (nt 816-835) and
5'-GCATACACTTTCTCCCGCAG -3' (nt 1566-1585) for
RXR; 5'-CTCAGGAAAGCACTACGGGG-3' (nt 465-484) and
5'-CAGGGTCATTTGTCGAGTTC-3' (nt 804-823) for RXR; 5'-CTGGACTTCGAGCAAGAGAT-3' (nt 702-721) and
5'-TCGTCATACGCCTGCTTGCT-3' (nt 1132-1113) for
-actin. The samples were denatured at 95°C for 5 minutes, then
subjected to 35 cycles at 95°C for 1 minute, at 54°C for 1 minute, and at 72°C for 1 minute, with a final 10 minutes of
extension at 72°C in a Gene Amp PCR System 9600 (Perkin-Elmer Cetus, Norwalk, CT). PCR products (10 µL) were
analyzed on a 1.5% agarose gel in TAE buffer (40 mmol/L Tris,40 mmol/L
sodium acetate,1 mmol/L EDTA, pH 8.4) using a DNA ladder 100-base pair
(bp) marker.
Assay of histamine, tryptase, and cytokine levels
Histamine concentrations in cell lysates obtained by the treatment
of the cultured cells with 0.5% Nonidet P-40 and in
supernatant were measured by the histamine radioimmunoassay (RIA) kit
(Immunotech, SA). The detection limit was 0.05 ng/mL, and
cross-reactions with t-methylhistamine or histidine were very low.
Intra-assay CV and interassay CV were 7.6% to
8.4% and 8.2% to 11.5%, respectively. The tryptase
concentrations in the cell lysates were measured with a
fluoroenzymeimmunoassay (UniCAP Tryptase, Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden). The concentrations of
GM-CSF, IL-4, IL-6, and TGF-1 in the supernatant of the
cultured cells were measured by an enzyme-linked immunosorbent assay
(ELISA) (Amersham International, Buckinghamshire, UK). All assays were conducted in triplicate.
Statistical analysis
All experiments were carried out at least 3 times and were shown to
be reproducible. Values are expressed as means ± SD. One-way analysis of variance, followed by post hoc contrasts with Bonferroni limitation, was used for more than 3 independent groups.
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Results |
Effects of all-trans retinoic acid or 9-cis retinoic
acid on the growth and properties of cultured mast cells supported by
stem cell factor
To examine the effects of ATRA or 9-cis RA on the human mast
cell development, we first used 10-week cultured cells generated with
10 ng/mL of SCF from CD34+ cord blood cells as target
cells. Immunocytochemical staining showed that almost all of
these cultured cells were positive for tryptase, as described
previously.14 One × 104 10-week
cultured cells were incubated for 2 weeks in wells containing SCF at 100 ng/mL with or without ATRA or 9-cis RA at
concentrations ranging from 1011 mol/L to
107 mol/L. The results are shown in Figure
1. The addition of either ATRA or
9-cis RA to the culture with SCF gave rise to a dose-dependent decrease in the numbers of progeny. The maximal inhibition
with both RAs was observed at a level of at least
108 mol/L. As shown in Figure
2, the number of cultured mast cells grown
in SCF+107 mol/L ATRA was similar to the value
obtained by SCF+107 mol/L 9-cis RA. It is of
particular interest that, at these optimal concentrations, the RAs
inhibited the SCF-dependent mast cell generation to a greater extent
than IL-4 or IL-6, alone or in combination.
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| Fig 1.
Dose response to ATRA or 9-cis RA of mast cell
growth supported by SCF.
The 1 × 104 10-week cultured mast cells were
incubated in wells containing SCF at 100 ng/mL with either ATRA or
9-cis RA at 1011 mol/L to
107 mol/L. After 2 weeks, the viable cells were
enumerated. The results shown are from 1 representative experiment of
3. Similar results were obtained in the other 2 experiments.
Significantly different from SCF alone (*P < .0005,
**P < .0001).
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| Fig 2.
Comparison of effects of RAs with those of IL-4, IL-6, or
IL-4+IL-6 on mast cell growth supported by SCF.
The 1 × 104 10-week cultured mast cells were
incubated in wells containing 100 ng/mL of SCF, ATRA or 9-cis
RA at 107 mol/L, 20 ng/mL of IL-4, or 50 ng/mL of
IL-6, alone or in combination. After 2 weeks, the viable cells were
enumerated. Results shown are the mean ± SD of 3 experiments.
Significantly different from ATRA (*P < .002,
**P < .0001) and from 9-cis RA
(#P < .001,
##P < .0001).
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To clarify whether RA-induced inhibition represented a decrease in
proliferation rate or a decreased survival, we examined the time course
of the proliferative response and the cell cycle status of 10-week
cultured mast cells exposed to SCF alone or SCF+ATRA. As shown in
Figure 3, the total viable cell number was unchanged in the culture containing SCF+ATRA at 107
mol/L. In the absence of SCF, the inhibitory effects of ATRA were
either absent or present at low levels. The flow cytometric analysis
revealed that the addition of ATRA to the culture with SCF caused a
decrease in the percentage of S plus G2/M
cells on day 2 (10.4% in SCF alone vs 3.0% in
SCF+ATRA). However, there was no significant appearance of a
sub-G1 peak of the cultured cells in the presence or
absence of ATRA. Similar results were obtained on days 4 and 7.
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| Fig 3.
ATRA reduces the proliferation rate of mast cells under
stimulation with SCF.
The 5 × 104 10-week cultured mast cells were
incubated in the presence or absence of SCF at 100 ng/mL for 1 week.
ATRA was used at 107 mol/L. (A) The viable cells
were enumerated every day. SCF alone, open circles; SCF+ATRA, closed
circles; no factors, open squares; ATRA alone, closed squares. (B) The
DNA distribution was examined by labeling of the cells with PI on day
2. FSC, forward light-scatter characteristics; SSC, side-scatter
characteristics.
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Next, we examined whether ATRA and 9-cis RA influenced the
size, intracellular histamine level, and chymase expression of the
cultured mast cells grown in SCF. The results are presented in Table
1. The RAs equivalently diminished the mean
diameter and histamine content of the cultured mast cells, whereas IL-4 and IL-6 each increased these 2 parameters. The histamine level in the
cultured cells grown in SCF plus ATRA or 9-cis RA was
approximately 50% of that in the cultured cells grown in SCF alone.
The histamine concentration in the supernatant of the cultured mast
cells exposed to SCF+107 mol/L ATRA and in those
exposed to SCF+107 mol/L 9-cis RA was one
seventh and one sixth of the level obtained with SCF alone,
respectively. At the initiation of culture, 4.0% ± 1.4% of the
cells were positive for chymase. After 2 weeks, the percentage of
chymase+ cells in the cultured mast cells increased in the
presence of SCF+IL-4 or SCF+IL-6, compared with the level obtained with
SCF alone, as described previously.14 On the other hand,
neither ATRA nor 9-cis RA influenced the chymase expression. In
addition, the content of tryptase was not markedly reduced by
107 mol/L ATRA (4.3 pg per cell in SCF alone vs 3.5 pg per cell in SCF+107 mol/L ATRA).
Because GM-CSF, TGF-1, IL-4, and IL-6 were reported to suppress the
SCF-dependent mast cell growth by us or other
investigators,14,28-31 it is possible that the effects of
RAs on the mast cell development are mediated by these cytokines. The
concentrations of all the factors in the supernatant of the 10-week-old
mast cells after the 2-week incubation with SCF alone or
SCF+107 mol/L ATRA were at levels below detectable
limits. None of the neutralizing anti-GM-CSF Ab at 2 µg/mL,
anti-TGF-1 Ab at 100 µg/mL, anti-IL-4 Ab at 10 µg/mL,
and anti-IL-6 Ab at 10 µg/mL affected the progeny generation under
stimulation with 100 ng/mL of SCF or 100 ng/mL of SCF
+107 mol/L ATRA (data not shown).
Expression of nuclear retinoid receptors in cultured mast cells
grown in stem cell factor
To elucidate whether ATRA and 9-cis RA exerted their action
through the receptor(s), we examined the expression of messenger RNAs
(mRNAs) for the retinoid receptor subtypes using RT-PCR analysis. Thirty-five cycles were used for amplification. As positive controls, we used the human pancreatic carcinoma cell line (Panc-1)32 for the expression of RAR, and the human myeloma cell line (RPMI 8226)27 for the expression of the other receptors. The
molecular sizes of RT-PCR products obtained with the primers were
compatible with the expected molecule size: 800 bp for RAR, 481 bp
for RAR, 537 bp for RAR, 833 bp for RXR, 770 bp for RXR,
and 359 bp for RXR. The expression of RAR, RAR, RXR, and
RXR was observed in 10-week cultured mast cells, as shown in Figure
4.
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| Fig 4.
Expression of nuclear retinoid receptors in cultured mast
cells grown in SCF.
The cDNA of the nuclear retinoid receptors in cultured mast cells grown
in SCF alone or SCF+ATRA at 107 mol/L was amplified
by 35 cycles. As positive controls, Panc-1 cells were used for the
expression of RAR, and RPMI 8226 cells exposed to ATRA at
107 mol/L for 24 hours were used for the expression
of the other receptors. (A) Mast cells grown in SCF at 100 ng/mL, (B)
mast cells exposed to SCF at 100 ng/mL plus ATRA at
107 mol/L, and (C) positive controls.
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Selective involvement of RAR in the regulation of stem cell
factor-dependent mast cell development
We attempted to determine which of the 2 retinoid responsive
pathways is mainly involved in the regulation of the mast cell development, using 2 synthetic retinoids that selectively bind and
activate RARs and RXRs, respectively. As shown in Figure
5A, the addition of Ro 13-7410 (RAR
agonist) at 1010 mol/L to 107
mol/L decreased the number of progeny grown by SCF, with a maximal inhibition of approximately 85% at a level of at least
108 mol/L. There was no difference in the number of
the progeny between SCF+ Ro 13-7410 at 108 mol/L or
107 mol/L and SCF+ATRA at 107
mol/L. On the other hand, Ro 25-7386 (RXR agonist) at
1011 mol/L to 108 mol/L failed to
inhibit the mast cell growth, though the number of mast cells grown by
SCF was slightly reduced at 107 mol/L. The addition
of Ro 25-7386 at 107 mol/L to the culture containing
SCF plus Ro 13-7410 at concentrations of 1010 mol/L
to 107 mol/L did not further enhance the inhibition
of the progeny production (Figure 5B). Both the mean diameter and the
intracellular histamine content of the cultured mast cells were
significantly attenuated by 107 mol/L Ro 13-7410, but not 107 mol/L Ro 25-7386 (Table 1). These
results imply that the growth and properties of cultured mast cells are
regulated through activation of the RAR-RXR response pathway rather
than the RXR-RXR response pathway.
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| Fig 5.
Effects of RAR- and RXR-selective retinoids on
SCF-dependent mast cell growth.
(A) Dose response to RAR-selective retinoid (Ro 13-7410) or
RXR-selective retinoid (Ro 25-7386) of mast cell growth supported by
SCF. The 1 × 104 10-week cultured mast cells were
incubated in wells containing SCF at 100 ng/mL with various
concentrations of Ro 13-7410, Ro 25-7386, or ATRA. After 2 weeks, the
viable cells were enumerated. Results shown are the mean ± SD of
3 experiments. Significantly different from SCF alone
(*P < .0001). (B) No cooperative effects of Ro 13-7410 at
concentrations of 1010 mol/L to
107 mol/L and Ro 25-7386 at concentrations of
109 mol/L to 107 mol/L on the
growth of mast cells supported by SCF. Significantly different from no
Ro 25-7386 (*P < .0001).
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Next, we analyzed which of the endogenous RAR subtypes is involved in
the RA-dependent regulation of mast cell generation, using Ro 40-6055 (RAR agonist), Ro 19-0645 (RAR agonist), and Ro 44-4753 (RAR
agonist). As presented in Figure 6A, the
addition of Ro 40-6055 at concentrations of at least
1010 mol/L to the culture with SCF markedly
decreased the number of progeny grown under stimulation with SCF. The
inhibitory ability of Ro 40-6055 at a level of at least
109 mol/L was comparable to that of
107 mol/L ATRA. Neither Ro 19-0645 nor Ro 44-4753 suppressed the mast cell growth, except for 107
mol/L Ro 19-0645. There were no cooperative effects between Ro 40-6055 at concentrations of 1011 mol/L to
107 mol/L and Ro 19-0645 at a concentration of
107 mol/L in the suppression of the mast cell growth
(Figure 6B).
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| Fig 6.
Effects of RAR-, RAR-, and RAR-selective
retinoids on SCF-dependent mast cell growth.
(A) Dose response to RAR-selective retinoid (Ro 40-6055),
RAR-selective retinoid (Ro 19-0645), or RAR-selective retinoid
(Ro 44-4753) of mast cell growth supported by SCF.
One × 104 10-week cultured mast cells were
incubated in wells containing SCF at 100 ng/mL with various
concentrations of Ro 40-6055, Ro 19-0645, Ro 44-4753, or ATRA. After 2 weeks, the viable cells were enumerated. The results shown are from 1 representative experiment of 3. Similar results were obtained in the
other 2 experiments. Significantly different from SCF alone
(*P < .0001). (B) No cooperative effects of Ro 40-6055 at
concentrations of 1011 mol/L to
107 mol/L and Ro 19-0645 at concentrations of
109 mol/L to 107 mol/L on the
growth of mast cells supported by SCF. Significantly different from no
Ro 19-0645 (*P < .0001).
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To confirm the selective involvement of RAR, we examined whether Ro
41-5253 (RAR antagonist) counteracted the inhibition of the mast
cell growth induced by ATRA, 9-cis RA, or Ro 40-6055. The
results are presented in Figure 7. Ro
41-5253 (107 mol/L to 105 mol/L)
showed no effects on the SCF-dependent generation of mast cells. The
addition of 1 000- to 10 000-fold excess concentrations of Ro 41-5253 significantly increased the number of mast cells grown in the presence
of SCF plus ATRA or 9-cis RA at a concentration of
109 mol/L or 108 mol/L. The
suppression of the mast cell generation mediated by Ro 40-6055 at
either 1010 mol/L or 109 mol/L
was also counteracted by the treatment with Ro 41-5253 at
105 mol/L (data not shown).
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| Fig 7.
Effects of RAR-selective antagonist on mast cell
growth under stimulation with SCF+ATRA or SCF+9-cis RA.
The effects of Ro 41-5253 (RAR antagonist) on the progeny
generation from 1 × 104 10-week cultured mast
cells in the presence of SCF plus ATRA or 9-cis RA were
examined. Significantly different from no Ro 41-5253 (*P < .0005, **P < .0001).
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Effects of all-trans retinoic acid on the early phase of
mast cell development
We then analyzed whether ATRA exerted its effects at the early phase
of mast cell development. Ten thousand CD34+ cells were
plated in wells containing 100 ng/mL of SCF with or without ATRA at
109 mol/L to 107 mol/L. As
presented in Figure 8, all the
concentrations of ATRA significantly decreased the cell production by
CD34+ cells under stimulation with SCF. Almost all the
4-week cultured cells were positive for tryptase, both in the culture
containing SCF alone and in the culture with SCF+107
mol/L ATRA. Similar results were obtained by the addition of 9-cis RA at 109 mol/L to
107 mol/L (data not shown). When 5000 CD34+c-kit+ cells sorted by a
FACStarplus flow cytometer were used as target cells, the
numbers of the viable cells were
5.4 ± 0.4 × 104 cells at 2 weeks, and
16.7 ± 1.3 × 104 cells at 4 weeks in 100 ng/mL of SCF alone; 3.1 ± 0.1 × 104 cells at
2 weeks, and 3.7 ± 0.5 × 104 cells at 4 weeks
in 100 ng/mL of SCF+107 mol/L ATRA.
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| Fig 8.
Effects of ATRA on mast cell growth by
CD34+ cord blood cells.
CD34+ cord blood cells (1 × 104) were
cultured with 100 ng/mL of SCF and/or ATRA at 109
mol/L to 107 mol/L. The viable cells were enumerated
until 4 weeks. The results shown are from 1 representative experiment
of 3. Similar results were obtained in the other 2 experiments.
Significantly different from SCF alone (*P < .0001).
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We attempted to elucidate the direct action of ATRA on the
SCF-dependent generation of mast cells by hematopoietic progenitors, using single-cell culture studies. The results are presented in Table
2. One sixth of the
CD34+c-kit+ cells responded to SCF to form
colonies at 4 weeks of culture, in which almost all constituent cells
reacted with antitryptase MoAb. The addition of ATRA diminished the
number of mast cell colonies grown by SCF at 4 weeks. However, the
inhibitory effects of 109 mol/L to
108 mol/L ATRA were modest at the early stage of
culture.
Finally, we examined whether ATRA exerted an inhibitory effect at the
mast cell-committed precursor level. The 10-week cultured cells were
plated at 5000 cells per dish containing serum-deprived methylcellulose
culture medium supplemented with SCF or SCF+ATRA, ranging from
1010 mol/L to 107 mol/L. As shown
in Figure 9, SCF alone supported the
formation of 105.5 ± 20.0 mast cell colonies and
201.0 ± 35.9 clusters. The addition of ATRA at a level of at
least 109 mol/L caused a significant reduction of
the SCF-dependent growth of mast cell colonies and clusters.
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| Fig 9.
Effects of ATRA on the early phase of mast cell
development.
Five thousand 10-week cultured mast cells were plated per dish
containing serum-deprived methylcellulose culture medium supplemented
with SCF, SCF+ATRA, SCF+IL-4, or SCF+IL-6. After 2 weeks, aggregates
consisting of 30 or more cells were scored as mast cell colonies, and
those of 10 to 29 cells as mast cell clusters. SCF, 100 ng/mL; ATRA,
1010 mol/L to 107 mol/L; IL-4, 20 ng/mL; IL-6, 50 ng/mL. Numbers of mast cell colonies (black bars) and
mast cell clusters (gray bars) are shown. The results shown are from 1 representative experiment of 3. Similar results were obtained in the
other 2 experiments. Significantly different from SCF alone
(*P < .002, **P < .0001).
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Discussion |
We previously reported that SCF alone could support the selective
growth of mast cells from CD34+ human cord blood cells in
serum-deprived cultures.14 When 10-week-old cultured mast
cells were used as target cells, both ATRA and 9-cis RA
suppressed the generation of progeny under stimulation with SCF in a
dose-dependent manner. At the optimal concentration, ATRA was as potent
an inhibitor as 9-cis RA.
RA-mediated inhibition may be based on a decrease in proliferation rate
rather than an increase in apoptotic death. On the basis of the clonal
cell culture, the inhibitory effects of RA appear to be exerted at the
mast cell-committed precursor level. Furthermore, the addition of
109 mol/L to 107 mol/L ATRA
decreased the SCF-dependent mast cell production by CD34+
or CD34+c-kit+ cord blood cells. The
single-cell culture study showed a direct action of ATRA on the growth
of mast cell colonies by the CD34+c-kit+ cells.
The inhibitory effects of 109 mol/L to
108 mol/L ATRA, however, were modest at the early
phase of culture. These results suggest that RAs are able to inhibit
the production of human mast cells under stimulation with SCF, whereas
the early steps in mast cell development appear to be less sensitive to RA.
It has been demonstrated that GM-CSF, TGF-1, IL-4, and IL-6 are
potent inhibitors for the mast cell growth supported by
SCF.14,28-31 The concentrations of the 4 cytokines in the
supernatant of the cultured mast cells under stimulation with SCF alone
or SCF+ATRA were at levels below detectable limits. Moreover, none of
the neutralizing antibodies for the factors influenced the progeny generation in the presence of SCF alone or SCF+ATRA. Therefore, the
effects of ATRA on the mast cell development are unlikely to be
mediated by these cytokines. RT-PCR analysis showed that RAR,
RAR, RXR, and RXR mRNA were expressed in 10-week cultured mast
cells. The addition of Ro 13-7410 at 1010 mol/L to
107 mol/L decreased the number of mast cells grown
by SCF. The activity of the RAR selective agonist at a level of at
least 108 mol/L was comparable to that of ATRA at
107 mol/L. On the other hand, all concentrations of
Ro 25-7386, except for 107 mol/L, failed to inhibit
the mast cell growth. The addition of the RXR selective agonist at
107 mol/L to the culture containing SCF plus Ro
13-7410 at concentrations of 1010 mol/L to
107 mol/L did not further enhance the reduction of
the progeny production. These results imply that the antiproliferative
effects of RAs on the SCF-dependent mast cell growth are mainly induced
through RAR/RXR heterodimers. Among 3 of the synthetic retinoids
selective for RAR subtypes, when used at 109 mol/L
to 107 mol/L, only the RAR agonist was equivalent
to ATRA (107 mol/L) in the inhibition of the mast
cell growth. Conversely, the addition of 1 000- to 10 000-fold excess
concentrations of the RAR antagonist significantly counteracted the
ATRA-, 9-cis RA-, and RAR agonist-mediated reduction of the
number of mast cells grown under stimulation with SCF. Thus, RAR
appears to be the major endogenous RAR subtype for RA-dependent
regulation of mast cell production. In our previous study, the addition
of IL-6 or IL-4 resulted in a substantial decrease in the number of
cultured mast cells grown in SCF.14 Nevertheless, the
cultured mast cells exposed to SCF+IL-6 or SCF+IL-4 for 2 weeks showed an apparent increment in the intracellular histamine level compared with those exposed to SCF alone. Additionally, the percentage of
chymase+ cells was higher in the culture containing IL-6 or
IL-4. This may be due to the enhancement of chymase expression mediated
by these cytokines, because either IL-6 or IL-4 was required for the
induction of chymase+ cells from 4-week cultured cells
under stimulation with SCF (unpublished data). In this study, both ATRA
and 9-cis RA, when used at 107 mol/L,
inhibited the SCF-dependent mast cell generation to a greater extent
than IL-4 or IL-6, alone or in combination. It is of particular
interest that both of the RAs (107 mol/L) decreased
the histamine concentration in the cultured mast cells by approximately
50%. In addition, the RAs did not influence the chymase expression.
Therefore, RA appears to function as a negative regulator for both the
multiplication and intracellular histamine level of human mast cells.
Our results may provide a new strategy targeting the production process
of mast cells for prophylactic treatment of allergic disorders.
| |
Footnotes |
Submitted September 23, 1999; accepted January 3, 2000.
Supported by grants-in aid nos. 11670753 and 09041178 from the Ministry
of Education of Japan.
Reprints: Kenichi Koike, Department of Pediatrics, Shinshu
University School of Medicine, 3-1-1, Asahi, Matsumoto, 390-8621, Japan.
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.
| |
References |
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Kirshenbaum AS, Worobec AS, Davis TA, Goff JP, Semere T, Metcalfe DD.
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Douer D, Koeffler HP.
Retinoic acid enhances growth of human early erythroid progenitor cells in vitro.
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1982;69:1039-1041.
3.
Tobler A, Dawson MI, Koeffler HP.
Retinoids: structure-function relationship in normal and leukemic hematopoiesis in vitro.
J Clin Invest.
1986;78:303-309.
4.
Smeland EB, Rusten L, Jacobsen SE, et al.
All-trans retinoic acid directly inhibits granulocyte colony-stimulating factor-induced proliferation of CD34+ human hematopoietic progenitor cells.
Blood.
1994;84:2940-2945 |
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Abstract
Introduction