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* Wihuri Research Institute, FIN-00140 Helsinki, Finland;
Protein Chemistry Laboratory, Institute of Biotechnology, FIN-00014 University of Helsinki, Finland; and
Departments of Pathology and Virology, Haartman Institute, FIN-00014 University of Helsinki, Finland
1Correspondence: Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki, Finland. E-mail: ken.lindstedt{at}wri.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: recombinant chymase • proteolysis • TGF-ß activation • TGF-ß receptors • gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Unregulated tissue repair, however,
can result in pathological conditions such as pulmonary fibrosis, liver
cirrhosis, systemic sclerosis, and keloids, all having the
characteristics of increased TGF-ß1 expression and fibrosis
(2)
. In transgenic mice, overexpression of a
constitutively active TGF-ß1 under control of regulatory
sequences of phosphoenolpyruvate carboxykinase resulted in fibrosis of
the liver, kidney, and adipose tissue (3)
.
Mature TGF-ßs (25 kDa) are secreted constitutively by most tissues
and cells in a latent dimeric form (4)
. Latency is caused
by the amino-terminal prodomain LAP (latency-associated protein), which
is cleaved from the carboxyl-terminal active TGF-ß1 in the secretory
pathway but remains noncovalently associated with the mature protein
(5)
. A major fraction of the small latent TGF-ß1 forms a
larger complex with the latent TGF-ß1 binding protein (LTBP), which
binds to the LAP part of the small latent TGF-ß1 with a disulfide
bond (6)
. LTBPs aid in the processing and secretion of
latent TGF-ß1 (7)
and bind avidly to the extracellular
matrix, thereby attaching the large latent TGF-ß1 complex to the
matrix (8)
. To become physiologically active, the mature
TGF-ß1 has to be released from the matrix through proteolytic,
physicochemical, or conformational processing of LTBP and further
activated by selective removal of LAP (8
, 9)
. Active
TGF-ß1 is then capable of triggering local autocrine and paracrine
cellular responses by activating its specific high-affinity type I
(TßRI) and type II (TßRII) serine/threonine kinase receptor
systems.
Mast cells are also known to express TGF-ß1 (10
, 11)
,
and thus have been implicated as potential effector cells in
pathological processes of which typical hallmarks are inflammatory and
fibrogenic events, such as systemic sclerosis (12)
,
pulmonary fibrosis (13)
, myocardial infarction
(14)
, and myocardial fibrosis of the transplanted heart
(15)
. In these diseases, the number of mast cells is
increased; more important, they show signs of activation and
degranulation (14
, 15)
. When mast cells are activated in
vivo or in vitro through IgE-mediated cross-linking of Fc
RI
(16)
, by neighboring T lymphocytes (17)
or
macrophages (18)
, by the complement system (C3a, C5a)
(19)
, or by an experimental degranulating agent such as
compound 48/80, they expel their cytoplasmic secretory granules. In the
process of degranulation, the granules (consisting of preformed
mediators bound to a network of negatively charged heparin and
chondroitin sulfate proteoglycans) become swollen and their individual
membranes fuse to form tubular degranulation channels in which the
granules lie in chains (20)
. The degranulation channels
then open to the extracellular fluid and the soluble components of the
granules diffuse away, whereas the heparin proteoglycans and the mast
cell-specific neutral proteases (e.g., chymase and CPA) remain tightly
bound to each other, forming proteolytically active extracellular
‘granule remnants’ (21)
. These remnants are eventually
phagocytosed by adjacent phagocytes, such as macrophages and smooth
muscle cells (22
, 23)
. The degranulated mast cells replace
their lost preformed mediators through rapid onset of de novo
synthesis, followed by formation of new secretory granules. Eventually,
the granules are ready to participate in a new degranulation process
(24)
. However, the mechanism by which TGF-ß1 is
expressed, synthesized, and secreted by mast cells has remained vague.
Chymase, one of the components of the exocytosed granule remnants, has
previously been suggested to participate in TGF-ß metabolism by
releasing large latent TGF-ß complexes from the extracellular matrix
of cultured human endothelial cells through specific proteolytic
cleavage of LTBP-1 (25)
. We show here in a physiological
system that serosal mast cells also contain large latent TGF-ß1,
which, in contrast to the situation in other TGF-ß1-producing cells
in tissues, is stored in their cytoplasmic secretory granules together
with rat chymase 1, the major form of chymase in these cells. It was
recently shown that rat serosal mast cells also express significant
amounts of mRNA for rat mast cell protease-5 (RMCP-5) and trace amounts
of mRNA for RMCP-2 (26)
. However, the authors were unable
to identify a translation product for these two relatives of rat
chymase 1 (26)
. Furthermore, rat serosal mast cells are
completely void of other known chymase relatives, such as RMCP-3 and
RMCP-4 (26)
.
Since activation of latent TGF-ß1 is critical for its biological activity, it is important to delineate the physiological mechanisms involved in this activation process. Here we show that upon stimulation, both in vivo and in vitro, rat serosal mast cells cosecrete chymase 1 and latent TGF-ß1 in a rapid process of degranulation in which active TGF-ß1 is produced through the proteolytic action of the colocalized chymase 1. The mast cell-derived active TGF-ß1 then exerts an immediate paracrine effect on adjacent TßR-containing cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Control
rats received an intraperitoneal injection of 1 ml of PBS. The
peritoneal cells were allowed to recover for 1 h, when they were
isolated as described earlier (28)
and used for RNA
isolation and the reverse transcriptase-polymerase chain reaction
(RT-PCR).
Isolation and stimulation of rat serosal mast cells in vitro
Rat serosal cells consisting of mast cells and mononuclear cells
(mostly macrophages) were isolated from the peritoneal and pleural
cavities of male Wistar rats by lavage as described earlier
(28)
. Highly purified mast cells (> 98%) were obtained
by using a Percoll purification system (29)
. The mast
cells were stimulated with compound 48/80 (1 µg/ml) at 37°C and
incubated for increasing periods of time, as indicated in Fig. 3
. To
obtain macrophages, the peritoneal cells were seeded onto plastic
dishes and incubated in a humidified CO2
incubator at 37°C for 1 h. The adherent cells (mostly
macrophages) were removed and tested for the presence of TßRI,
TßRII, and PAI-1, as described below.
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Preparation of mast cell releasate and granule remnants
After mast cell stimulation and degranulation, the supernatant,
i.e., the mast cell ‘releasate’ containing all the material released
from the stimulated mast cells, was separated as described earlier
(28)
. The experiments were performed in the presence of
inhibitors of serine proteases, soybean trypsin inhibitor (TRINH,
Sigma), and phenylmethylsulfonyl fluoride (PMSF, Sigma), as indicated.
The mast cell releasate was separated from the mast cells by low-speed
centrifugation (150 g), then further separated into granule
remnants and granule remnant-free supernatant by higher-speed
centrifugation (15000 g). Granules with intact membranes
were prepared as described previously (31)
, using mild
sonication in a Kerry water bath sonicator. The membrane-covered
granules were then treated with 0.1% (v/v) Triton X-100 to remove the
membranes and produce granule remnants similar to those released by
stimulated mast cells. The Triton X-100 treatment of the intact
granules was performed in the presence or absence of chymostatin
(Sigma), a specific inhibitor of chymotrypsin-like enzymes.
Purification of rat mast cell chymase 1
Rat mast cell chymase 1 was purified from granule remnants as
described earlier (30)
. Granule remnants were resuspended
in 10 mM phosphate buffer, pH 7.0, containing 2 M KCl to dissociate
chymase from heparin proteoglycans. The components in the high-salt
mixture were separated on a Sephacryl S-200 column (1x50 cm, Pharmacia
LKB Biotechnology) using the same buffer at a flow rate of 5 ml/h at
4°C. The fractions (900 µl/fraction) containing the major part of
the chymase 1 activity (fractions 32–37), as determined using
N-benzoyl-L-tyrosine ethyl ester (BTEE) as substrate, were
diluted with 10 mM phosphate buffer to give a final concentration of
0.5 M KCl. Having a high net positive charge (+19.1) vs. other chymase
relatives, notably RMCP-5 (+14.1) and RMCP-2 (+5.2) (26)
,
chymase 1 was further affinity purified on a HiTrap Heparin column (1
ml, Pharmacia LKB Biotechnology), using the SMARTTM system (Pharmacia
LKB). The HiTrap Heparin column was eluted with a linear KCl gradient
(0.5–2 M), and chymase 1 that was recovered as a single peak at 
1 M
KCl, was stored at -70°C until used. To verify the purity of chymase
1, the enzyme was alkylated with 4-vinylpyridine and further purified
by reversed-phase (RP) chromatography on a 0.21 x 15.0 cm column
(Poros R2, PerSeptive Biosystems). The chymase 1, which eluted from the
RP column as a single peak both before and after alkylation, was then
dried and digested with trypsin, and the resulting peptides were
analyzed by matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry. The peptide masses were used to identify
the protein from sequence databases by peptide mass fingerprinting and
the protein was identified as rat chymase 1.
RT-PCR analysis of mast cell and macrophage gene expression
Total RNA was prepared from rat serosal mast cells and
macrophages, using an ultra-pure TRIzol reagent according to the
procedure described by the manufacturer (Gibco-BRL, Grand Island,
N.Y.). The RNA from mast cells was treated with heparinase I
(32)
and transcribed into cDNA using a SuperscriptTM
preamplification system (Gibco-BRL). This cDNA was further amplified by
PCR with specific primers for rat genes, as described in Table 1
, and the PCR fragment sequences obtained were verified by specific
restriction enzyme treatment.
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Competitive RT-PCR analysis of TGF-ß1 and chymase 1 expression
in rat mast cells
For competitive RT-PCR, a competitor DNA for TGF-ß1 was
prepared by insertion of a 239 bp external DNA fragment into the
BamHI site. The PCR products for target TGF-ß1 and its
competitor were 446 bp and 685 bp, respectively. A competitor DNA was
also obtained for rat chymase 1 by deleting a 173 bp fragment using the
restriction enzymes HindIII and StuI. The PCR
products for rat chymase 1 and its competitor were 645 and 467 bp,
respectively. The amounts of these PCR products were quantified by
competitor dilutions.
Northern blot analysis
For Northern blotting, total RNA (20 µg) was denatured using
glyoxal and dimethyl sulfoxide, separated by electrophoresis on a 1.2%
agarose gel, and blotted onto a Hybond N+ nylon
membrane (Amersham, Arlington Heights, IL) using standard methods
(33)
. The above-described 446 bp rat TGF-ß1 cDNA
fragment (100 ng) was labeled with 32P using the
Rediprime DNA labeling kit (Amersham) and used as a probe. The
membranes were hybridized at 65°C in 10 mg/ml BSA, 70 mg/ml SDS, 0.5
M sodium phosphate buffer (pH 6.8) containing 1 mM EDTA and the
32P-labeled DNA probe. Washing was
performed at 65°C in 5 mg/ml BSA, 50 mg/ml SDS, 40 mM sodium
phosphate buffer (pH 6.8), and 1 mM EDTA, then in 10 mg/ml SDS, 40 mM
sodium phosphate buffer (pH 6.8), and 1 mM EDTA. The Hybond filters
were exposed to Reflection film (DuPont, NEN, Wilmington, DE) with
intensifying screens at -80°C.
Immunocytochemistry of TGF-ß1, LTBP-1, and TßRII in rat
peritoneal cells
Rat peritoneal cells (1.5x104) were spun
onto cytoslides using a cytocentrifuge (Cytospin) and fixed in methanol
for 30 min in the presence of 0.8% hydrogen peroxide to inactivate
endogenous peroxidase. The cytoslides were further treated with 0.1%
saponin in PBS to allow the antibodies to penetrate the plasma
membrane, then preincubated in buffer A (1% skimmed milk containing
3% normal goat serum and 0.1% saponin) at room temperature for 1 h to inhibit nonspecific binding sites. The primary antibodies
(polyclonal antibodies against TGF-ß1 (Santa Cruz Biotechnology,
Santa Cruz, CA), LTBP-1 (34)
, and TßRII (Santa Cruz) or
equal amounts of nonimmune serum or PBS (controlling for the
specificity of primary, secondary, and tertiary antibodies) were
applied to the cytoslides in buffer A and incubated at room temperature
for 1 h. The cytoslides were washed extensively with PBS and
further incubated with biotinylated goat anti-rabbit IgG (Santa Cruz)
in buffer A at room temperature for 1 h. After extensive washing,
the immunoglobulin complexes formed were detected by incubating the
cytoslides with horseradish peroxidase-labeled streptavidin in buffer A
(without normal goat serum) and visualized with an aminoethylcarbazol
(AEC) developing solution. The color reaction was followed for up to 20
min and terminated by washing the cytoslides in water. The stained
cytoslides were mounted with an aqueous mounting media (Faramount,
DAKO) and viewed under an Olympus BH-2 light microscope.
Sodium deoxycholate polyacrylamide gel electrophoresis, SDS-PAGE,
and immunoblotting
LTBP-1, ß1-LAP, and TGF-ß1 were detected using gradient
(4–20% polyacrylamide) sodium deoxycholate-PAGE (DOC-PAGE) as
described previously (25
, 34)
. The use of DOC-PAGE in
analyzing TGF-ß1 allows us to detect TGF-ß1 in its native forms.
Samples in the corresponding gel were transferred to Probind 45
membranes (Amersham) and the membranes were treated at 80°C for
2 h to fix the transferred proteins. TGF-ß1, ß1-LAP, and
LTBP-1 were immunodetected essentially as described (35)
.
The filters were incubated in PBS containing 5% nonfat milk, 1% BSA,
and 1% Triton X-100 to prevent nonspecific reactivity and the
immunoreactive bands were identified by polyclonal antibodies against
TGF-ß1 (Santa Cruz), ß1-LAP, and LTBP-1 (34)
, using
biotin-streptavidin amplification and ECLTM Western blotting (Amersham
Pharmacia Biotech). TßRII was analyzed in detergent extracts from
purified mast cells and macrophages by SDS-PAGE and immunoblotting with
polyclonal antibodies against TßRII (Santa Cruz).
Enzyme-linked immunosorbent assay (ELISA) for active TGF-ß1
Human TGF-ß-soluble type II receptor protein (an sRII/Fc
chimera from R&D Systems (Abingdon, Oxon, UK) that cross-reacts with
rat TGF-ß1) was coated onto ELISA plates by incubating the plates at
room temperature overnight. Nonspecific binding sites were saturated by
further incubating the plates in PBS containing 5% Tween-20, 5%
sucrose, and 0.05% NaN3 as recommended by the
manufacturer. Isolated mast cells were stimulated with compound 48/80
(1 µg/ml) at 37°C for 15 min to produce mast cell releasates.
Pericellular matrix was prepared by incubating subconfluent rat aortic
smooth muscle cells (SMCs) in serum free medium for 24 h, after
which the SMCs were removed by 0.5% sodium deoxycholate in the
presence of protease inhibitors PMSF (1 mmol/l), aprotinin, leupeptin,
and pepstatin A (2 µg/ml each) as described earlier
(36)
. To detect active TGF-ß1, the mast cell releasate
or the culture media from rat chymase 1-treated pericellular matrix
were incubated on the receptor-containing ELISA plates at room
temperature for 4 h. After extensive washing, the receptor-bound
active TGF-ß1 was detected using polyclonal antibodies against
TGF-ß1 (R&D Systems), followed by a biotin-streptavidin amplification
system and peroxidase staining.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) for TGF-ß1,
rat chymase 1, basic fibroblast growth factor (bFGF), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The peritoneal cells
obtained from control rats expressed only small amounts of TGF-ß1
(Fig. 1
, lane 1) whereas those obtained from rats treated with compound 48/80
showed high amounts of TGF-ß1 expression (Fig. 1
, lane 2). The
observed difference in TGF-ß1 mRNA expression (10-fold) resembled
the degranulation-induced expression of two mast cell-derived,
preformed granule-associated molecules, the neutral serine protease rat
chymase 1 (lanes 3 and 4), and the bFGF (lanes 5 and 6). Expression
levels of GAPDH, a housekeeping gene, are at comparable levels in the
resting and stimulated peritoneal cell populations (lanes 7 and
8).
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Localization of TGF-ß1 to mast cell granules
To localize the TGF-ß1 translation product in the peritoneal
cell population, we next prepared cytoslides containing cells obtained
by peritoneal lavage and performed an immunocytochemical analysis using
polyclonal antibodies against TGF-ß1 and LTBP-1. All the mast cells
stained positively for TGF-ß1 (Fig. 2A
) and LTBP-1 (Fig. 2B
), but little reactivity was
present in the other cells. Some (30%) of the mast cells showed
strong positive staining for TGF-ß1, whereas all the mast cells
stained strongly for LTBP-1. If nonimmune serum was used instead of the
specific antibodies, no positive staining was found in the mast cells
(data not shown). Furthermore, both TGF-ß1 and LTBP-1 showed a
typical granular staining pattern in the cytoplasm, revealing their
presence in the secretory granules of the mast cells. The two major
granule components—rat chymase 1 (Fig. 2C
) and heparin
(Fig. 2D
) —also showed the typical granular staining
pattern. These results are compatible with the hypothesis that TGF-ß1
is a preformed granule-associated mediator present in the mast cells as
a large latent complex, in contrast to most other cells in tissues,
which secrete latent TGF-ß1 in a constitutive fashion.
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Purified rat mast cells overexpress TGF-ß1 and chymase 1 after
stimulation and degranulation
To characterize the regulation of TGF-ß1 expression in
mast cells, we performed competitive RT-PCR on cDNA prepared from
purified peritoneal mast cells that had been stimulated in vitro with
compound 48/80 and allowed to recover for increasing periods. TGF-ß1
expression increased dramatically (10-fold) at 1 h after
stimulation and slowly returned to the basal level within 4–8 h after
stimulation (Fig. 3A
, upper panel). The expression pattern of TGF-ß1 was
similar to that of rat chymase 1 (Fig. 3A
, lower panel),
which also increased after mast cell stimulation and degranulation. The
presence of TGF-ß1 mRNA in mast cells was further verified using
Northern blot analysis. The level of TGF-ß1 mRNA expression was found
to have increased 1 h after stimulation (Fig. 3B
),
after which it slowly returned to the basal level. In addition to the
predominant 2.4 kb mRNA, we also found a smaller transcript of 1.9 kb
that earlier had been shown to be an alternatively spliced TGF-ß1
transcript and to be strongly expressed at sites of injury
(37)
.
Proteolytic processing of large latent TGF-ß1 by rat chymase 1
Since TGF-ß1 and LTBP-1 were found to colocalize with chymase 1
and heparin in the cytoplasmic granules of rat serosal mast cells, we
next studied the fate of TGF-ß1 after mast cell stimulation and
degranulation. The stimulated and degranulated mast cells were first
separated (150 g) from the mast cell releasate, which was
then subjected to repeated centrifugation (15000 g) to
obtain granule remnants and granule remnant-free supernatant. Mast cell
stimulation and degranulation resulted in rapid secretion of mature (25
kDa) TGF-ß1, which was found to cosediment with the granule remnants
(Fig. 4
, bottom panel, lane 2). A minor fraction of the mature TGF-ß1 was
found in the granule remnant-free supernatant (bottom panel, lane 1) in
addition o some small latent TGF-ß1 particles (middle panel, lane 1).
The absence of large latent TGF-ß1 particles (top panel, lanes 1 and
2) suggested a rapid and specific degradation of these particles by rat
chymase 1, with subsequent release of small latent TGF-ß1 particles
and formation of mature TGF-ß1. In the presence of protease
inhibitors known to inhibit rat chymase 1 (38)
, we
detected two slightly different molecular forms of large latent
TGF-ß1: a larger one bound to the granule remnants (top panel, lane
4) and a smaller one in the granule remnant-free supernatant (top
panel, lane 3). These results suggested the involvement of rat chymase
1 in the production of mature TGF-ß1. In a control experiment, we
found that rat serosal mast cells do not secrete TGF-ß1 in a
constitutive manner (data not shown).
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Isolation of TGF-ß1-containing mast cell granules
Even in the presence of protease inhibitors, considerable amounts
of mature (25 kDa) TGF-ß1 were detected in the granule remnant
fraction (Fig. 4
, bottom panel, lane 4). These results suggest that the
experimental conditions in this experiment (Fig. 4)
, in which the
protease inhibitors were added after mast cell stimulation (in order
not to interfere with the process of mast cell stimulation and
degranulation), did not bring about immediate and complete inhibition
of rat chymase 1. Thus, to verify the role of granule-bound rat chymase
1 in the metabolism of mast cell-derived TGF-ß1, we prepared
membrane-covered intact granules (31)
that maintain their
acidic milieu and thus contain rat chymase 1 in an inactive form. These
intact granules did not contain mature (25 kDa) TGF-ß1 (Fig. 5
, lanes 1 and 2). Removal of granule membranes with detergents, which
exposed the granules to a neutral milieu, led to the formation of
granule remnants containing active rat chymase 1 and to the release of
mature TGF-ß1 (Fig. 5
, lanes 3 and 4). In the presence of
chymostatin, a specific inhibitor of chymotrypsin-like serine
proteases, the proteolytic activity of rat chymase 1 was fully
inhibited, and so was the formation of mature TGF-ß1 (lanes 5 and 6).
The degree of rat chymase 1 inhibition was independently monitored in
all granule fractions by analyzing the chymase-specific degradation of
angiotensin I (39)
(data not shown).
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Purified rat chymase 1 activates native large latent rat TGF-ß1
To further investigate the ability of rat chymase 1 to activate
latent TGF-ß1, we purified this enzyme from the granule remnants and
analyzed its purity using RP-HPLC and MALDI-TOF mass spectrometry
analysis. As shown in Fig. 6A
, alkylated rat chymase 1 eluted from the RP column as a
single peak, suggesting the presence of only a single protein in the
sample. After treating the RP-purified chymase with trypsin, the
peptide masses (Fig. 6B
) were used to identify the protein
from sequence databases by peptide mass fingerprinting and the chymase
protein was identified as rat chymase 1. The purified rat chymase 1 was
then incubated with native large latent TGF-ß1 from rat aortic smooth
muscle cells of synthetic phenotype (s-SMCs). The pericellular matrix
of s-SMCs was obtained by treating monolayers of s-SMCs with 0.5%
sodium deoxycholate in the presence of protease inhibitors to remove
the cells completely (36)
. The remaining pericellular
matrix contained native large latent TGF-ß1 and was incubated in the
presence or absence of purified rat chymase 1. The amount of active
TGF-ß1 in the culture medium was determined by a functional in vitro
TßRII ELISA assay, which allowed us to detect physiologically active
TGF-ß1. As shown in Table 2
, active TGF-ß1 (106±5 pg/106 s-SMCs) was
present in the culture medium only in the presence of rat chymase 1
(right lane). This result indicates that rat chymase 1 is capable of
activating native large latent TGF-ß1.
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Recombinant human chymase activates recombinant human latent
TGF-ß1
However, since rat serosal mast cells have been shown to express
mRNA for RMCP-5 and RMCP-2 (26)
and because of the
catalytic nature of proteases, it is impossible to completely exclude
the possibility of a contaminating enzyme in the chymase 1 preparation.
To verify that chymase is indeed capable of activating latent TGF-ß1,
we repeated the experiment in a pure in vitro system using recombinant
human chymase and recombinant human latent TGF-ß1. As shown in
Fig. 7
, human recombinant chymase was capable of activating recombinant human
latent TGF-ß1 (-PMSF) as a function of the added amount of enzyme.
Furthermore, the chymase-mediated activation of recombinant human
latent TGF-ß1 was inhibited in the presence of PMSF (+PMSF), a potent
inhibitor of chymase activity.
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Mast cell-derived TGF-ß1 is also physiologically active
To further study whether the mature TGF-ß1 in the granule
remnants and in the granule remnant-free supernatant (see Fig. 4
) was
physiologically active (i.e., was able to bind to its receptor), we
analyzed the two fractions in a TßRII ELISA assay. Both the granule
remnants and the granule remnant-free supernatant obtained from
compound 48/80 stimulated mast cells (>80% histamine release) were
found to contain active TGF-ß1 (Fig. 8
). The major part of the active TGF-ß1 (32
pg/106 mast cells) was bound to the granule
remnants and the rest (6 pg/106 mast cells) was
present in the granule remnant-free supernatant. In the absence of
compound 48/80, mast cells were found to secrete small quantities of
active TGF-ß1, which was in proportion to the low degree of
spontaneous histamine release (5%) induced by mechanical handling.
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Mast cells do not express TßRI, TßRII, or PAI-1, suggesting a
paracrine effect
We next analyzed whether the mast cell-derived TGF-ß1 would
exert autocrine or paracrine effects. Activated mast cells were found
to overexpress TGF-ß1 (Fig. 9A
, lane 2) at a level comparable to that observed in the
macrophages (Fig. 9B
, lane 2). The levels of TßRI (Fig. 8A
, lanes 3 and 4), TßRII (Fig. 9A
, lanes 5 and
6), and PAI-1 (Fig. 9A
, lanes 7 and 8) mRNA in the mast
cells, as well as the level of TßRII protein (Fig. 9D
,
left lane), were below the detection limit. In contrast, the peritoneal
macrophages expressed both TßRI and TßRII mRNA (Fig. 9B
,
lanes 3 and 5), which were further enhanced in the presence of mast
cell releasates (Fig. 9B
, lanes 4 and 6). The mast
cell-specific stimulator, compound 48/80, used to produce the mast cell
releasate did not have a direct effect on the expression of TGF-ß1,
TßRI, TßRII, or PAI-1 in macrophages (data not shown). However,
TGF-ß1 (Fig. 9B
, lanes 1 and 2) and PAI-1 (Fig. 9B
, lanes 7 and 8) mRNA expression was also increased in the
peritoneal macrophages as a consequence of mast cell stimulation and
degranulation. Since PAI-1 is a sensitive bioindicator of TGF-ß1
signaling, these results suggest the presence of physiologically active
TGF-ß1 in the mast cell releasate and a functional TßRI and TßRII
signaling system in the peritoneal macrophages. To further validate the
biological significance of our in vitro results, we stimulated the mast
cells in vivo in the rat peritoneal cavity and analyzed the level of
mRNA expression in the total peritoneal cell population by RT-PCR. Mast
cell stimulation and degranulation in vivo resulted in increased
expression of all the gene products (TGF-ß1, TßRI, TßRII and
PAI-1) involved in the above-described mast cell-mediated TGF-ß1
signaling system (Fig. 9C
). To verify the absence of the
TßRII-mediated TGF-ß1 signaling system in the mast cells, we
performed an immunocytochemical analysis of rat peritoneal cells with
polyclonal anti-TßRII antibodies. All the mononuclear cells (mainly
macrophages), but no mast cells, were positive for TßRII (Fig. 10A
, C
), the staining being localized to distinct parts of the
plasma membranes of the positive cells (Fig. 10B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Numerous experimental approaches have been carried out to identify
natural mechanisms for TGF-ß activation (8)
and many
physiologically relevant proteases have been analyzed for their
capacity to activate latent forms of TGF-ß1. The first successful
one, the wide-spectrum protease plasmin (40)
, can activate
latent TGF-ß1 as later shown in numerous experimental settings.
However, in contrast to TGF-ß1 knockout mice, which die shortly after
weaning because of a massive invasion of inflammatory cells
(41)
, loss of plasminogen activator (u-PA and t-PA)
function in mice does not severely affect the immune system
(42)
. These observations suggest that mechanisms other
than the plasmin are also responsible for the in vivo activation of
TGF-ß1. Indeed, gelatinases A and B (MMP-2 and MMP-9)
(43)
and collagenase 3 (MMP-13) (44)
have
recently been seen to bring about TGF-ß1 activation. Furthermore,
several proteases, such as neutrophil elastase and human mast cell
chymase, can degrade LTBP-1 and release the truncated large latent
TGF-ß1 from the extracellular matrix (25
, 34)
. However,
chymase purified from human skin (in contrast to plasmin) was unable to
activate purified recombinant large latent TGF-ß1 (25)
.
In the physiological system of the present study in which rat chymase 1
and TGF-ß1 were allowed to remain in their natural forms (i.e., bound
to the heparin proteoglycan matrix of the granule remnants), rat
chymase 1 was able to activate TGF-ß1. In addition, we could show
that native large latent TGF-ß1, bound to the pericellular matrix
produced by rat aortic s-SMCs, was also activated by purified rat
chymase 1. In contrast to chymase purified from human skin
(25)
, we found that recombinant human chymase was capable
of activating recombinant human latent TGF-ß1. Thus, although human
chymase and rat chymase 1 belong to different subgroups within the
chymase family (the 
- and ß-chymases, respectively) and show
differences in their ability to selectively hydrolyze substrates, such
as angiotensin I (45
46
47)
, they both seem to be able to
activate latent TGF-ß1. Even with regard to angiotensin I cleavage
specificity, the - and ß-chymases are not strictly dichotomous
(47)
. The exact cleavage site(s) on latent TGF-ß1, as
well as the observed differences between purified human skin chymase
and recombinant human chymase, remains to be shown in future
experiments.
It has been shown recently that heparin proteoglycans, in contrast to
low-sulfated mucosal heparan sulfates, potentiate the biological
activity of TGF-ß1 by protecting it from inactivation by
2-macroglobulin (48)
. The
protective effect was obtained through strong and specific binding of
TGF-ß1 to the glycosaminoglycan (GAG) part of the heparin
proteoglycan. Other classes of GAGs, such as chondroitin and dermatan
sulfates, were relatively ineffective in protecting and potentiating
the biological activity of TGF-ß1 (48)
. Since mast cells
are the only natural source of heparin proteoglycans, the formation of
a mast cell-derived macro complex of heparin proteoglycans and active
TGF-ß1 suggests a unique role for mast cells in the rapid production
and maintenance of an active and locally bioavailable pool of TGF-ß1.
It has been observed that mast cell activation induces in the activated
cells the expression of chymase, TNF-, and M-CSF (49
, 50)
. We find here that TGF-ß1 mRNA is also highly
overexpressed in mast cells because of their activation and
degranulation. Compared with macrophages, resting mast cells express
only low levels of TGF-ß1 mRNA, suggesting a difference in TGF-ß1
gene regulation between these two cell populations. Indeed, in contrast
to macrophages, mast cells do not express and secrete TGF-ß1 in a
constitutive manner, but store the translation product in their
intracellular granule compartment and release it rapidly upon
degranulation. Since the TGF-ß1 expression level was rapidly
increased as a consequence of mast cell stimulation, it appears that
the expression is regulated by factors capable of inducing mast cell
stimulation and degranulation. Thus, repeated mast cell activation and
subsequent degranulation, as it may occur in chronically inflamed
tissues, may be a mechanism creating a vicious circle involving chronic
elevation of TGF-ß1 expression in mast cells.
Although mast cell stimulation and degranulation seem to be
prerequisites for induced expression of growth factors in mast cells,
it is not known whether the induction is triggered by specific
stimulatory mechanisms leading to degranulation or merely by
resynthesis of lost preformed mediators. Mast cells derived from
cultured human umbilical cord blood expressed and secreted TGF-ß1 in
a constitutive manner, and stimulation of mast cells with the calcium
ionophore A23187 did not affect the level of TGF-ß1 expression
(51)
. This result showed that a mere increase in the level
of intracellular calcium, with ensuing degranulation, was not enough to
induce TGF-ß1 expression. Our present results with the basic
polyamine, compound 48/80, which stimulates mast cells to degranulate
in a receptor-mimetic fashion (27)
similar to the
physiological compounds, such as anti-IgE, C5a, C3a, and neurotensin,
suggest that the mechanism of physiological mast cell stimulation is
important for the onset of de novo TGF-ß1 expression.
It has been suggested that TGF-ß1 can inhibit rat peritoneal mast
cell stimulation in an autocrine manner (52)
. However, in
the present study we did not observe the presence of TßRI or TßRII
responsible for mediating the cellular responses to active TGF-ß1 in
rat serosal mast cells. Furthermore, neither rat serosal mast cells nor
human mast cells (53)
expressed PAI-1, a sensitive
bioindicator of TGF-ß1-mediated cellular responses (54
, 55)
, which suggests a paracrine rather than an autocrine role
for mast cell-derived TGF-ß1. In addition, mast cell stimulation and
degranulation induced the expression of TßRI and TßRII mRNA in
macrophages, suggesting an enhancement of TGF-ß1-mediated activity in
these cells. Thus, the mast cells seem to be unique in that they can
store latent TGF-ß1 in an intracellular granule compartment and, upon
stimulation, can rapidly secrete active TGF-ß1, which affects
neighboring cells in a paracrine fashion. A challenge for further
studies is to delineate the potential role of mast cells in
pathological processes characterized by inflammatory and fibrotic
events that are triggered by the multiple effects of active
TGF-ß1.
|
ACKNOWLEDGMENTS |
|---|
Received for publication May 8, 2000.
Revision received August 28, 2000.
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x174-HincII. B) For Northern blot
analysis, total RNA (20 µg) was denatured using glyoxal and dimethyl
sulfoxide, separated by electrophoresis on a 1.2% agarose gel, and
blotted onto a Hybond N+ nylon membrane. Hybridization was
performed with a 32P-labeled 446-nucleotide fragment of rat
TGF-ß1 cDNA.
