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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 187-195
Interleukin-4 Promotes the Development of Tryptase and Chymase
Double-Positive Human Mast Cells Accompanied by Cell Maturation
By
Hano Toru,
Mitsuoki Eguchi,
Ryoji Matsumoto,
Makoto Yanagida,
Junichi Yata, and
Tatsutoshi Nakahata
From The Department of Pediatrics, School of Medicine, Tokyo Medical
and Dental University, Tokyo, Japan; The Second Department of
Pediatrics, Dokkyo University School of Medicine, Tochigi, Japan; The
Department of Bacterial Infection, The Institute of Medical Science,
The University of Tokyo, Tokyo, Japan; The Pharmaceutical Development
Laboratory, Kirin Brewery Co, Ltd, Gumma, Japan; The Department of
Clinical Oncology, The Institute of Medical Science, The University of
Tokyo, Tokyo, Japan.
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ABSTRACT |
Human cultured mast cells (HCMCs) grown from cord blood mononuclear
cells in the presence of stem cell factor (SCF) and interleukin-6
(IL-6) expressed tryptase but no or low chymase in their cytoplasm. The
addition of IL-4 to these cells strikingly increased chymase
expression. Consequently, the activity of chymase was significantly
higher in IL-4-treated mast cells than that in IL-4-nontreated mast
cells, whereas the activity of tryptase and histamine content were
comparable in both cells. Electron microscopic immunocytochemistry also
showed that secretary granules containing chymase increased in
IL-4-treated mast cells. Interestingly, the IL-4-induced increase of
chymase expression in HCMCs was accompanied by morphological maturation
of the cells. Cytoplasmic projections were few in IL-4-nontreated
HCMCs, and a small number of secretary granules were observed, most of
which were empty or partially filled with discrete scrolls with rough
particles showing immaturity. In contrast, IL-4-treated HCMCs had
extremely abundant cytoplasmic projections and had many secretary
granules filled with electron-dense crystal materials. Taken together,
immature HCMCs grown only with SCF and IL-6 expressed tryptase with no
or a low amount of chymase, and addition of IL-4 promoted cell
maturation together with the expression of both tryptase and a high
amount of chymase. Our findings will raise a possibility of a linear
pathway of human mast cell development from tryptase single positive
mast cells into tryptase and chymase double positive mast cells as the
cells mature and will suggest that this maturation process is promoted
by IL-4.
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INTRODUCTION |
ON THE BASIS OF protease expression,
human mast cells have been classified into two
phenotypes.1-4 One phenotype, which is designated
MCT (tryptase single positive mast cells), contains
tryptase but not chymase, while another phenotype, designated
MCTC (tryptase and chymase double positive mast cells),
expresses both tryptase and chymase, cathepsin G, and carboxypeptidase
A. MCT and MCTC have also been reported to be
distinguishable ultrastructurally, mainly by the morphological pattern
of contents in their granules.5,6 Discrete scrolls are
associated with MCT, and crystal, grating, or lattice
substructures are associated with MCTC. However, it has
been currently unknown whether MCT can differentiate into
MCTC or vice versa.
Protease expression in in vitro human mast cells has been examined by
several investigators.7-10 The majority of human mast cells
developed from cord blood cells by stem cell factor (SCF) expressed
tryptase but not chymase, indicating that SCF is not sufficient for the
development of MCTC.7,10 In addition, these
mast cells did not reach full maturity based on granule-filling
criteria even after 14-week culture.7,11-14 In contrast,
more than 90% of human mast cells developed in a coculture of cord
blood mononuclear cells with 3T3 fibroblasts were the MCTC
phenotype and had ultrastructurally mature features with many crystal
granules.8 These reports suggested that SCF was sufficient
for neither the development of MCTC phenotype nor full
maturation of human mast cells. Moreover, these observations suggested
that there might be a relation between human mast cell maturation and
protease expression and that there would be a factor(s) upregulating
chymase expression along with promotion of human mast cell maturation.
The maturation and phenotype differentiation of mast cells has been
extensively studied in mice. Phenotype classification of murine mast
cells is based on the pattern of expression of the proteases. In murine
mast cells, five types of chymase (mouse mast cell protease [MMCP]-1,
-2, -3, -4, and -5) and one mast-cell carboxypeptidase and two types of
tryptase (MMCP-6 and -7) have been reported.15-22 The
plasticity of the protease expression in murine mast cells has been
shown.23-29 Bone-marrow-derived immature mast cells
cultured with interleukin-3 (IL-3) express predominantly MMCP-5 and
mast-cell carboxypeptidase mRNA. Addition of SCF enhanced the
expression of MMCP-4, MMCP-6 mRNA, and heparin proteoglycan in the
IL-3-dependent mast cells.23-25 Replacement of IL-3 with
IL-10 resulted in the expression of MMCP-1 and -2
mRNAs.26,27 In addition, several studies showed that murine
mast cell heterogeneity was the result of the differentiation and
maturation of common mast cell-committed progenitor cells regulated by
cytokines and/or other tissue-specific factors in the
microenvironment.30-32 Accordingly, regulation of human
mast cell protease expression and maturation by cytokines would be
suggested.
Previously, we have reported that human cultured mast cells (HCMCs)
grown from cord blood mononuclear cells in the presence of SCF and IL-6
express few or no high affinity IgE receptors (Fc RI),33
which is one of the aspects of immature mast cells.34 We
reported that IL-4 induced FcRI on HCMCs, resulting in high
histamine releasing activity on crosslinking of FcRI in IL-4-primed
mast cells.33 In addition, IL-4 has various biological
effects on HCMCs, including upregulation of intercellular adhesion
molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1
(LFA-1) expression and suppression of c-kit.35-37 All of
these results strongly suggested that IL-4 is a maturation and
differentiation factor for human mast cells. In this report, we found
that most HCMCs grown from cord blood mononuclear cells in the presence
of SCF and IL-6 expressed tryptase but low or no chymase in their
cytoplasm and that IL-4 strongly increased the chymase expression.
Consequently, significantly higher chymase activity was observed in
HCMCs cultured with IL-4 than those cultured without IL-4. Electron
microscopic immunocytochemistry showed that both tryptase and chymase
were detected in the granules of IL-4-treated HCMCs, whereas tryptase
but little chymase was detected in their granules in IL-4-nontreated
HCMCs. Remarkably, along with the increase of chymase expression, IL-4
promoted morphological maturation of HCMCs. These data suggest that
IL-4 promotes the development of MCTC accompanied by
morphological maturation as well as functional maturation of the cells.
Our finding will lead to the proposal that there is a linear pathway of
MCTC via MCT along with maturation of the cells
and that this maturation process is strongly promoted by IL-4.
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MATERIALS AND METHODS |
HCMCs.
HCMCs were obtained as previously described10 with some
modification. Briefly, cord blood mononuclear cells were grown in
tissue culture flasks (Becton Dickinson, Lincoln Park, NJ) in  -MEM
(GIBCO-BRL, Life Technologies Inc, Gaithersburg, MD) supplemented with
20% fetal calf serum (Hyclone Sterile Systems, Inc, Logan, UT) in the
presence of SCF (100 ng/mL; Amgen, Thousand Oaks, CA) and IL-6 (80
ng/mL; Ajinomoto Co, Ltd, Tokyo, Japan) for 10 weeks. Adhesive cells
like macrophages were eliminated by transferring nonadhesive cells to
fresh culture flasks. Then the 10-week cultured HCMCs were divided into
two aliquots. One aliquot was cultured with IL-4 (10 ng/mL; a generous
gift from DNAX, Palo Alto, CA) in the presence of SCF (100 ng/mL) and
IL-6 (80 ng/mL), and the other aliquot was cultured without IL-4 in the
presence of SCF (100 ng/mL) and IL-6 (80 ng/mL). Both aliquots were
cultured in 24-well flat-bottomed plates (Becton Dickinson; 5 ×
105 cells/mL/well), and half of the media was changed
weekly for fresh media supplemented with cytokines.
Immunocytochemical assays.
HCMCs cultured with or without IL-4 in the presence of SCF and IL-6 for
indicated periods were cytocentrifuged onto each glass slide, after the
total cell number was counted, and fixed with the fixing solution (Muto
Pure Chemicals Ltd, Tokyo, Japan) consisting of formaldehyde (8.75%)
and acetone (45%) for 1 minute, and the samples were washed three
times with Tris-buffered saline. Then the samples were blocked with
rabbit serum for 10 minutes followed by incubation with mouse antihuman
tryptase or mouse antihuman chymase monoclonal antibodies (MoAbs; 10
µg/mL; Chemicon International, Inc, Temecula, CA) at room temperature
(RT) for 1 hour. After washing three times with Tris-buffered saline,
the samples were reacted 1:50 with rabbit antimouse IgG (MBL, Nagoya,
Japan) at RT for 1 hour and then washed three times. The samples were
further reacted 1:50 with soluble complexes of alkaline phosphatase and
mouse monoclonal antialkaline phosphatase (DAKO Co Ltd, Glostrup,
Denmark) at RT for 1 hour and washed three times. Finally, they were
developed with chromogenic substrate at 37°C for 30 minutes.
Measurement of chymase and tryptase activities
HCMCs (1 × 106) cultured with or without IL-4 (10
ng/mL) for 21 days were lysed by sonication in 500 µL of the
appropriate reaction solution. The activity of tryptase was measured at
22°C by the cleavage of 1.0 mmol/L tosyl-L-arginine methyl ester
(Sigma Chemical Co, St Louis, MO) in the reaction buffer containing
0.04 mol/L Tris with 0.01 mol/L CaCl2 at pH 8.1 with
continuous spectrophotometrical monitoring of absorbance at 247 nm. The
activity of chymase was measured at 22°C by the cleavage of
benzoyl-L-tyrosine ethyl ester (0.54 mmol/L; Sigma Chemical Co) in the
reaction mixture containing 0.04 mol/L Tris with 0.05 mol/L
CaCl2, and 25% (vol/vol) methanol at pH 7.8 with
continuous monitoring of absorbance at 256
nm.38,39 One unit of enzyme cleaved 1 µmol
of substrate per minute. The enzymatic activity was expressed in units
per 1 × 106 mast cells.
Assay for histamine content.
HCMCs (2 × 105 cells in 200 µL) cultured with or
without IL-4 (10 ng/mL) for 21 days were lysed with Triton X (1%;
Sigma Chemical Co). The histamine content in the supernatant was
measured by an automated fluorometric histamine analyzer (Auto Analyzer
II; BRAN & LUEBBE Co Ltd, Tokyo, Japan).
Electron microscopic analysis.
HCMCs grown for 10 weeks in the presence of SCF and IL-6 were divided
into two aliquots and cultured with or without IL-4 (10 ng/mL),
respectively, in the presence of SCF and IL-6 for 28 days. Then the
cells were fixed with 2.5% glutaraldehyde at 4°C for 30 minutes
and postfixed in 1% osmium tetroxide in cacodylated buffer at 4°C
for 1 hour. After that, the samples were dehydrated in ethanol and
embedded in epoxy resin. Thin sections, 60 to 100 nm, were cut on an
ultramicrotome from tissue blocks. These sections were collected on
mesh copper grids, stained with uranyl acetate and lead citrate, and
then examined under an electron microscope (JEOL100B; JEOL, Tokyo,
Japan).
Electron microscopic immunocytochemistry.
Electron microscopic immunocytochemistry of tryptase and chymase in
HCMCs was performed as previously described.40 HCMCs grown
for 10 weeks in the presence of SCF and IL-6 were divided into two
aliquots and cultured with or without IL-4 (10 ng/mL), respectively, in
the presence of SCF and IL-6 for 28 days. Then, the cells were fixed
with 2.5% glutaraldehyde at 4°C for 30 minutes. HCMCs fixed with
glutaraldehyde were dehydrated without postfixation with osmium and
embedded in L R White resin (London Resin Co, London, UK). The
ultrathin sections mounted on the nickel grids were immersed in 1%
bovine serum albumin (Sigma Chemical Co) in phosphate buffered saline
(PBS) for 5 minutes. The sections were then reacted with primary
antibodies (mouse antihuman tryptase or mouse antihuman chymase;
Chemicon International, Inc; 2 µg/mL in PBS) for 2 hours, washed
twice with PBS, and immersed further in 0.1% bovine serum albumin in
PBS for 30 minutes. The sections were then placed in protein A-gold
solution (10 nm in diameter; Sigma Chemical Co), diluted 20-fold with
PBS for 1 hour, washed with PBS and distilled water, stained with
uranyl acetate and lead citrated, and examined under an electron
microscope. For an immunocytochemical control, the above procedure was
performed using subtype-matched mouse IgG1 instead of mouse antihuman
tryptase or antihuman chymase.
Statistical analysis.
Statistical analysis was performed by a paired two-way Student's
t-test. Data were expressed as the mean ± standard
deviation (SD). A P value of <.05 was considered
statistically significant.
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RESULTS |
IL-4 increased the chymase expression in HCMCs.
HCMCs were obtained by culturing cord blood mononuclear cells in the
presence of SCF (100 ng/mL) and IL-6 (80 ng/mL) for 10 weeks. The
cultured cells expressed c-kit (>99%), contained
histamine,33 and over half of the cells had polylobed
nuclei and the rest had single-lobed nuclei
(Fig 1). More than 99% of the cells also
expressed high amounts of tryptase determined by immunocytochemical
staining using MoAb specific for human tryptase (Fig 1a). In contrast,
most of the HCMCs expressed no or an extremely low amount of chymase
(MCT or MCTClow) when
stained with antihuman chymase MoAb and classified based on
Table 1 (Fig 1b). We examined whether IL-4
affected chymase expression in HCMCs. Whereas HCMCs cultured without
IL-4 stayed predominantly MCT or
MCTClow (Fig 1d and f), addition of IL-4 (10
ng/mL) in the presence of SCF and IL-6 increased HCMCs expressing a
high amount of chymase (MCTChigh) (Fig 1e and
g). The increase of MCTChigh was observed as
early as 5 days after addition of IL-4 (Fig 1e), and a significantly
increased number of MCTChigh was observed on
day 56 (Fig 1g).
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| Fig 1.
Immunocytochemical staining of tryptase and chymase in
HCMCs cultured with or without IL-4. HCMCs grown in the presence of SCF
(100 ng/mL) and IL-6 (80 ng/mL) for 10 weeks (defined as day 0 mast
cells) were further cultured with or without IL-4 (10 ng/mL) in the
presence of SCF and IL-6 for 56 days. Day 0 mast cells were stained
with antitryptase (a), antichymase (b), subclass-matched control mouse
IgG1 (c). Day 5 and day 56 mast cells cultured without IL-4 (d and f)
or with IL-4 (e and g) stained with antichymase are shown. Typical
chymase high positive mast cells (MCTChigh;
arrow head) and chymase low positive mast cells
(MCTClow; arrow) are indicated (e).
This experiment was repeated three times with similar results.
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To confirm the effect of IL-4 on the development of
MCTChigh, the percentage of
MCTChigh was determined at various culture
periods after the addition of IL-4 (10 ng/mL;
Fig 2a). Before IL-4 was added, only 2.5%
± 0.7% (mean ± SD of three independent experiments) of the
cells was MCTChigh (Day 0 in Fig 2a) and 27.2%
± 1.2% was MCTClow, and more than 70% of
the mast cells was MCT. Although in the absence of IL-4,
the percentage of MCTChigh and
MCTClow increased slightly, and on day 56 they
reached 14.3% ± 3.5% (Fig 2a) and 45.6% ± 3.6%,
respectively. In contrast, addition of IL-4 significantly resulted in
the predominance of MCTChigh as shown in Fig 1e
and g and Fig 2a. The increase of the percentage of
MCTChigh could be observed on day 5 after IL-4
was added to the culture (12.5% ± 3.5%). The percentage of
MCTChigh constantly increased and reached
81.5% ± 7.4% on day 28 and 93.5% ± 5.7% on day 56,
respectively, and most of the rest of the cells were
MCTClow. When IL-4 was withdrawn on day 21, the
increase of the percentage of MCTChigh stopped;
however, the percentage of MCTChigh stayed
64.8% ± 5.1%, even on day 56 (Fig 2a). The minimum concentration
of IL-4 to induce maximum chymase expression in HCMCs was 10 ng/mL (Fig
2b). The presence of IL-4 did not affect expression of tryptase, and
more than 99% of both IL-4-treated and IL-4-nontreated cells in each
observation period expressed tryptase. No significant increase or
decrease of the total cell number was observed in either IL-4-treated
or IL-4-nontreated HCMCs through the observation period, although a
slight increase in the cell number in IL-4-treated cells was observed
on day 7 (5.5 ± 0.2 × 105 cells/mL/well), which
was the peak cell number through the culture period, and by day 28 the
cell number gradually returned to the starting cell number (5.0 ×
105 cells/mL/well). No remarkable increase or decrease of
the cell number may indicate that the increase of the percentage of
MCTChigh in IL-4-added HCMCs is not caused by
induction of selective proliferation of the few
MCTChigh observed on day 0 or selective death
of a large population of MCT. These data suggested that
IL-4 strongly promoted the phenotypic change of MCT and
MCTClow into MCTChigh.
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| Fig 2.
Time kinetic and dose response analysis of the
IL-4-induced development of MCTChigh. HCMCs
grown in the presence of SCF (100 ng/mL) and IL-6 (80 ng/mL) for 10
weeks (defined as day 0 mast cells) were further cultured with (closed
circle) or without (open circle) IL-4 (10 ng/mL) in the presence of SCF
and IL-6. In some experiments, IL-4 was withdrawn (open triangle) after
21-day culture with IL-4. In time kinetic analysis (a) the cells were
cytocentrifuged after indicated periods of culture, and chymase was
detected immunocytochemically with MoAb specific for human chymase.
IL-4-treated mast cells showed statistically significant increased
number of MCTChigh compared with
IL-4-nontreated mast cells (*P < .05, **P < .01,
***P < .001). In dose-response analysis (b), 10-week cultured
HCMCs were cultured with various concentrations of IL-4 for 21 days and
then chymase was detected immunocytochemically (***P < .001).
At least 500 cells were counted in each sample under a microscope. The
data shown are the mean of three independent experiments.
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IL-4 increased the activity of chymase in HCMCs.
Next, we examined the activity of chymase and tryptase and the
histamine content in HCMCs cultured with or without IL-4
(Table 2). The activity of tryptase and the
histamine content did not differ between IL-4-treated and
IL-4-nontreated cells, whereas the activity of chymase in
IL-4-treated mast cells was significantly higher than that in
IL-4-nontreated cells. High chymase activity observed in IL-4-treated
mast cells is consistent with the data obtained by immunocytochemical
assay in which MCTChigh cells
predominated when cultured with IL-4.
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Table 2.
Histamine Content, and Tryptase and Chymase
Activities in Human Mast Cells Cultured With or Without IL-4 in
Addition to SCF and IL-6
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IL-4 promoted morphological maturation and increased chymase positive
granules in HCMCs.
We then performed ultrastructural analysis to examine whether IL-4
induced morphological changes in HCMCs, because MCT and
MCTC have been reported to be also distinguishable
ultrastructurally, mainly by the pattern of contents in their
granules.5,6 In both IL-4-treated and IL-4-nontreated
HCMCs, the cells were round or oval shaped, the nuclei were indented or
lobulated, and nuclear chromatin was finely condensed at the margin of
nuclear membrane (Fig 3a and d). HCMCs
cultured without IL-4 had a large and bright nucleolus, suggesting
immaturity (Fig 3a). Remarkable morphological differences were noted in
cytoplasmic projections and cytoplasmic organelles between HCMCs
cultured with IL-4 and those cultured without IL-4. Fewer cytoplasmic
projections were observed in HCMCs cultured without IL-4 (Fig 3a). They
contained many large empty granules and partially filled granules with
discrete scrolls and lamellar structures, and the latter may have been
longitudinal sections of discrete scrolls (Fig 3a, b, and c). Granules
containing a few rough particles together with discrete scrolls could
be observed (Fig 3c). Dense crystal granules were extremely rare in
HCMCs cultured without IL-4 (Fig 3a, b, and c). The Golgi apparatus was
poorly developed in these mast cells (Fig 3a). The morphology observed
in the granules of HCMCs cultured without IL-4 corresponded to the
previously reported features of MCT5,6 as well
as to those of immature mast cells containing numerous empty or partly
full granules which have particles at an early stage, scrolls later,
and rarely contain crystals.11-14 In contrast, HCMCs
cultured with IL-4 had more abundant cytoplasmic projections, had a
remarkably developed Golgi apparatus, and the secretary granules were
increased in number (Fig 3d). Most granules in HCMCs cultured with IL-4
were filled with dense, crystal materials as well as compact
electron-dense nondiscrete scrolls (Fig 3e and f). Granules containing
discrete scrolls were rarely observed in HCMCs cultured with IL-4.
Thus, the morphology of the IL-4-treated mast cells corresponded to
the ultrastructure of previously reported
MCTC5,6 and to the ultrastructure observed in
mature mast cells, which have numerous small, dense, crystal
granules.11 Therefore, these morphological analyses
suggested that IL-4 promotes maturation of human mast cells.
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| Fig 3.
Ultrastructure of human mast cells cultured in the
presence or absence of IL-4. HCMCs grown in the presence of SCF (100
ng/mL) and IL-6 (80 ng/mL) for 10 weeks were further cultured with (d,
e, and f) or without IL-4 (a, b, and c; 10 ng/mL) in the presence of
SCF and IL-6 for 28 days. HCMCs cultured without IL-4 has few
cytoplasmic projections and has an immature nucleus with a large,
bright nucleolus (a). Discrete scrolls and lamellar structures with a
few rough particles (P in c) are found in the same granules (a and c),
and some granules have irregular lamellar structure (b). HCMCs cultured
with IL-4 has abundant cytoplasmic projections, well-developed Golgi
apparatus (G in d) and numerous granules, which contain dense, crystal
materials (d and e), as well as compact electron-dense nondiscrete
scrolls (f). Representative results are shown. (Original
magnifications: a, ×9,000; b, ×20,000; c, ×20,000; d, ×6,000;
e, ×16,000; f, ×22,000.)
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We finally examined electron microscopic immunocytochemistry using MoAb
specific for human tryptase or chymase. The same level of the
immunogold accumulation was detected in the granules of mast cells with
or without IL-4 treatment when MoAb to human tryptase was used
(Fig 4a and b). In contrast, in many mast
cells cultured with IL-4, chymase reactivity was highly positive in
granules compared with those of IL-4-nontreated mast cells (Fig 4c and
d). These data are consistent with the results obtained by light
microscopic observation and protease activity assay in which IL-4
increases MCTChigh. Taken together, these
results indicated that IL-4 promotes the development of
MCTChigh, which accompanied morphological
maturation of the cells.
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| Fig 4.
Detection of chymase and tryptase in HCMCs by electron
microscopic immunocytochemistry. Electron microscopic
immunocytochemistry of tryptase and chymase was performed on HCMCs
cultured with or without IL-4 (10 ng/mL) for 28 days in the presence of
SCF (100 ng/mL) and IL-6 (80 ng/mL). The mast cell cultured without
IL-4 stained with antitryptase (a) shows the same reactivity as the
mast cell cultured with IL-4 (b). In contrast, the mast cell cultured
without IL-4 stained with antichymase (c) shows weak reactivity
compared with the mast cell cultured with IL-4 (d). Immunocytochemical
control treated with mouse IgG1 instead of antitryptase or
antichymase is shown (e). Representative results are shown. (Original
magnification × 30,000.)
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DISCUSSION |
In this report, we showed that IL-4 promoted the development of
MCTChigh in HCMCs accompanied by morphological
maturation of the cells. Immunocytochemical staining showed that IL-4
increased the percentage of MCTChigh in HCMCs
from 2.5% to 93.5%. Electron microscopic
immunocytochemistry also confirmed this result. Consistently, activity
of chymase was significantly higher in IL-4-treated mast cells than in
IL-4-nontreated mast cells, whereas the activity of tryptase and
histamine content in both cells were comparable. Remarkably, along with
the increase of chymase expression in HCMCs by IL-4, these cells
achieved morphological maturity.
It has remained unknown whether two phenotypes of human mast cells,
MCT and MCTC, develop from common precursor
cells or whether they are derived from two different precommitted
cells. K. Tsuji (Department of Clinical Oncology, The Institute of
Medical Science, The University of Tokyo) and T. Nakahata
(unpublished data) have observed that both MCT and
MCTC were found in the same colony derived from a single
cord blood CD34+ cell, suggesting that MCT and
MCTC develop from common precursor cells. In the murine
system, it is known that two phenotypes of murine mast cells
(mucosal-type mast cells and connective tissue-type mast cells)
expressing different sets of protease are derived from common precursor
cells,30-32 and that final stages of mast cell
differentiation are regulated by cytokines in the tissue
microenvironment.23-32 Although it has been
currently unknown whether MCT can differentiate into
MCTC, or vice versa, development of MCTC via
MCT is possible by the observation that tryptase single
positive cells first appeared at week 2 of the culture and tryptase
chymase double positive cells at week 7 of the culture by weekly
analysis of cultured mast cells grown from cord blood CD34+
cells.10 Consistently, there is a report that tissue mast
cells in neonates and fetus contain a less amount, if any, of chymase
than the mast cells in adults, instead of comparable levels of
histamine and tryptase content with those in adult mast
cells.39 Thus, although the possibility that the divergent
pathways of differentiation of MCTC and MCT
cannot be thoroughly excluded, these observations may suggest a linear
pathway of development of MCTC cells through the
MCT stage.
Consistent with this hypothesis, we showed here that IL-4-induced
development of MCTChigh was accompanied by
morphological maturation of the cells. Previously, Dvorak et
al11-14 reported by sequential ultrastructural studies of
human cord-blood-derived mast cells cultured with 3T3 fibroblasts that
immature cultured mast cells contained fewer granules, which were empty
or partially filled with particles in the earliest mast cells and later
with scrolls, like immature mast cells in situ. While maturing, these
cultured mast cells have numerous small, dense granules like those most
frequently present in human skin mast cells.11-14 Based on
this granule-filling criteria, Mitui et al7 showed that
human mast cells developed from cord blood cells by SCF did not reach
full maturity even after a 14-week culture, and furthermore, the
majority of them expressed tryptase but not chymase. Consistently, in
our observation, the morphology of the HCMCs cultured only with SCF and
IL-6, which predominated MCT and
MCTClow and contained many empty or partially
filled granules, corresponded to the morphology of immature type mast
cells, whereas the morphology of the HCMCs cultured with IL-4, which
predominated MCTChigh and contained numerous
crystal granules, corresponded to that of mature mast cells. Thus,
these results indicated that immature mast cells expressed tryptase
alone, and as the cells matured, they produced both tryptase and
chymase. In this context, it is suggested that IL-4 promotes maturation
of human mast cells that produce both tryptase and chymase.
Mast cell chymase is a serine protease, which has important
proinflammatory effects. Chymase has been shown to rapidly convert
angiotensin I to angiotensin II four times more efficiently than
angiotensin-converting enzyme.41-43 Angiotensin II has an
important effect on regulation of microcirculation including
contraction of smooth muscle44 and enhancement of vascular
permeability in vitro.45 Chymase also attacks the lamina
lucida of the basement membrane at the dermal-epidermal junction of
human skin46 causing recruitment of inflammatory cells into
epidermis. The importance of mast cell chymase for allergic disorder
was further shown by the genetic association between variants of
mast-cell chymase gene and onset of eczema.47 Thus,
increased production and release of such an active protease from
activated mast cells with other vasoactive mediators may efficiently
contribute to amplification of allergic reaction. Because IL-4 is known
to play an important role in the onset of allergic disorder by
increasing IgE production by B cells and because increased IL-4
production by T cells from allergic patients has been
reported,48,49 our observation that IL-4 increases the
content of chymase in human mast cells may have a significant meaning
in the enhancement of allergic inflammation.
As shown in this report and others,7,33,35,50
HCMCs grown from cord blood mononuclear cells in the presence of SCF
have been shown to have many immature features including low expression
of FcRI, low histamine releasing activity, and the immature
morphology shown by the ultrastructural analysis, although they express
c-kit and contain a significant amount of tryptase and histamine. We
and others have previously shown that IL-4 promotes maturation and
differentiation of human mast cells in several aspects. IL-4 induces
FcRI expression in HCMCs as well as in murine mast cells, resulting
in high histamine-releasing activity on crosslinking of
FcRI.33,51 IL-4 suppresses c-kit
expression35,37 and also strongly induces LFA-1 and ICAM-1
expression.35,36 In this report, we have shown that IL-4
promotes morphological maturation of HCMCs in accordance with the
increase of chymase expression. Taken together, all these observations
would suggest that IL-4 is a key factor for human mast cell
differentiation and maturation.
| |
FOOTNOTES |
Submitted June 16, 1997;
accepted August 27, 1997.
Address reprint requests to Hano Toru, MD, Department of Pediatrics,
Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo
113, 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.
| |
ACKNOWLEDGMENT |
We thank Dr H. Ishida for providing cytokines, M. Toriyama and I.
Tanaka for technical assistance, and Drs S. Sasaki, I. Kamiyama, and
the staff of Matsushima Obstetric and Pediatric Hospital for providing
human cord blood. We thank Dr S. Nonoyama for critical review of the
manuscript.
| |
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Abstract
Introduction
Abstract