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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 1055-1061
Stem Cell Factor Influences Mast Cell Mediator Release in
Response to Eosinophil-Derived Granule Major Basic Protein
By
Glenn T. Furuta,
Steven J. Ackerman,
Lei Lu,
Rachel E. Williams, and
Barry K. Wershil
From The Combined Program in Pediatric Gastroenterology and
Nutrition, The Children's Hospital and Massachusetts General Hospital,
and the Division of Experimental Pathology, Beth Israel Deaconess
Medical Center, and Harvard Medical School, Boston, MA; and the
Department of Biochemistry and Molecular Biology, The University of
Illinois at Chicago, Chicago, IL.
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ABSTRACT |
Stem cell factor (SCF) is an important mast cell growth,
differentiation, and survival factor. We investigated whether SCF influenced the response of mouse mast cells to an IgE-independent stimulus, eosinophil-derived granule major basic protein (MBP). Mouse
bone marrow cultured mast cells (BMCMC) were derived in either
concanavalin-stimulated mouse spleen conditioned medium (CM) or SCF.
The cloned growth, factor-independent mast cell line Cl.MC/C57.1 was
also studied. BMCMC in SCF exhibited cytochemical staining properties,
protease and histamine content, and increased serotonin uptake
consistent with more mature differentiated mast cells as compared with
BMCMC in CM or Cl.MC/ C57.1 cells. BMCMC in SCF released serotonin,
14C-labeled arachidonic acid metabolites and tumor necrosis
factor- (TNF-) on stimulation with MBP, while no response was
seen from either BMCMC in CM or Cl.MC/C57.1 cells. All three mast cell
populations released mediators on stimulation with the cationic MBP
analog, poly-L-arginine, indicating that the cationic charge did not
explain the selective response of BMCMC in SCF to eosinophil-derived
granule MBP. These findings show that SCF significantly influences mast cell differentiation and the responsiveness of mast cells to
eosinophil-derived granule MBP.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
MAST CELLS PRODUCE a variety of
biologically active mediators that play an important role in
hypersensitivity and other allergic inflammatory reactions. Mast cell
degranulation is accompanied by the release of preformed mediators,
such as histamine, serotonin (in murine species), proteases,
proteoglycans, and tumor necrosis factor- (TNF-). As well, mast
cells can generate newly synthesized mediators, such as products of
arachidonic acid metabolism, platelet activating factor, and a variety
of multifunctional cytokines.1
The activation of mast cells by antigen cross-linking of IgE antibodies
bound to high-affinity IgE receptors (Fc RI) on the cells' surface is one of the most extensively studied mechanisms of
mast cell degranulation.2 However, a number of alternative pathways that are independent of IgE antibodies can elicit mast cell
mediator release. For example, complement fragments, bradykinin, bacterial products, and certain neuropeptides, such as substance P, can
induce mast cell degranulation.3,4 As well, certain proteins contained within the cytoplasmic granules of eosinophils have
actions on mast cells and basophils. The eosinophil-derived granule
major basic protein (MBP) has been shown to elicit histamine release
from rodent mast cells and human basophils.5,6 Thus, mast
cell activation by IgE-independent mechanisms may be important in the
pathogenesis of a number of allergic or inflammatory conditions.
The response of mast cells to stimuli not involving IgE antibodies is
critically dependent on the population of mast cells examined. Mast
cells in different anatomic locations in rodent species and humans
exhibit significant phenotypical and functional heterogeneity.7 Mast cells in rodent species can be broadly divided into at least two populations: mast cells located at mucosal surfaces termed mucosal-type mast cells and mast cells in the skin,
peritoneal cavity, and submucosa of the intestinal tract termed
connective tissue-type mast cells. These mast cell populations differ
in a number of characteristics, including biochemical and histochemical
properties, the types of mediators produced upon degranulation, as well
as their functional response to agents that induce mast cell
degranulation through IgE-independent mechanisms.8 Thus,
the response of mast cells at one anatomic site to a given stimulus
does not necessarily predict the response of mast cells at a different
anatomic location to the same stimulus.
The factors controlling mast cell heterogeneity have not been
completely defined. A number of cytokines have been shown to influence
mast cell proliferation and maturation, including interleukin-3 (IL-3),
IL-4, IL-9, IL-10, and nerve growth factor, but recent studies suggest
that stem cell factor (SCF) plays a critical role in mast cell
development and survival. For example, mice genetically deficient in
SCF have an absence of mast cells in both connective tissues and at
mucosal surfaces.9 SCF acting alone can induce mast cell
proliferation and maturation in vitro10 and influence the
development of both mucosal and connective tissue-type mast cells in
vivo.11 In addition to its effects on mast cell
differentiation, SCF also has profound effects on mast cell function.
For example, while SCF has little effect on mast cell mediator release
in vitro, we reported that the intradermal injection of SCF elicits
mast cell degranulation and mast cell-dependent cutaneous
inflammation.12
In the current study, we investigated whether soluble recombinant rat
SCF influences the response of mast cells to an IgE-independent stimuli. We now report that bone marrow cultured mast cells (BMCMC) derived in SCF acquire the ability to release preformed and newly synthesized mediators in response to eosinophil-derived granule MBP.
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MATERIALS AND METHODS |
Cells.
Studies were conducted with three different populations of murine mast
cells. A cloned, growth factor-independent mast cell line
(Cl.MC/C57.1) that was originally derived from BMCMC isolated from
BALB/c mice13 was maintained in Dulbecco's modified
Eagle's medium (GIBCO, Grand Island, NY) with 10% heat-inactivated
fetal calf serum (Intergen, Purchase, NY), 50 µmol/L
2-mercaptoethanol, and 2 mmol/L L-glutamine at 37°C in 5%
CO2. In addition, femoral bone marrow cells were
obtained from BALB/c mice and maintained in medium as above, but
supplemented with either Concanavalin A-stimulated mouse
spleen cell-conditioned medium (BMCMC in conditioned medium [CM]), as
previously described,14 or recombinant rat SCF164 (50 ng/mL). Mast cells derived in 20%
WEHI-3-conditioned medium (a source of IL-3) were also examined. After
4 to 6 weeks, mast cells represented more than 98% of the total cells
as determined by neutral red staining.
Histochemical analysis and histamine content of different mast cell
populations.
The different mast cell populations were washed and suspended at 3 × 106 cells/mL in their respective media.
Cytocentrifuge slides were prepared from each mast cell population and
stained in 0.5% alcian blue (Rowley Biochemical Institute, Rowley,
MA), pH 0.1 at 60°C for 10 minutes. Slides were then immediately
counterstained in 0.1% safranin (Rowley Biochemical Institute) for 5 minutes at room temperature. The slides were then rinsed in water, 95%
ethanol, 100% ethanol, and xylene.
One-milliliter aliquots (3 × 106 mast cells) were
taken and centrifuged at 800 rpm for 10 minutes. Pellets and
supernatants were taken for determination of specific histamine release
as previously described.15
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of
mast cell protease expression.
Total RNA was isolated from mast cells using Ultraspec RNA
(Biotecx Laboratories Inc, Houston, TX) and treated with 1 U of Heparinase I (Sigma Chemical Co, St Louis, MO) per microgram RNA and 40 U of RNase inhibitor (Sigma Chemical Co) at 25°C for 2 hours. One
microgram of total RNA from each sample was reverse transcribed with
oligo dT and then 1/20 of this product was amplified with 100 pmol of
primers for individual mast cell proteases in 50 µL of PCR Supermix
(GIBCO-BRL, Grand Island, NY). The primer sequences for
murine mast cell protease 2 (mMCP2), murine mast cell protease 4 (mMCP4), and murine mast cell carboxypeptidase A (mMCCPA) were as
follows: 5 primers: 5-GTGATGACT GCTGCACACTG, 5-GTAATTCCTCTGCCTCGTCCT, 5-ACACAGGATCGAATG TGGAG;
3 primers: 5-CTTGAAGAGTCTGACTCAGG, 5-ACCCAGGGTTAT
CAGAGCTC, 5-TAATGCAGGACTTCATGAGC, respectively. PCR was
performed as follows: 94°C, 3 minutes; 30 cycles of 94°C,
1 minute; 52°C, 2 minutes; 72°C, 2 minutes; 72°C, 8 minutes. The PCR products were identified by electrophoresis in a 1.5%
agarose gel and verified by molecular size.
Isolation of human eosinophils and purification of MBP.
Human eosinophils and eosinophil granule proteins were isolated as
previously described.16 Briefly, whole blood was obtained from healthy and hypereosinophilic donors in the Clinical Research Center at the Beth Israel Deaconess Medical Center (Boston, MA). Venipuncture procedures were approved by and performed within the
guidelines of the Institutional Care and Use Committee at the Beth
Israel Deaconess Medical Center. The buffy coat was collected and
separated by density centrifugation in Ficoll-Paque (Pharmacia, Piscataway, NJ). Red blood cells were lysed in
NH4Cl, and the remaining granulocytes were washed in
phosphate-buffered saline (PBS) with 2% fetal bovine serum, and
counted. Eosinophils were then isolated by negative selection using the
magnetic cell separation system.17 The
granulocytes were incubated with CD 16 antibodies conjugated to
magnetic particles (50 µL of particles per 5 × 107
cells) for 30 minutes at 6°C, then passed through a separation syringe containing iron fibers. Eosinophil number and purity were assessed by Randolph's stain with eosinophil preparations of greater than 95% purity typically obtained. The eosinophils were lysed in
hypertonic sucrose (0.25 mol/L) and centrifuged at 400g for 10 minutes. The cellular lysates were then centrifuged at 13,000g for 20 minutes to pellet eosinophil granules. The granules were lysed
in 10 mmol/L HCl and the lysates were fractionated by column chromatography using Sephadex G-50 (Pharmacia)
equilibrated with 0.025 mol/L acetate buffer (pH 4.3), 0.15 mol/L NaCl.
MBP was identified by its distinctive chromatographic filtration
pattern. The purity of MBP was assessed by protein gel electrophoresis and the concentration determined by spectrophotometric absorbance at
277 nm.16
The effects of MBP or poly-L-arginine on the release of
3H-5-hydroxytryptamine from murine mast cells.
The effect of either MBP or poly-L-arginine, a highly charged synthetic
analog of eosinophil-derived granule proteins,18 on mast
cell mediator release was determined using a
3H-5-hydroxytryptamine (3H-5HT) release assay,
as previously described.19 Briefly, mast cells were
incubated with 1 mCi/mL of 5[1,2-3H](N)-hydroxytryptamine
creatine sulfate (NEN Dupont, Boston, MA) for 2 hours. The cells were
washed three times and then challenged with their respective media
alone (unstimulated) or different concentrations of either MBP or
poly-L-arginine (Sigma Chemical Co) for 10 minutes at 37°C. Each
mast cell population was maintained in its respective medium throughout
the incubation, wash, and challenge periods. The cells were then
centrifuged at 1,000 rpm for 10 minutes. Radioactive counts were
measured in the supernatants and pellets, and the percentage of
specific 3H-5-HT release was calculated as follows: % Specific Release = 100 × [(cpm Stimulated Supernatants/cpm
Stimulated Cells + Stimulated Supernatants)  (cpm Unstimulated
Supernatants/cpm Unstimulated Cells + Unstimulated
Supernatants)]. In some experiments after 3H-5HT incubation, mast cells were centrifuged and
radioactive counts determined as a measure of total serotonin uptake.
Effects of MBP, poly-L-arginine, or IgE/antigen on arachidonic acid
mediator release from BMCMC in SCF.
We also examined the effects of MBP or poly-L-arginine on the release
of 14C-labeled arachidonic acid from BMCMC in SCF as
previously described.20 The mast cells were incubated with
14C arachidonic acid (1 mCi/mL) for 12 hours at 37°C.
In some experiments, murine monoclonal IgE antidinitrophenyl (DNP)
antibodies (3 µg/mL) were added for the final 2 hours of incubation.
The cells were washed three times, then incubated with different
concentrations of either MBP, poly-arginine, 20 ng/mL
DNP30-40-human serum albumin (Sigma Chemical Co),
or calcium ionophore (Sigma Chemical Co) for 10 minutes. Radioactive
counts were measured in the supernatants and pellets and the percent
specific release of 14C-labeled arachidonic acid was
calculated as follows: % Specific Release = 100 × [(cpm
Stimulated Supernatants/cpm Stimulated Cells + Stimulated
Supernatants) (cpm Unstimulated Supernatants/cpm Unstimulated
Cells + Unstimulated Supernatants)].
Effects of MBP or poly-L-arginine on the production of TNF- by
mast cells.
Mast cells were incubated with medium alone or different concentrations
of poly-L-arginine or MBP for 30 minutes at 37°C. The supernatants
were collected and immediately stored at 80°C. Immunoreactive TNF- protein was measured by enzyme-linked
immunosorbent assay (ELISA) (Endogen, Woburn, MA).
Statistical analysis.
The results of differences in TNF- production between the MBP or
poly-L-arginine and unstimulated cells or specific
3H-5HT release from different mast cell populations
were analyzed for statistical significance (defined as P < .05) by the Student's t-test (two-tailed).
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RESULTS |
BMCMC in SCF exhibit positive staining with safranin, mCCPA, mMCP-2,
and mMCP-4 protease expression, increased histamine content, and
increased uptake of 3H-serotonin.
We initially compared the phenotypic characteristics of
BMCMC grown in SCF as the only exogenous growth factor (BMCMC
in SCF) with BMCMC derived in Concanavalin A-stimulated spleen
conditioned medium, a source of multiple growth factors (BMCMC in CM),
or a cloned, growth factor-independent mast cell line, Cl.MC/C57.1. We
used a histochemical approach for defining mast cell heterogeneity by
comparing the staining characteristics with alcian blue and safranin.21 Such an approach can identify mucosal-type mast cells by their cytoplasmic staining with alcian blue and their absence
of cytoplasmic staining with safranin, while connective tissue-type
mast cells exhibited strong cytoplasmic staining with safranin. We
found that BMCMC in CM (Fig 1A) or
conditioned medium and Cl.MC/C57.1 mast cells (not shown) exhibited
cytoplasmic staining with alcian blue, but failed to stain with
safranin. In contrast, BMCMC in SCF exhibited heterogeneous cytoplasmic
staining characteristics (Fig 1B). The majority of cells exhibited
mixed alcian blue and safranin staining of their cytoplasmic granules
(55% ± 6% of total mast cells), while cells with either
predominantly alcian blue cytoplasmic staining cells or predominantly
safranin staining accounted for the remainder of the cells (25% ± 5% and 20% ± 4%, respectively).
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| Fig 1.
Histochemical staining properties of different
populations of mast cells in vitro. Cytocentrifuge preparations of
BMCMC grown in 20% Concanavalin A stimulated spleen conditioned medium
(BMCMC in CM, [A]) or BMCMC grown in 50 ng/mL of SCF (BMCMC in SCF,
[B]) were stained with alcian blue and safranin O. Photomicrographs were taken at 400× magnification. White arrow denotes alcian blue positive (safranin negative) mast cell, black arrow denotes safranin positive mast cell (alcian blue negative), and black arrowhead denotes
mast cells with mixed alcian blue and safranin positive granules. The
growth factor independent cloned mast cell line, Cl.MC/C57.1 exhibited
similar staining characteristics as BMCMC in CM (data not shown).
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Mast cell heterogeneity has also been defined by the expression of
various neutral proteases.22 Therefore, we examined whether the differences in histochemical staining characteristics between BMCMC
in SCF and BMCMC in CM were accompanied by changes in protease gene
expression. We found that BMCMC in CM expressed mCCPA and mMCP-2, but
not mMCP-4 (Fig 2A), consistent with
previous reports.23,24 In contrast, BMCMC in SCF expressed
mCCPA, mMCP-2, and mMCP-4 (Fig 2A). Thus, in addition to the changes in
histochemical staining properties, BMCMC in SCF also exhibited a change
in protease gene expression.
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| Fig 2.
Mast cell protease expression, histamine content, and
uptake of 3H-5HT in different populations of mast cells in
vitro. RT-PCR analysis of mast cell protease expression was performed
on BMCMC in CM and BMCMC in SCF for the murine mast cell proteases
(mMCP)-2 and -4, and the exopeptidase carboxypeptidase A (mMCCPA) (A). Total histamine content of Cl.MC/C57.1, BMCMC in CM, and BMCMC in SCF
was determined by fluorometric assay and expressed as pg of histamine
per cell (B). The ability of the three mast cell populations to take up
3H-5HT was assessed. Mast cells were incubated with
3H-5HT for 2 hours, then washed. Total radioactive counts
in the cell pellets were measured and expressed as cpm (C). Data in (B and C) are shown as mean ± SEM (n = 2 to 4/group). Similar results were obtained in eight repeat experiments.
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We also determined the histamine content of these mast cell populations
and the ability of the different mast cell populations to take up
exogenous 5-hydroxytryptamine (5-HT). BMCMC in CM and the Cl.MC/C57.1
line contained approximately 0.1 pg of histamine per cell, which is
consistent with previous reports,10,25 but the BMCMC in SCF
contained approximately 25 times more histamine on a per cell basis
(Fig 2B). In addition, we found that BMCMC in SCF took up approximately
10 times the amount of 5-HT compared with either BMCMC in CM or the
Cl.MC/C57.1 line (Fig 2C). The increased histamine content and uptake
of exogenous 5-HT exhibited by BMCMC in SCF are consistent with a
maturational/phenotypical change in the cells, as previously reported
for BMCMC in CM that had been changed and maintained in medium with SCF
as the only exogenous growth factor for 4 to 6 weeks.10
MBP induces 3H-5HT release from mouse mast cells grown in
SCF.
We next examined the ability of eosinophil granule MBP to induce mast
cell degranulation and preformed mediator release from the different
mast cell populations. MBP elicited a dose-dependent release of
3H-5HT from BMCMC in SCF, but had no effect on either the
Cl.MC/C57.1 line, BMCMC in CM (Fig 3A), or
BMCMC derived in WEHI-3-conditioned medium, a source of IL-3 (not
shown). Cell viability, as determined by Trypan blue exclusion, was not
significantly changed in BMCMC in SCF challenged with MBP
compared with cells challenged with a similar dilution of column
elution buffer (98% v 96%, respectively, at the highest
concentration of MBP). Thus, MBP, at the concentrations used, had no
inherent cytotoxic effects on any of the mast cell populations
examined.
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| Fig 3.
Effect of MBP on 3H-5HT release in different
populations of murine mast cells. BMCMC grown in stem cell factor
(BMCMC in SCF), BMCMC grown in Concanavalin A stimulated spleen
conditioned medium (BMCMC in CM), or the growth factor independent
cloned mast cell line, Cl.MC/C57.1, was incubated with
3H-5HT for 2 hours, washed and then challenged with
different concentrations of MBP (A) or poly-L-arginine (B) for 10 minutes. Specific 5-HT release was calculated as described in Materials
and Methods. Results are expressed as mean ± SD (n = 3).
***P < .001 by the two-tailed paired Student's
t-test. Similar results were obtained in a repeat experiment.
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Poly-L-arginine stimulates the release of 3H-5HT from
mouse mast cells.
MBP has a high content of arginine residues (15%) in its amino acid
sequence that results in a highly charged cationic protein. Several
laboratories have reported that some of the stimulatory effects of MBP
are related to its charge and these effects can be imitated by
poly-L-arginine, a highly charged cationic analog of
MBP.26,27 Therefore, we examined the effect of
poly-L-arginine on mast cell mediator release.
In contrast to the response with MBP, all three populations of murine
mast cells examined released 3H-5HT after stimulation with
poly-L-arginine (Fig 3B), and there was no adverse effect on mast cell
viability (data not shown). The concentration of poly-L-arginine
necessary to elicit 5-HT release was high (10 5
mol/L) and there appeared to be a threshold effect in that almost no
specific serotonin release occurred at lower concentrations. Nevertheless, this effect of poly-L-arginine was seen in each of the
mast cell populations tested (Fig 3B).
MBP and poly-L-arginine stimulates the release of arachidonic acid
metabolites and TNF- from mast cells grown in SCF.
In addition to preformed mediators such as serotonin and histamine,
immunologically stimulated murine mast cells can generate products of
arachidonic acid metabolism and a number of multifunctional cytokines.
Therefore, we investigated the effects of MBP on the release of newly
synthesized arachidonic acid mediators and cytokine production. Like
the histochemical and biochemical heterogeneity exhibited by mast
cells, the pathways of arachidonic acid metabolism used by mast cells
is characteristic of different mast cell populations. Mucosal mast
cells use the lipoxygenase pathway and produce mainly leukotriene
C4, while the cyclooxygenase pathway
predominates in connective tissue-type mast cells, resulting in the
production of prostaglandin D2.28
To define whether arachidonic acid metabolites derived from either
pathway were produced, we prelabeled BMCMC in SCF with
14C-arachidonic acid and determined if MBP or
poly-L-arginine elicited the release of arachidonic acid mediators. We
found that MBP induced a dose-dependent release of
14C-arachidonic acid from BMCMC in SCF
(Fig 4). MBP was more effective than
poly-L-arginine in eliciting 14C-arachidonic acid release
(Fig 4). The release of 14C-arachidonic acid from BMCMC in
SCF by MBP at 10-7 mol/L was similar to that seen when the
cells were sensitized with IgE anti-DNP antibodies and
challenged with 20 ng/mL of DNP30-40-human serum albumin
(25.5% ± 0.2% specific release) or stimulated with calcium
ionophore at 4 µmol/L (28.1% ± 2% specific release).
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| Fig 4.
Effect of MBP or poly-L-arginine on 14C
arachidonic acid release from BMCMC maintained in SCF. BMCMC grown in
SCF (BMCMC in SCF) were incubated with 14C-arachidonic acid
for 12 hours. BMCMC in SCF were incubated with different concentrations
of MBP or poly-L-arginine for 10 minutes. The data are expressed as
mean ± SD (n = 3). Specific arachidonic acid release was calculated
as described in Materials and Methods.
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We also examined the effect of MBP or poly-L-arginine on the production
of TNF- in all three mast cell populations. We found that MBP
induced significant TNF- release from BMCMC in SCF, but had little
effect on TNF- production in either BMCMC in CM or the Cl.MC/C57.1
cell line (Fig 5A). In contrast,
poly-L-arginine induced the production of TNF- in all three mast
cell populations (Fig 5B).
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| Fig 5.
Production of TNF- from different populations of mast
cells stimulated with either MBP or poly-L-arginine. BMCMC in SCF, BMCMC in CM, or Cl.MC/C57.1 mast cells were stimulated with either MBP
(106 mol/L) (A) or poly-L-arginine (10-5
mol/L) (B) for 30 minutes. Supernatants were then collected and analyzed for TNF- by ELISA. Cell viability was greater than 90% as
determined by Trypan blue exclusion. The data are expressed as mean ± SD (n = 3). ***P < .05 by the two-tailed paired
Student's t-test.
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DISCUSSION |
The mechanisms involved in mast cell activation by stimuli other than
IgE antibodies have not been completely defined. We found that SCF is
an important factor in the development of mast cell responsiveness to
the eosinophil granule-associated protein MBP. Now, in addition to its
role as a growth and survival factor for mast cells in vitro and in
vivo,10,11,29 our study shows that SCF also influences mast
cell/eosinophil interactions.
The population of mast cells derived in SCF from the bone marrow of
BALB/c mice had several phenotypical characteristics of mature
connective tissue-type mast cells. The mast cells exhibited positive
staining for safranin, increased histamine content, increased uptake of
5-HT compared with the negative safranin staining, low histamine and
5-HT uptake typically seen in BMCMC in CM. These findings are similar
to a previous report in which BMCMC in CM were transferred into medium
containing SCF as the only exogenous growth factor.10 After
4 weeks in culture, the mast cells exhibited alterations in their
phenotype similar to the BMCMC in SCF, but not identical to peritoneal
mast cells.10 However, in this study, mast cell function
was not examined.
As well, we found that BMCMC in SCF expressed the protease mMCP-4,
which is typically found in connective tissue-type mast cells and not
mucosal-type mast cells.30,31 Thus, phenotypically mature
mast cells could be derived directly from mouse bone marrow cells using
soluble SCF as the only exogenous growth factor. However, it is
important to note that while these cells had characteristics similar to
mature connective tissue-type mast cells, BMCMC in SCF represent a
heterogeneous population of mast cells. Thus, BMCMC in SCF are similar,
but not identical to connective tissue mast cells derived from the
peritoneal cavity of mice.
We found that MBP elicited the release of a mast cell
granule-associated mediator (serotonin) and newly synthesized mediators (arachidonic acid metabolites) from BMCMC in SCF. In addition, MBP
induced significant TNF- production by BMCMC in SCF. These responses
did not occur in BMCMC in CM. Thus, in addition to the biochemical and
morphological changes seen in BMCMC in SCF, the mast cells also
underwent a functional change in their responsiveness to MBP.
Ogasawara, et al32 reported that the coculture of
BM-derived mast cells with Swiss 3T3 fibroblasts for 4 to 6 days
resulted in the cells acquiring responsiveness to polycationic
activators, such as compound 48/80 or substance P, but that short-term
culture with soluble SCF (6 days) did not confer responsiveness to
compound 48/80. We have found that BMCMC in CM that were switched into SCF-containing medium and maintained for 6 weeks acquired
responsiveness to substance P also release mediators in response to
challenge with substance P (manuscript in preparation).
Thus, SCF either as a soluble dimer and, presumably in its membrane
bound form associated with fibroblasts or smooth muscle cells, can
alter mast cell responses to certain agents that activate mast cells.
MBP is thought to damage cell membranes33,34 and is a toxin
to helminths and mammalian cells in vitro.35 But in our
study, MBP did not affect mast cell viability. This is similar to other reports demonstrating noncytolytic mediator release from rodent mast
cells and human basophils5,6 by MBP. It has been suggested that the effects of MBP are the result, at least in part, of the high
cationic charge of MBP.26 However, cationic charge alone cannot explain the effects of MBP on mast cells. If cationic charge only was responsible for mast cell activation, then MBP would be
predicted to elicit mediator release from each of the different mast
cell populations examined. However, we found that MBP induced mediator
release only from BMCMC in SCF and not BMCMC in CM or Cl.MC/C57.1
cells. This is in contrast to a synthetic MBP analog, poly-L-arginine,
which elicited mediator release from all populations. This suggests
that poly-L-arginine is not an ideal alternative to authentic native
MBP for the study of MBP/mast cell interactions.
Previous studies have shown that MBP stimulates the preformed mediator
release from connective tissue-type mast cells isolated from the
peritoneal cavity of rats.5,6 Our findings show that SCF is
an important factor in the development of mast cell responsiveness to
MBP. At this time, it is not clear whether SCF induces the growth of a
population of mast cells that respond to MBP or, alternatively, induces
a direct change in MBP unresponsive mast cells that leads to
responsiveness, such as by the induction of an as yet unidentified MBP
receptor or receptor-like complex.36,37 We favor the former
hypothesis, because BMCMC in SCF after 3 weeks in culture (mast cells
represented >75% of the total cell population) did not release
serotonin to challenge with MBP (data not shown). This suggests that
SCF is not simply facilitating the action of MBP, but may, in fact, be
driving the proliferation and differentiation of a population of mast
cells that have the capacity to respond to MBP.
The findings of our study may have important implications regarding
mast cell/eosinophil interactions in vivo. As previously discussed, SCF
has important effects on mast cell growth and differentiation. The
responsiveness of mast cell populations under the influence of SCF to
eosinophil-derived granule proteins may be important in inflammatory
and allergic reactions. For example, MBP-induced mast cell
degranulation may lead to an amplification of the inflammatory response
through the generation of preformed and newly synthesized mediators by
mast cells. As well, granule release by mast cells may promote the
downregulation of the inflammatory response by the release of heparin
from connective tissue-type mast cells, which has been shown to
neutralize the biological activity of MBP.38,39 Thus, the
ability of mast cells to release mediators in response to MBP may have
multiple, complex biological consequences.
In conclusion, our study shows that BMCMC in SCF express phenotypic
characteristics similar to mature connective tissue mast cells as
defined by histochemical staining, protease expression, histamine
content, and 5-HT uptake. BMCMC in SCF release preformed 5-HT, products
of arachidonic acid metabolism, and TNF- in response to MBP. These
findings suggest that SCF is an important factor in the development of
a functional response of mast cells to eosinophil-derived granule MBP.
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FOOTNOTES |
Submitted January 29, 1998;
accepted April 6, 1998.
Supported by National Institutes of Health Grants No. DK33506 and Core
B (Morphology), DK40561 (Clinical Nutrition Research Center at
Harvard), DK46819 (to B.K.W.) and AI25230 (to S.J.A.), and a Career
Development Award from the Crohn's and Colitis Foundation of America
(to G.T.F.).
Address reprint requests to Barry K. Wershil, MD, Combined Program in
Pediatric Gastroenterology and Nutrition, Massachusetts General
Hospital, 149 13th St (1493404), Charlestown, MA 02129.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We thank Dr Keith E. Langley of AMGEN, Inc (Thousand Oaks,
CA) for providing rrSCF164.
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Abstract
Introduction
Abstract