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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 496-508
Interleukin-6 Directly Modulates Stem Cell Factor-Dependent
Development of Human Mast Cells Derived From CD34+
Cord Blood Cells
By
Tatsuya Kinoshita,
Nobukuni Sawai,
Eiko Hidaka,
Tetsuji Yamashita, and
Kenichi Koike
From the Department of Pediatrics, Shinshu University School of
Medicine, and Central Clinical Laboratories, Shinshu University
Hospital, Matsumoto, Japan; and Research & Development, Mitsubishi
Kagaku Bio-Clinical Laboratories, Inc, Tokyo, Japan.
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ABSTRACT |
In the present study, we attempted to clarify the effects of
interleukin-6 (IL-6) on the growth and properties of human mast cells
using cultured mast cells selectively generated by stem cell factor
(SCF) from CD34+ cord blood cells. The addition of IL-6
to cultures containing mast cells resulted in a substantial reduction
of the number of progenies grown by SCF in the liquid culture. This
IL-6-mediated inhibition of mast cell growth may be due in part to the
suppression at the precursor level, according to the results of a
clonal cell culture assay. Moreover, a flow cytometric analysis showed
that the cultured mast cells grown in the presence of SCF+IL-6 had decreased c-kit expression. The exposure of cultured mast cells to
SCF+IL-6 also caused substantial increases in the cell size, frequency of chymase-positive cells, and intracellular histamine level
compared with the values obtained with SCF alone. The flow cytometric
analysis showed low but significant levels of expression of IL-6
receptor (IL-6R) and gp130 on the cultured mast cells grown with SCF.
The addition of either anti-IL-6R antibody or anti-gp130 antibody
abrogated the biological functions of IL-6. Although IL-4 exerted an
effect similar to that of IL-6 on the cultured mast cells under
stimulation with SCF, the results of comparative experiments suggest
that the two cytokines use different regulatory mechanisms. Taken
together, the present findings suggest that IL-6 modulates
SCF-dependent human mast cell development directly via an IL-6R-gp130 system.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MAST CELLS PLAY an important role as the
primary effector cells in immediate-type hypersensitivity reactions.
There are at least two phenotypically distinct subpopulations of mast cells in rodents,1,2 ie, mucosal mast cells and connective tissue mast cells. In humans, two types of mast cells have been identified on the basis of protease expression: one phenotype is
positive only for tryptase, and the other is positive for both tryptase
and chymase.3,4 To elucidate the physiological and pathological characteristics of human mast cells, numerous
investigators5-10 have attempted to establish human mast
cell cultures, because only limited numbers of mast cells can be
obtained from human tissues. Stem cell factor (SCF) has been
demonstrated to be a pivotal growth factor that promotes the
development of mast cells6-10 as well as the proliferation
and differentiation of various hematopoietic progenitors. However, the
purity of cultured mast cells grown by SCF alone has ranged from
approximately 40% to 85%.6,8,9
Interleukin-4 (IL-4) has been demonstrated to diminish the number of
mast cells that develop in response to SCF.11,12 In addition, IL-4 has various biological effects on human cultured mast
cells, including the upregulation of the expressions of functional high-affinity IgE receptor (Fc RI), intercellular adhesion
molecule-1, and lymphocyte function-associated
antigen-1.13-15 Toru et al16 recently reported
that IL-4 promotes the morphologic maturation of human cultured mast
cells in accordance with the increase of chymase expression.
In contrast to the relative abundance of information regarding the role
of IL-4 in the regulation of development and function of human mast
cells, the effects of IL-6 remain unclear. In the present study, we
attempted to clarify the effects of IL-6 on the development and
properties of human mast cells by using cultured mast cells generated
by SCF from CD34+ cord blood cells. Because fetal bovine
serum (FBS) is a potential endogenous source of hematopoietic growth
factors,17 we used serum-deprived cultures in this study.
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MATERIALS AND METHODS |
Factors and antibodies.
Human recombinant SCF and IL-3 were generously provided by Kirin
Brewery Co Ltd (Takasaki, Japan). Human recombinant IL-4 and
recombinant transforming growth factor- 1 (TGF-1) were purchased from R&D Systems (Minneapolis, MN). Human recombinant IL-6 was kindly
provided by Ajinomoto Co (Kawasaki, Japan).
For the experiments of neutralization of IL-6 activity at the
receptors, we used antihuman IL-6 receptor antibody (anti-IL-6R Ab;
R&D Systems) and antihuman gp130 Ab (B-R3; Biosource International, Camarillo, CA). The polyclonal anti-IL-6R Ab was made by immunizing goats with recombinant human IL-6 soluble R derived from the insect cell line Sf21. The monoclonal antibody (MoAb) against gp130
was made by immunizing mice with natural soluble gp130. These Abs recognize IL-6R and gp130, respectively, and can neutralize IL-6 activity. The neutralization dose50 (ND50) of
the anti-IL-6R Ab was determined to be approximately 1 to 4 µg/mL in
the presence of 20 ng/mL of IL-6 using the M1 cell line. Approximately
25 ng of the anti-gp130 Ab neutralized 0.2 ng of IL-6 activity by 50% in an XG-1 cell proliferation bioassay. The polyclonal rabbit antihuman
IL-6 Ab (1.2 mg/mL) was a gift from Ajinomoto Co. It was made by
immunizing rabbits with recombinant IL-6 in complete Freund's
adjuvant.18 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 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 TGF- 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. The MoAb
for CD2 (T11) was from Coulter (Miami, FL); the MoAbs for CD11b
(2LPM19c), CD15 (C3D-1), CD19 (HD37), and glycophorin A (JC159) were
from Dako (Glostrup, Denmark). The MoAb for eosinophil peroxidase
(MAB1087) was obtained from Chemicon International Inc. Control isotype
mouse MoAbs were purchased from Dako.
For the flow cytometric analysis, the MoAbs for c-kit (95C3,
phycoerythrin [PE]) and CD9 (ALB6, fluorescein isothiocyanate [FITC]) were purchased from Immunotech S.A. (Marseilles, France); the
MoAbs for CD34 (HPCA-2 FITC), CD33 (LeuM9 PE), CD11a (LFA1 FITC),
and CD11b (Leu15 PE) were from Becton Dickinson Immunocytometry Systems
(Mountain View, CA); and the MoAbs for CD45 (T29/33 FITC), CD61 (Y2/51
FITC), and CD68 (KP1, FITC) were from Dako. The MoAb for human FcRI
(CRA-1) was obtained from Kyokuto Pharmaceutical Industria Co
(Takahagi, Japan). For the analysis of IL-6R and gp130 expressions on
the cultured mast cells grown with SCF, we used the PE-conjugated MoAb
against human IL-6R (M91; Immunotech) and the anti-gp130 MoAb described
above, 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 were separated by density centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ), washed
twice, and suspended in Ca2+- and Mg2+-free
phosphate-buffered saline (PBS) containing 1 mmol/L EDTA-2Na and 2.5%
FBS (Hyclone, Logan, UT). After treatment with Silica (Immuno-Biological Laboratories, Fujioka, Japan) for 30 minutes at
37°C, CD34+ cells were enriched using a Dynal CD34
Progenitor Cell Selection System (Dynal A.S., 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 performed for 45 minutes at room temperature. The detached beads
were removed by the magnet, and the cells were collected as
CD34+ cells. As shown in Fig 1,
greater than 90% of the isolated cells were CD34+, as
determined by FACScan flow cytometry (Becton Dickinson).
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| Fig 1.
CD34 expression on cord blood cells separated by
immunomagnetic beads coated with anti-CD34 monoclonal antibody. ( )
Labeled with FITC-conjugated anti-CD34 MoAb. (···) Labeled with
FITC-conjugated mouse IgG.
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Suspension cultures.
Serum-deprived liquid cultures were performed in 6-well culture plates
(#3046; Becton Dickinson) using a modification of the technique
described previously.20 CD34+ cells (1 × 105) were cultured in each well containing 10 mL of
-medium (Flow Laboratories Inc, Rockville, MD) supplemented with 1%
deionized bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO),
300 µg/mL fully iron-saturated human transferrin (~98% pure;
Sigma), 16 µg/mL soybean lecithin (Sigma), 9.6 µg/mL cholesterol
(Nakalai Chemicals Ltd, Tokyo, Japan), 10 ng/mL of SCF, 100 U/mL of
IL-3, and 50 ng/mL of IL-6, 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 cells and culture medium was replaced weekly with fresh
medium containing the cytokine(s). The number of viable cells was
determined by a trypan-blue exclusion test using a hemocytometer. For
the examination of the effects of IL-6, IL-4, and IL-3 on the
SCF-dependent development of the cultured mast cells, 1 to 2 × 104 cells at various culture stages were incubated for 1 to
2 weeks in 24-well culture plates (#3047; Becton Dickinson) containing 10 or 100 ng/mL of SCF, 50 ng/mL of IL-6, 20 ng/mL of IL-4, and 100 U/mL of IL-3, alone or in combination.
Serum-containing cultures contained 10% FBS or 10% pooled cord blood
sera instead of a combination of BSA, transferrin, lecithin, and
cholesterol. To examine the effects of FBS on SCF-dependent or
SCF+IL-6-dependent mast cell growth, we used different sources of FBS,
ie, Hyclone, GIBCO BRL (Grand Island, NY), Sigma, Stem Cell
Technologies (Vancouver, British Columbia, Canada).
Serum-deprived single-cell culture.
Single-cell sorting was performed by two-step sorting. Cord blood
mononuclear cells (2 × 106) were stained with 20 µL
of FITC-conjugated anti-CD34 MoAb. After two washes, CD34+
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, and 10 ng/mL of SCF, using the FACStarplus
flow cytometer equipped with an automatic cell deposition unit (Becton
Dickinson), as described previously.21,22 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. Then, colonies consisting of more than 50 cells were
individually lifted with an Eppendorf micropipet, and the constituent
cells of colonies were stained with antitryptase MoAb.
Clonal cell cultures.
The mast cell colony assay was performed in 35-mm Lux suspension
culture dishes (#171099; Nunc, Naperville, IL) using a modification of
the technique described previously.23 The culture consisted of 5,000/mL 10-week-old cultured cells grown by 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 50 ng/mL of IL-6. Dishes were incubated at
37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2, and 90% N2. On days 7 and 14, aggregates consisting of 30 or more cells were scored as mast
cell colonies, and those consisting of 10 to 29 cells were scored as
mast cell clusters. To confirm the in situ identification of mast
cells, 60 individual colonies and clusters were lifted with a 3-µL
Eppendorf micropipette, spread on glass slides using a Cytospin II
(Shandon Southern, Sewickly, PA), and stained with the antitryptase
MoAb or mouse IgG1 using the alkaline phosphatase-antialkaline
phosphatase (APAAP) technique. Almost all of the constituent cells were
positive for tryptase.
Cytochemical staining.
Cultured cells (5 × 103) were spread on glass slides
using a Cytospin II and stained with May-Grünwald-Giemsa,
toluidine blue, and Bieblich scarlet. Cytochemical reactions with
peroxidase and -naphthyl butyrate esterase were performed by the
conventional methods.
Immunocytochemical staining.
Reactions with mouse MoAbs against tryptase, chymase, eosinophil
peroxidase, and CD11b were detected using the APAAP method (Dako APAAP
Kit System; Dako Corp, Carpinteria, CA), as described previously.24 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 three times, the samples were reacted with a mouse MoAb for 30 minutes at room temperature in a humidified chamber. After three more
washes with PBS, the samples were incubated with rabbit antimouse IgG
antibody, washed three 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. Five hundred cells were examined.
The diameter of the mast cells was measured by calculating the average
of two perpendicular diameters of tryptase+ cells on glass
slides, using a microscope equipped with an ocular micrometer.
Flow cytometric analysis.
For the analysis of surface markers on the cultured cells, 1 to 2 × 105 cells were collected in plastic tubes and
incubated with an appropriately diluted FITC- or PE-conjugated MoAb, as
described previously.21,22 After the cells were washed
twice, their surface markers were analyzed with a FACScan flow
cytometer using the Lysis 2 software program. Based on our preliminary
results obtained by the incubation of nonfixed mast cells with
propidium iodine, viable cells were gated according to their forward
scatter characteristics and side scatter characteristics. The
proportion of positive cells was determined by comparison to cells
stained with an FITC- or PE-conjugated mouse isotype-matched IgG (Dako).
For the analysis of FcRI and gp130 on the cultured mast
cells, 1 × 105 cells were incubated with 20 µL anti-FcRI MoAb or anti-gp130 MoAb for 30 minutes at 4°C.
The mouse isotype MoAb was used as a control. The cells were washed
three times and stained with FITC-conjugated goat antimouse Ig (GAM;
Becton Dickinson) for 15 minutes.
For the assay of the intracellular CD68 expression of the cultured
cells grown with SCF, the cells were treated with 2 mL of ORTHO
PermeaFix (Ortho Diagnostic Systems, Raritan, NJ) for 40 minutes at
room temperature and then incubated with the FITC-conjugated anti-CD68
MoAb, as described previously.20 An FITC-conjugated isotype
MoAb was used as a control.
Ultrastructural study.
For the ultrastructural examination, the cells were fixed with 1.25%
glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.2) for 2 hours and
postfixed in 1% osmium tetroxide, as described previously.24 The specimens were then dehydrated in alcohol and embedded in Araldite (Nissin Co, Tokyo, Japan). Ultrathin sections
were stained with uranyl acetate and lead citrate. These sections were
then examined with an electron microscope (H-300; Hitachi, Tokyo, Japan).
Assay of histamine 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 a radioimmunoassay (RIA; Immunotech). The GM-CSF concentrations in the supernatant of the cultured cells were measured by an enzyme-linked immunosorbent assay (Amersham International, Buckinghamshire, UK). We also measured the concentrations of GM-CSF, IL-4, IL-6, and TGF-1 in pooled cord blood sera using this assay. All assays were conducted in triplicate.
Statistical analysis.
All experiments were performed at least two times and were shown to be
reproducible. Values are expressed as the means ± SD. The
Student's t-test was used to determine the significance of differences between two independent groups. One-way analysis of variance, followed by post hoc contrasts with Bonferroni limitation, was used for more than three independent groups.
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RESULTS |
SCF alone stimulates the selective growth of mast cells from
CD34+ cord blood cells.
CD34+ cord blood cells (1 × 105)
separated by immunomagnetic beads were plated in wells containing 10 mL
of serum-deprived culture medium supplemented with 10 ng/mL of SCF.
Half of the cultured cells and culture medium was replaced weekly with
fresh culture medium containing 10 ng/mL of SCF. Cultured cells were
nonadherent throughout the culture period. As shown in
Fig 2A, a progressive, steady increase of
cell production was achieved, but the cell growth abated after 26 weeks. The cumulative cell number reached 1010-fold the
input quantity at 50 weeks of culture. There was no cell growth in the
absence of SCF. The May-Grünwald-Giemsa staining showed that
almost all of the cultured cells generated after 4 weeks had a round or
oval nucleus and contained prominent granules in the cytoplasm, as
shown in Fig 3A. The cells became larger and contained more granules in the cytoplasm with prolongation of the
culture period (Fig 3B). The cultured cells grown beyond 4 weeks were
negative for peroxidase, -naphthyl butyrate esterase, and Biebrich
scarlet stainings. The cells with other lineage-specific markers (CD2,
CD19, CD11b, CD15, or glycophorin A) were at negligible levels
according to the immunocytochemical staining. The granules showed
metachromasia, as determined by toluidine-blue staining. As shown in
Table 1, tryptase-positive cells appeared
at 1 week of culture. After 4 weeks, almost all of the cultured cells
reacted with antitryptase MoAb, but were negative for chymase. At 10 weeks of culture, a part of the cultured cells weakly reacted with
antichymase MoAb (Fig 3E). After 36 weeks, a vast majority of the cells
were positive for both types of protease, as shown in Fig 3D and F. When 1 × 104 CD34+ cord blood cells
sorted by the FACStarplus flow cytometer were used as
target cells, significant cell production was also observed: 3.5 ± 0.3 × 104 cells at 1 week, 9.5 ± 0.3 × 104 cells at 2 weeks, 23.3 ± 2.6 × 104 cells at 3 weeks, and 59.5 ± 2.6 × 104 cells at 4 weeks. Almost all of the 4-week cultured
cells were positive for tryptase.
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| Fig 2.
Time course of mast cell development from
CD34+ cord blood cells in serum-deprived liquid cultures.
(A) CD34+ cord blood cells (1 × 105) were
cultured in each well containing 10 mL of serum-deprived liquid culture
medium supplemented with 10 ng/mL of SCF. The number of viable cells
was serially counted, and the results presented are corrected for
demipopulation. Similar results were obtained in the other two
experiments. Values are expressed as the mean ± SD. (B) The single
CD34+ cord blood cells were sorted into the individual
wells of a 96-well culture plate containing 10 ng/mL of SCF, as
described in Materials and Methods. The number of cells in each well
was serially counted until 4 weeks. The cell growth of a total of 15 colonies that generated more than 50 cells positive for tryptase at 4 week is shown.
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| Fig 3.
Cytological characteristics of 10-week and 40-week
cultured cells grown by SCF. Cytochemical and immunologic stainings of
the cultured cells grown by 10 ng/mL of SCF were performed on
cytocentrifuged samples. Staining of 10-week cultured cells (A) and
40-week cultured cells (B) with May-Grünwald-Giemsa. Staining of
10-week cultured cells (C) and 40-week cultured cells (D) with an MoAb
for tryptase. Staining of 10-week cultured cells (E) and 40-week
cultured cells (F) with an MoAb for chymase. (Original magnification × 1,000.)
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To elucidate the cellular growth in the early phase of culture, we
performed single CD34+ cell culture studies. The cell
growth of a total of 15 colonies that generated more than 50 cells at 4 week is shown in Fig 2B. The number of cells in each well was 21 ± 12 (range, 5 to 52) cells at 1 week, 59 ± 53 (range, 4 to 202)
cells at 2 weeks, 189 ± 140 (range, 10 to 467) cells at 3 weeks,
and 525 ± 578 (range, 74 to 1821) cells at 4 weeks. Almost all of
the 4-week cultured cells in the individual wells reacted with
antitryptase MoAb. On the other hand, 29% of CD34+ cells
showed an apparent, but transient progeny generation and eventually
degenerated. The remaining CD34+ cells underwent no cell division.
The electron microscopic analysis showed that the 40-week cultured
cells had more granules in their cytoplasm compared with the 10-week
cultured cells. The complete scrolls reported for lung
tryptase+ mast cells by Craig et al25,26 were
not observed in the cells at either timepoint. Instead, the granules
were similar to electron-dense cores, as described for immature
tryptase+ chymase+ mast cells in tissues.
The cultured cells had a significant amount of intracellular histamine.
The histamine levels in the cell lysates increased with the culture
period: 1.05 ± 0.08 pg/cell at 4 weeks, 1.53 ± 0.11 pg/cell at
10 weeks, 2.57 ± 0.13 pg/cell at 30 weeks, and 5.05 ± 0.1 pg/cell at 40 weeks. The flow cytometric analysis showed that, whereas
both the 10-week and 40-week cultured cells displayed high c-kit
antigen density, the mean intensity was substantially lower in the
40-week cells. Both of the cells were positive for CD33 and CD45
antigens and weakly positive for FcRI. The percentages of CD9 and
CD61 were low and those of CD11a and CD11b were virtually negative.
Moreover, an intracellular expression but not surface expression of
CD68 was observed. Thus, the present results indicated that SCF alone
could stimulate the selective growth of mast cells from
CD34+ human cord blood cells in serum-deprived culture conditions.
Since Durand et al10 reported that a cocktail of SCF and
IL-3 is necessary for human mast cell differentiation from
CD34+ cord blood cells, we examined whether IL-3 influenced
SCF-dependent mast cell development from CD34+ cord blood
cells in our culture condition. As shown in
Fig 4A, IL-3 alone could not support the
generation of tryptase+ cells. The frequency of
tryptase+ cells grown under stimulation with SCF+IL-3 was
10.5% to 13.5% at 4 weeks. Consequently, there was no difference in
the absolute numbers of tryptase+ cells between SCF and
SCF+IL-3. These results were confirmed by the reaction with anti-c-kit
MoAb (Fig 4B). Significant numbers of the cultured cells grown by IL-3
or SCF+IL-3 were identified to be eosinophils and basophils by
toluidine blue staining and immunocytochemical staining. Next, we
examined the effects of IL-3 on the development of 10-week-old cultured
mast cells grown by SCF. After 2 weeks, the 10-week cultured cells did
not grow/survive in the presence of IL-3 alone. In addition, IL-3
failed to influence SCF-dependent progeny generation by 1 × 104 10-week-old cultured cells: the numbers of progenies
after 2 weeks were 1.08 ± 0.16 × 105 in SCF alone
and 1.11 ± 0.15 × 105 in SCF+IL-3. No difference
in the percentages of chymase+ cells was observed between
the two groups.
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| Fig 4.
Effects of IL-3 on mast cell growth by
CD34+ cord blood cells. (A) CD34+ cord
blood cells (1 × 103 to 1 × 105) were
cultured with 10 ng/mL of SCF and/or 100 U/mL of IL-3. The number of
viable cells was counted at 4 weeks, and then the percentage of
tryptase+ cells ( ) was determined by
immunocytochemical staining. Values are expressed as the mean ± SD.
(B) Expression of c-kit on the 4-week cultured cells was analyzed by
flow cytometry. The percentage of c-kit+ cells in the
cultured cells grown by SCF+IL-3 was 12.7%. () Labeled with
PE-conjugated anti-c-kit MoAb. (···) Labeled with PE-conjugated
mouse IgG.
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Effects of IL-6 on the growth and properties of cultured mast cells
supported by SCF.
We examined the effects of IL-6 on the growth and properties of
cultured mast cells generated by SCF from CD34+ cord blood
cells. Ten-week-old cultured mast cells (1 × 104)
were incubated for 2 weeks in wells containing IL-6 at concentrations ranging from 0.01 to 100 ng/mL, with SCF at 100 ng/mL. The results are
shown in Fig 5A. The addition of IL-6 to
the culture with SCF gave rise to a significant decrease in the numbers
of progenies. Maximum inhibition was seen with greater than 10 ng/mL of
IL-6. In subsequent experiments, IL-6 at 50 ng/mL was used. To examine whether IL-6 exerted an inhibitory effect at the mast cell precursor level, 10-week cultured cells were plated at 5,000 cells per dish containing serum-deprived methylcellulose culture medium supplemented with SCF or SCF+IL-6. As shown in Fig 5B, SCF alone supported the
formation of 98.0 ± 20.0 mast cell colonies and 195.5 ± 35.1 clusters. The addition of IL-6 resulted in a marked reduction of
SCF-dependent mast cell colony growth (1.5 ± 1.3 colonies and 51.0 ± 14.4 clusters). The ratio of colonies to clusters changed with
time in culture; at 1 week, 1.0 ± 0.8 colonies and 69.0 ± 8.3 clusters were grown in the culture with SCF alone, whereas no colonies
and 0.8 ± 0.5 clusters were formed in the culture with SCF+IL-6.
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| Fig 5.
Effects of IL-6 on SCF-dependent mast cell development.
(A) Dose response to IL-6 or IL-4 of mast cell growth supported by SCF.
Ten-week cultured mast cells (1 × 104) were incubated in
wells containing either IL-6 at 0.01 to 100 ng/mL or IL-4 at 0.01 to 80 ng/mL with SCF at 100 ng/mL. After 2 weeks, the number of viable cells
was determined. Significantly different from SCF alone (*P < .0005, **P < .0001). (B) Effects of IL-6 on the formation of
mast cell colonies and clusters supported by SCF. Five thousand 10-week
cultured mast cells were plated per dish containing serum-deprived
methylcellulose culture medium supplemented with SCF or SCF+IL-6.
After 14 days, aggregates consisting of 30 or more cells were scored as
mast cell colonies and those of 10 to 29 cells were scored as mast cell
clusters. SCF, 100 ng/mL; IL-6, 50 ng/mL. Numbers of mast cell colonies
() and mast cell clusters ( ). Results shown are the mean ± SD
of three experiments. Significantly different from SCF alone
(*P < .0001). (C) Effects of IL-6 on the cell growth by
CD34+ cord blood cells under stimulation with SCF.
CD34+ cord blood cells (1 × 104) were
cultured with 10 ng/mL of SCF or 10 ng/mL of SCF + 50 ng/mL of IL-6.
The number of viable cells was determined every week. Significantly
different from SCF alone (*P < .0001).
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Next, we examined the stage of differentiation of hematopoietic
progenitors into mast cell lineage on which IL-6 exerted the inhibitory
action. IL-6 was added together with CD34+ cord blood cells
and SCF on day 0. As shown in Fig 5C, the number of viable cells
generated by SCF+IL-6 was comparable to the value obtained by SCF alone
until 3 weeks. However, significant differences were observed after 4 weeks. Even when SCF was added on day 1 to the culture initiated with
IL-6, SCF-dependent cell growth was not affected during the early
culture period. There was no difference in the frequency of
tryptase+ cells in the 2-week cultured cells between SCF
alone and SCF+IL-6 (70% v 74%).
Nakahata et al27 reported that IL-6 is a requisite for the
growth of sufficient numbers of pure mast cells from cord blood cells
in a serum-containing liquid culture medium supplemented with SCF.
Thus, we compared the SCF-dependent (10 ng/mL) and SCF (10 ng/mL)+IL-6
(50 ng/mL) -dependent progeny production by 1 × 104
cultured mast cells at the age of 10 weeks in the two different liquid
culture conditions. In the serum (Hyclone's FBS)-containing cultures,
SCF alone generated 0.19 ± 0.03 × 104 cells and
SCF+IL-6 generated 1.59 ± 0.27 × 104 cells after
2 weeks. In the serum-deprived cultures, SCF alone yielded 3.13 ± 0.18 × 104 cells and SCF+IL-6 yielded 1.99 ± 0.15 × 104 cells. Because similar results were obtained by
the other sources of FBS, we attempted to identify a negative
regulator(s) in FBS using the cord blood sera of normal human neonates
because of the unavailability of antibovine cytokine
antibodies. The addition of 10% pooled cord blood sera to the culture
containing the 10-week cultured cells and 100 ng/mL of SCF decreased
the number of progenies by 35% after 1 week. According to the
conventional enzyme immunoassay, the concentrations of GM-CSF, IL-4,
IL-6, and TGF-1 in pooled cord blood sera were less than 2 pg/mL,
less than 0.5 pg/mL, 8.46 pg/mL, and 55.4 ng/mL, respectively. None of
neutralizing anti-GM-CSF antibody at 2 µg/mL, anti-IL-4 antibody at
10 µg/mL, and anti-IL-6 antibody at 10 µg/mL influenced the
progeny generation in the presence of SCF plus cord blood sera, whereas
the addition of anti-TGF- antibody at 200 µg/mL partially but
significantly restored the cell production. Furthermore, 5 to 10 ng/mL
of TGF-1 substantially inhibited SCF-dependent mast cell growth.
Along with the cell growth retardation, SCF+IL-6 caused a significant
increment in the mean diameter of cultured mast cells: 15.3 ± 2.2 µm (range, 11.5 to 20 µm; n = 100) in 10 ng/mL of SCF alone versus
22.4 ± 4.6 µm (range, 12 to 32 µm; n = 100) in 10 ng/mL of SCF + 50 ng/mL of IL-6 (P < .0001), and 21.0 ± 3.6 µm (range, 14 to 41 µm; n = 100) in 100 ng/mL of SCF alone versus 28.9 ± 5.4 µm (range, 18 to 50 µm; n = 100) in 100 ng/mL of SCF + 50 ng/mL of IL-6 (P < .0001). Furthermore, IL-6 substantially increased the frequency of chymase+ cells in the cultured
mast cells grown by SCF, as shown in Fig 6.
At the initiation of culture, almost 100% of the 10-week cultured cells were positive for tryptase, but only 4.0% ± 1.4%
of them reacted with antichymase MoAb. After 2 weeks, in the case of 10 ng/mL of SCF, the percentage of chymase+ cells increased to
5.0% ± 1.2% in SCF alone and 49.7% ± 8.1% in SCF+IL-6. In
the case of 100 ng/mL of SCF, the percentages of chymase+
cells were 34.7% ± 8.6% in SCF alone and 81.8% ± 10.4% in
SCF+IL-6. Next, we examined the effect of IL-6 on the intracellular
histamine levels of the cultured mast cells. As shown in Fig 6, the
addition of IL-6 to the culture containing 10 ng/mL or 100 ng/mL of SCF substantially increased the histamine content compared with SCF alone.
Similar results were obtained in cultures with 25-week cultured mast
cells (data not shown).
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| Fig 6.
Effects of IL-6 on SCF-dependent development and
intracellular histamine levels of cultured mast cells. Ten-week
cultured mast cells (1 × 104) were plated in
wells containing SCF or SCF+IL-6 for 2 weeks. After the numbers of
viable cells were counted, the percentages of tryptase+
cells and chymase+ cells were determined by
immunocytochemical staining. At the same time, the intracellular
histamine concentrations were measured by the RIA. Results shown are
the mean ± SD of three experiments. SCF, 10 or 100 ng/mL; IL-6, 50 ng/mL. Tryptase+chymase cells (),
tryptase+chymase+ cells ().
Significantly different from SCF alone (*P < .05, #P < .001).
|
|
Expressions of IL-6 receptor and gp130 on cultured mast cells grown
by SCF.
To elucidate whether IL-6 exerted its effect through the receptor, we
examined the expressions of IL-6R and gp130 on cultured mast cells
grown by SCF using flow cytometry. The cultured mast cells showed low
but significant expressions of IL-6R and gp130 (Fig 7). The addition of IL-6 to the
cultures containing SCF resulted in a decrease in the expressions of
the two receptors on cultured mast cells.
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| Fig 7.
Expressions of IL-6 receptor, gp130, and c-kit on
cultured mast cells grown by SCF. Expressions of IL-6R, gp130, and
c-kit on 10-week mast cells exposed to SCF, SCF+IL-6, or SCF+IL-4
for 2 weeks were analyzed by flow cytometry according to the procedure
described in Materials and Methods. () Labeled with FITC-conjugated
anti-IL-6R MoAb, anti-gp130 MoAb followed by FITC-conjugated GAM, or
PE-conjugated anti-c-kit MoAb. (···) Labeled with
FITC-conjugated mouse IgG, mouse IgG followed by FITC-conjugated GAM,
or PE-conjugated mouse IgG. SCF, 100 ng/mL; IL-6, 50 ng/mL; IL-4, 20 ng/mL.
|
|
Next, we examined whether anti-IL-6R Ab or anti-gp130 Ab could
counteract the biological activity of IL-6. The results presented in
Fig 8 are from one representative
experiment of three. Similar results were obtained in the other two
experiments. Anti-IL-6R Ab or anti-gp130 Ab at 5 or 10 µg/mL was
added to the cultures containing SCF or SCF+IL-6. Anti-IL-6R Ab
neutralized the inhibitory effect of IL-6 on mast cell growth, although
the numbers of mast cells grown by SCF alone were not influenced.
Conversely, the anti-IL-6R Ab abrogated the stimulatory effect of IL-6
on the intracellular histamine content. Like anti-IL-6R Ab, anti-gp130 Ab neutralized both of these effects of IL-6.
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| Fig 8.
Neutralization of IL-6 activity by anti-IL-6R Ab or
anti-gp130 Ab. To examine whether anti-IL-6R Ab or anti-gp130 Ab
counteracted the biological effects of IL-6 on the cultured mast cells
supported by SCF, each Ab was added at 5 µg/mL () or 10 µg/mL
() to wells containing 1 × 104 10-week cultured cells
with SCF, SCF+IL-6, or SCF+IL-4. SCF, 100 ng/mL; IL-6, 50 ng/mL;
IL-4, 20 ng/mL. Significantly different from no Ab (*P < .0001).
|
|
Comparison of effects of IL-6 with those of IL-4 on mast cell
development supported by SCF.
Because several investigators11-16 have reported the effect
of IL-4 on the growth, c-kit expression, and functions of mast cells
using serum-containing liquid cultures, we compared the effects of IL-6
with those of IL-4 on the development of cultured mast cells supported
by SCF. The addition of IL-4 decreased the numbers of mast cells grown
by SCF, and maximum inhibition was seen with greater than 10 ng/mL of
IL-4 (Fig 5A). Moreover, the exposure of 10-week cultured mast cells to
SCF at 10 or 100 ng/mL plus IL-4 at 20 ng/mL caused an increase in the
histamine concentrations in the cell lysates compared with the values
obtained by SCF alone, as shown in Fig 9.
Similar results were obtained in the case of 40-week cultured mast
cells. It is of interest that IL-6 was a more potent stimulator of the
intracellular histamine content of cultured mast cells under
stimulation with SCF than was IL-4. In the cultures containing 10-week
cultured mast cells and 100 ng/mL of SCF, IL-6 more intensely inhibited
the mast cell growth compared with IL-4, which was consistent with the
results shown in Fig 5A. When SCF, IL-6, and IL-4 were used together,
both the inhibition of mast cell growth and the elevation of
intracellular histamine levels were further amplified, except for the
cultures containing 40-week cells and 10 ng/mL of SCF. We then examined whether neutralizing anti-IL-4 antibody and anti-IL-6 antibody influenced the action of IL-6 and IL-4 on SCF-dependent mast cell growth, respectively. In the experiments using anti-IL-4 antibody at
100 µg/mL, the numbers of viable cells grown by 1 × 104 10-week-old cultured cells after 2 weeks were: 8.1 ± 0.7 × 104 cells without the antibody and 8.3 ± 0.8 × 104 cells with the antibody in SCF (100 ng/mL) alone; 2.2 ± 0.6 × 104 cells without the
antibody and 2.1 ± 0.3 × 104 cells with the
antibody in SCF (100 ng/mL) +IL-6 (50 ng/mL); and 5.4 ± 0.7 × 104 cells without the antibody and 8.0 ± 0.8 × 104 cells with the antibody in SCF (100 ng/mL) +IL-4 (20 ng/mL). On the other hand, in the experiments using anti-IL-6 antibody at 60 µg/mL, the numbers of progenies were as follows: 8.6 ± 0.5 × 104 cells without the antibody and 8.8 ± 0.7 × 104 cells with the antibody in SCF alone; 2.2 ± 0.6 × 104 cells without the antibody and 8.6 ± 0.7 × 104 cells with the antibody in
SCF+IL-6; and 5.9 ± 1.0 × 104 cells
without the antibody and 5.8 ± 0.7 × 104 cells
with the antibody in SCF+IL-4.
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| Fig 9.
Comparison of effects of IL-6 with those of IL-4 on mast
cell development supported by SCF. Ten-week or 40-week cultured mast
cells (2 × 104) were incubated in culture wells
containing 10 or 100 ng/mL of SCF, 50 ng/mL of IL-6, or 20 ng/mL of
IL-4, alone or in combination. After 2 weeks, the number of viable
cells was determined, and the cells were then processed for
immunocytochemical staining with an antitryptase or antichymase MoAb.
At the same time, histamine amounts in cell lysates were analyzed by
the RIA. The results shown are from one representative experiment of
three. Similar results were obtained in the other two experiments.
Experiment groups using 10 ng/mL of SCF ( ) experiment groups using
100 ng/mL of SCF (); tryptase+ chymase
cells (); tryptase+chymase+ cells ().
Significantly different from SCF alone (*P < .0005, **P < .0001). Significantly different among the two- or
three-factor combinations ( P < .0005, P < .0001).
|
|
Finally, we compared the effects of IL-6 with those of IL-4 on the
c-kit expression of cultured mast cells grown by SCF. As shown in Fig
7, IL-6 reduced the c-kit expression to a greater extent than did IL-4.
| |
DISCUSSION |
The present study clearly showed that, even when SCF was used alone,
the selective growth of a large number of mast cells from
CD34+ cord blood cells could be supported in long-term
serum-deprived cultures. The possibility of basophil lineage of the
cultured cells was ruled out, because the cells were positive for
c-kit, tryptase, and CD68, but negative for CD11b. Single
CD34+ cell culture studies imply that SCF acts as a
proliferative rather than survival factor in mast cell development from
cord blood hematopoietic progenitors. Whereas SCF and IL-3 are required
for murine mast cell development,28 the present study
showed that IL-3 failed to stimulate the growth of mast cells from
CD34+ cord blood cells in the presence or absence of SCF.
These results may represent a species-specific growth factor requirement.
Nakahata et al27 demonstrated that IL-6 is a requisite for
the growth of sufficient numbers of almost 100% pure mast cells from
cord blood cells in a serum-containing liquid culture medium supplemented with SCF. Based on the present experiments in which the
effects of SCF or SCF+IL-6 on the growth of cultured mast cells were
compared between serum-containing and serum-deprived cultures, the
discrepancy in the results was due to the distinct liquid culture
systems. The results obtained by the addition of neutralizing
anticytokine antibodies suggest a possible role of TGF- as a
negative regulator present in serum.
The addition of IL-6 resulted in a substantial reduction of the number
of cultured mast cells grown by SCF. In the clonal cell culture, the
numbers of mast cell colony-forming cells and cluster-forming cells
were markedly lower in the cultures with SCF+IL-6 than in the cultures
with SCF. These results suggest that the decreased number of mast cells
by the addition of IL-6 to the culture containing SCF in the liquid
culture is attributable in part to the inhibition at the mast cell
precursor level. However, IL-6 failed to alter SCF-dependent cell
growth by CD34+ cord blood cells until 3 weeks. In
addition, there was no difference in the frequency of
tryptase+ cells in the 2-week cultured cells between SCF
alone and SCF+IL-6. Thus, IL-6 does not appear to affect the
differentiation process into mast cell lineage. The flow cytometric
analysis showed that the exposure of the cultured mast cells to
SCF+IL-6 attenuated the c-kit expression. The addition of IL-4 to the
culture with 10-week cultured mast cells and 100 ng/mL of SCF also
caused the retardation of the cell growth and the reduction of c-kit
expression, but both of these parameters were at lower magnitudes
compared with the values obtained by the addition of IL-6 (Figs 5A, 7, and 9). Furthermore, the 40-week cultured mast cells generated the
progenies in response to SCF to a lesser extent compared with the
10-week cultured mast cells (Fig 9). The mean intensity of c-kit
antigen density was substantially shifted to lower values on the
late-appearing mast cells. These results imply the association of the
proliferative potential to SCF of cultured mast cells with the c-kit
expression. Therefore, one possible explanation is that the
IL-6-mediated reduction of c-kit expression causes the growth retardation of mast cells. However, we cannot rule out the possibility that the decreased levels of c-kit are a consequence, but not a cause
of an intrinsically decreased cell proliferation potential.
The cultured mast cells exposed to SCF+IL-6 for 2 weeks also showed a
more substantial increase in the intracellular histamine levels than
those exposed to SCF alone. The stimulation of the 10-week cultured
mast cells with SCF+IL-6 for 24 hours did not influence the cell
number, but increased the histamine concentrations of both the cell
lysate and the supernatant compared with the values obtained with SCF
alone (unpublished data). Thus, the increased level of
intracellular histamine may reflect an upregulation of histamine
production rather than an increment in histamine storage.
Because GM-CSF was reported to suppress SCF-dependent mast cell
growth,29,30 it is possible that the effect of IL-6 on mast
cell development is mediated by GM-CSF. In the present study, the
levels of GM-CSF in the supernatant of the 10-week-old mast cells
cultured with 100 ng/mL of SCF and with 100 ng/mL of SCF + 50 ng/mL of
IL-6 for 2 weeks were 3.1 ± 0.3 pg/mL and 2.8 ± 0.1 pg/mL,
respectively. Moreover, the addition of neutralizing anti-GM-CSF Ab
did not influence the SCF-dependent or SCF+IL-6-dependent mast cell
growth (unpublished data). The present flow cytometric analysis showed low but significant expressions of IL-6R and gp130 on
the cultured mast cells grown by SCF. The addition of anti-IL-6R Ab
abrogated both the inhibition of mast cell growth and the elevation of
the intracellular histamine content induced by SCF+IL-6. The anti-gp130
Ab also neutralized the biological activity of IL-6. These results
imply that the effects of IL-6 on the cultured mast cells are exerted
directly via an IL-6R-gp130 system.
Both IL-6 and IL-4 decreased the numbers of cultured mast cells but
also increased intracellular histamine levels under stimulation with
SCF. Comparative assays of the effects of IL-6 with those of IL-4 on
SCF-dependent mast cell development showed that significantly higher
concentrations of histamine in cell lysates were obtained in the
cultures with SCF+IL-6. Thus, IL-6 may be a more potent stimulator of
the intracellular histamine content of mast cells supported by SCF than
is IL-4. When SCF, IL-6, and IL-4 were used together, both the
inhibition of the cell growth and the elevation of intracellular
histamine levels were further amplified compared with the values
obtained by the two-factor combinations except for the cultures
containing 40-week cultured mast cells and 10 ng/mL of SCF. In addition
to the lack of effects of the anti-IL-6R Ab and anti-gp130 Ab on the
biological activity of IL-4 (Fig 8), the neutralizing anti-IL-6 Ab did
not influence the numbers of cultured mast cells grown by SCF+IL-4. On
the other hand, the numbers of cultured mast cells grown by SCF+IL-6
were not affected by the addition of neutralizing anti-IL-4 Ab.
Therefore, IL-6 appears to modulate SCF-dependent human mast cell
development by a mechanism different from that of IL-4.
| |
ACKNOWLEDGMENT |
The authors are deeply indebted to Prof A. Komiyama (Department of
Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan)
for helpful comments and to Prof T. Katsuyama (Department of Laboratory
Medicine, Shinshu University School of Medicine and Central Clinical
Laboratory, Shinshu University Hospital) for valuable discussion on all
aspects of electron microscopy. We also thank Dr S. Ito (Blood
Transfusion Service, Shinshu University Hospital) for his excellent
technical assistance.
| |
FOOTNOTES |
Submitted December 3, 1998; accepted March 11, 1999.
Supported by Grants-in Aid No. 09670796 and 09770537 from the Ministry
of Education of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kenichi Koike, MD, Department of
Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi,
Matsumoto, 390-8621, Japan.
| |
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ABSTRACT
INTRODUCTION