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(The FASEB Journal. 2001;15:1377-1388.)
© 2001 FASEB

Activation of paracrine TGF-ß1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase

KEN A. LINDSTEDT*1, YENFENG WANG*, NAOTAKA SHIOTA*, JUHANI SAARINEN{dagger}, MARKO HYYTIÄINEN{ddagger}, JORMA O. KOKKONEN*, JORMA KESKI-OJA{ddagger} and PETRI T. KOVANEN*

* Wihuri Research Institute, FIN-00140 Helsinki, Finland;
{dagger} Protein Chemistry Laboratory, Institute of Biotechnology, FIN-00014 University of Helsinki, Finland; and
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As a source of transforming growth factor ß1 (TGF-ß1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-ß1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-ß1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-ß1, which is converted by chymase 1 into an active form recognized by the type II TGF-ß serine/threonine kinase receptor (TßRII). Thus, mast cells secrete active TGF-ß1 by a unique secretory mechanism in which latent TGF-ß1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-ß1 specifically by chymase was verified using recombinant human latent TGF-ß1 and recombinant human chymase. In isolated TßRI- and TßRII-expressing peritoneal macrophages, the activated TGF-ß1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TßRI, TßRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-ß1 in peritoneal mast cells and of TßRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-ß1 and by enhancing its response in target cells.—Lindstedt, K. A., Wang, Y., Shiota, N., Saarinen, J., Hyytiäinen, M., Kokkonen, J. O., Keski-Oja, J., Kovanen, P. T. Activation of paracrine TGF-ß1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase.


Key Words: recombinant chymase • proteolysis • TGF-ß activation • TGF-ß receptors • gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TRANSFORMING GROWTH FACTOR ßs (TGF-ßs) ARE MULTIPOTENT cytokines that regulate many biological functions, such as cell growth and tissue repair, that involve fibrosis (fibroblast proliferation and deposition of extracellular collagen), inflammatory responses, cell proliferation and migration, and angiogenesis (1 , 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{varepsilon}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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Stimulation of rat serosal mast cells in vivo
Rat serosal mast cells were stimulated in vivo by injecting 100 µg of compound 48/80 in 1 ml of phosphate-buffered saline (PBS) into the peritoneal cavity of the rat. Compound 48/80 (Sigma, St. Louis, MO) is a basic polyamine that stimulates mast cells in a receptor-mimetic fashion through activation of G-proteins (27) . 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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  		Figure 3. Time-dependent expression of TGF-ß1 and rat chymase 1 in stimulated rat serosal mast cells in vitro. Total RNA was isolated from mast cells at the times after mast cell stimulation indicated. A) Competitive RT-PCR was performed as described in Materials and Methods; the PCR fragments obtained were separated on a 1.2% agarose gel, stained with ethidium bromide, and photographed. The levels of TGF-ß1 (upper panel) and rat chymase 1 (lower panel) gene expression were analyzed at the indicated times. Marker = {phi}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.

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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  		Table 1. Primer sequences, annealing temperatures, and size of generated fragmentsa

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induced expression of TGF-ß1 and chymase 1 after rat mast cell stimulation in vivo
Rat serosal mast cells were stimulated to degranulate in vivo by injecting the mast cell-specific stimulator, compound 48/80, into the peritoneal cavity of rats. After incubation for 1 h, the peritoneal cells containing both mast cells and mononuclear cells (mostly macrophages) were recovered by lavage. The levels of gene expression in the isolated peritoneal cell population were analyzed with RT-PCR using specific oligonucleotides (see Table 1 ) 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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  		Figure 1. Analysis of TGF-ß1 and rat chymase 1 expression after mast cell stimulation in vivo. Rat serosal mast cells were stimulated in vivo by injecting 100 µg of compound 48/80 in 1 ml of PBS into the peritoneal cavity of the rat. After incubation for 1 h, the peritoneal cells were isolated (see Materials and Methods) and used for RNA extraction. The levels of gene expression of TGF-ß1 (lanes 1 and 2), rat chymase 1 (lanes 3 and 4), bFGF (lanes 5 and 6), and GAPDH (lanes 7 and 8) were analyzed by RT-PCR from the treated (+) and control (-) cells. Marker = {phi}x174-HincII.

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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  		Figure 2. Immunocytochemical detection of TGF-ß1 in rat peritoneal cells. Cytoslides containing isolated rat peritoneal cells were prepared and stained with antibodies against TGF-ß1 (A), LTBP-1 (B), and chymase (C) or with toluidine blue (metachromatic staining of mast cell heparin proteoglycans) (D) as described in Materials and Methods. The large cells are mast cells, and the small cells are mostly macrophages. The cells were further counterstained with Harris hematoxylin. Magnifications: A–C) 20x; D) 40x. Bar = 10 µm.

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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  		Figure 4. Immunoblot analysis of rat chymase 1-mediated activation of mast cell-derived large latent TGF-ß1. Isolated mast cells were stimulated with compound 48/80 (1 µg/ml) at 37°C. After complete degranulation (~1 min), incubation was continued for 10 min in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of PMSF (2 mM) and TRINH (100 µg/ml). The mast cell releasate formed, consisting of granule remnants (Granule R) (lanes 2 and 4) and granule remnant-free supernatant (Supernatant) (lanes 1 and 3), was then analyzed by DOC-PAGE and immunoblotting, using polyclonal antibodies against LTBP-1, ß1-LAP, and TGF-ß1, as indicated. LL-TGF-ß1 = large latent TGF-ß1; SL-TGF-ß1 = small latent TGF-ß1; M-TGF-ß1 = mature TGF-ß1.

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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  		Figure 5. Analysis of latent and active forms of TGF-ß1 in mast cell granules. Isolated and purified membrane-covered mast cell granules were incubated with (+) or without (-) chymostatin (50 µM), a chymase-specific protease inhibitor, and treated with Triton X-100 (0.1% v/v) to solubilize the intact granules. The granule remnants (Granule R) and granule remnant-free supernatants (Supernatant) thus formed were further analyzed by DOC-PAGE and Western blotting, using polyclonal antibodies against TGF-ß1. Recombinant human (rh)TGF-ß1 was used as a positive control (rightmost lane). Migration of the molecular mass markers (kDa) is shown on the left. SL-TGF-ß1 = small latent TGF-ß1; M-TGF-ß1 = mature TGF-ß1.

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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  		Figure 6. Analysis of purified rat chymase 1. The purity of rat chymase 1 was determined using RP-HPLC with a POROS R2 column (A) and the alkylated rat chymase 1 (arrow) was collected manually and dried in a vacuum centrifuge. Purified chymase 1 was subjected to MALDI-TOF mass spectrometry analysis (B) in conjunction with mass fingerprinting, i.e., matching the masses of tryptic peptides from the rat chymase 1 digest to theoretical peptides of known chymase relatives such as RMCP-2, -3, -4, and -5 from sequence databases. Each major and minor peak assigned with a m/z value in panel B represents one specific peptide produced by tryptic digestion of rat chymase 1. Angiotensin II (m/z 1046.54*) was used as an internal standard.


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  		Table 2. Purified rat chymase 1 activates native large latent TGF-ß1a

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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  		Figure 7. Activation of recombinant human latent TGF-ß1 by recombinant human chymase. Recombinant human latent TGF-ß1 (2 µg/ml, R&D Systems) was mixed with increasing amounts of recombinant human chymase (0–300 BTEE units/ml) (a kind gift from Dr. Kamimura, Teijin Ltd., Japan) in the presence (+) or absence (-) of PMSF, a potent inhibitor of chymase activity. The mixture was incubated for 15 min at 37°C in 20 µl of PBS containing 1 mg/ml BSA and 0.3 mg/ml of octyl-D-ß-glycopyranoside, as described previously (25) . The mature TGF-ß1 was detected by immunoblotting as described in Materials and Methods and further quantitated with a Gel Doc; 2000 gel documentation system (Bio-Rad). The plot is a representative of three individual experiments.

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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  		Figure 8. Detection of physiologically active TGF-ß1 after mast cell degranulation. Purified peritoneal mast cells were incubated in the presence of compound 48/80 (1 µg/ml) at 37°C for 15 min. Active TGF-ß1 was detected in the granule remnant (Granule R) fraction and the granule remnant-free supernatants (Supernatant), using human TGF-ß soluble receptor II ELISA plates and a polyclonal IgG against TGF-ß1 (R&D Systems). N.D. = not detectable.

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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  		Figure 9. Induction of the TGF-ß1 signaling system in peritoneal cells by mast cell stimulation in vitro and in vivo. Purified peritoneal mast cells (A) were incubated in the presence or absence of compound 48/80 (1 µg/ml) at 37°C for 15 min, after which the obtained releasates from the stimulated (+) or unstimulated (-) mast cells were added to the respective (+) and (-) plates of purified peritoneal macrophages (B). After continued incubation of the mast cells and macrophages at 37°C for 1 h, they were analyzed by RT-PCR for the expression of the genes involved in TGF-ß1 signaling. The experiment was repeated in vivo by stimulating the mast cells in the rat peritoneum for 1 h, after which the peritoneal cell population was isolated and analyzed by RT-PCR (C). Marker = {phi}x174-HincII. D) Purified mast cells (50 µg) and macrophages (50 µg) were also analyzed for the presence of TßRII by SDS-PAGE (4–20% polyacrylamide) under reducing conditions, followed by immunoblotting with polyclonal antibodies against TßRII. Migration of the molecular mass markers (kDa) is shown on the left. MC, mast cell; M{phi}, macrophage.



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  		Figure 10. Immunocytochemical detection of TßRII. Cytoslides containing rat peritoneal cells were prepared for immunocytochemistry as described in Materials and Methods. All mononuclear cells (curved arrows) except mast cells (stars) stained positively for TßRII (A, B). The mast cells were further stained with either toluidine blue (metachromatic staining of mast cell heparin proteoglycans) (B) or double stained with Harris hematoxylin (nuclear staining) and toluidine blue (metachromatic staining of mast cell heparin proteoglycans) (C). Note the negative staining of mast cells for TßRII. Bar = 1 µm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We find here that chymase 1-containing rat serosal mast cells are capable of expressing, secreting, and activating TGF-ß1 by a unique secretory mechanism in which the large latent TGF-ß1 and the activating enzyme (rat chymase 1) are coreleased. The mature TGF-ß1 is stored in a large latent form in the cytoplasmic secretory granules of the mast cells. Mast cell stimulation and degranulation lead to immediate secretion and activation of the large latent TGF-ß1 by the colocalized heparin proteoglycan-bound neutral protease, rat chymase 1, and subsequently to a rapid (within 1 h) increase in TGF-ß1 and rat chymase 1 mRNA expression both in vitro and in vivo. Furthermore, mast cell stimulation both in vitro and in vivo induced the expression of TßRI, TßRII, and PAI-1 mRNA in the peritoneal macrophage cell population. These results imply a novel role for activated mast cells in the paracrine regulation of TGF-ß1 signaling and provide an alternative proteolytic model of TGF-ß1 activation involving cosecretion of the latent TGF-ß1 and the activating protease rat chymase 1. In addition, these results present new aspects on the biological level of the regulation of function and action of TGF-ß1, namely, the specific process of mast cell stimulation and degranulation.

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 {alpha}- 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 {alpha}- 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 {alpha}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-{alpha}, 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
 
We appreciate the excellent technical assistance of Ms. Jaana Tuomikangas and Mr. Fridolin Seif. We are grateful to Dr. Kamimura, Teijin Ltd., Japan, for providing us with recombinant human chymase. K.A.L was supported with grants from the Aarne Koskelo Foundation and the Paavo Nurmi Foundation. J.K.-O. was supported with grants from the Academy of Finland and the Sigrid Juselius Foundation.

Received for publication May 8, 2000. Revision received August 28, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Roberts, A. B., Sporn, M. B., Assoian, R. K., Smith, J. M., Roche, N. S., Wakefield, L. M., Heine, U. I., Liotta, L. A., Falanga, V., Kehrl, J. H., Fauci, A. S. (1986) Transforming growth factor type-ß: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc. Natl. Acad. Sci. USA 83,4167-4171[Abstract/Free Full Text]
  2. Border, W. A., Ruoslahti, E. (1992) Transforming growth factor-ß in disease: the dark side of tissue repair. J. Clin. Invest. 90,1-7
  3. Clouthier, D. E., Comerford, S. A., Hammer, R. E. (1997) Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-ß1 transgenic mice. J. Clin. Invest. 100,2697-2713[Medline]
  4. Lawrence, D. A., Pircher, R., Julien, P. (1985) Conversion of a high molecular weight latent ß-TGF from chicken embryo fibroblasts into a low molecular weight active ß-TGF under acidic conditions. Biochem. Biophys. Res. Commun. 133,1026-1034[Medline]
  5. Gentry, L. E., Nash, B. W. (1990) The prodomain of pre-protransforming growth factor ß1 when independently expressed is a functional binding protein for the mature growth factor. Biochemistry 29,6851-6857[Medline]
  6. Kanzaki, T., Olofsson, A., Moren, A., Wernstedt, C., Hellman, U., Miyazono, K., Claesson-Welsh, L., Heldin, C.-H. (1990) TGF-ß1 binding protein: a component of the large latent complex of TGF-ß1 with multiple repeat sequences. Cell 61,1051-1061[Medline]
  7. Miyazono, K., Olofsson, A., Colosetti, P., Heldin, C.-H. (1991) A role of the latent TGF-ß1-binding protein in the assembly and secretion of TGF-ß1. EMBO J 10,1091-1101[Medline]
  8. Saharinen, J., Hyytiäinen, M., Taipale, J., Keski-Oja, J. (1999) Latent transforming growth factor-ß-binding proteins (LTBPs): structural extracellular matrix proteins for targeting TGF-ß action. Cytokine Growth Factor Rev 10,99-117[Medline]
  9. Taipale, J., Keski-Oja, J. (1997) Growth factors in the extracellular matrix. FASEB J 11,51-59[Abstract]
  10. Pennington, D. W., Lopez, A. R., Thomas, P. S., Peck, C., Gold, W. M. (1992) Dog mastocytoma cells produce transforming growth factor beta. J. Clin. Invest. 90,35-41
  11. Gordon, J. R., Galli, S. J. (1994) Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc{varepsilon}RI. Role for mast cell-derived transforming growth factor ß and tumor necrosis factor {alpha}. J. Exp. Med. 180,2027-2037[Abstract/Free Full Text]
  12. Seibold, J. R., Giorno, R. C., Claman, H. N. (1990) Dermal mast cell degranulation in systemic sclerosis. Arth. Rheum. 33,1702-1709[Medline]
  13. Broide, D. H., Smith, C. M., Wasserman, S. I. (1990) Mast cells and pulmonary fibrosis: identification of a histamine releasing factor in bronchoalveolar lavage fluid. J. Immunol. 145,1838-1844[Abstract]
  14. Patella, V., Marinò, I., Arbustini, E., Lamparter-Schummert, B., Verga, L., Adt, M., Marone, G. (1998) Stem cell factor in mast cells and increased mast cell density in idiopathic and ischemic cardiomyopathy. Circulation 97,971-978[Abstract/Free Full Text]
  15. Li, Q. Y., Raza-Ahmed, A., MacAulay, M. A., Lalonde, L. D., Rowden, G., Trethewey, E., Dean, S. (1992) The relationship of mast cells and their secreted products to the volume of fibrosis in posttransplant hearts. Transplantation 53,1047-1051[Medline]
  16. Ishizaka, T., Ishizaka, K. (1984) Activation of mast cells for mediator release through IgE receptors. Prog. Allergy 34,188-235[Medline]
  17. Sedgewick, J. D., Holt, P. G., Turner, K. J. (1981) Production of a histamine-releasing lymphokine by antigen- or mitogen-stimulated human peripheral T cell. Clin. Exp. Immunol. 45,409-418[Medline]
  18. Liu, M. C., Proud, D., Lichtenstein, L. M., MacGlashan, D. W., Schleimer, R. P., Adkinson, N. F., Kagey-Sobotka, A., Schulman, E. S., Plaut, M. (1986) Human lung macrophage-derived histamine-releasing activity is due to IgE-dependent factors. J. Immunol. 136,2588-2595[Abstract]
  19. Metcalfe, D. D., Kaliner, M., Donlon, D. A. (1981) The mast cell. Crit. Rev. Immunol. 3,23-74[Medline]
  20. Röhlich, P., Anderson, P., Uvnäs, B. (1971) Electron microscope observations on compound 48/80-induced degranulation in rat mast cells. Evidence for sequential exocytosis of storage granules. J. Cell Biol. 51,465-483
  21. Kovanen, P. T. (1993) The mast cell—a potential link between inflammation and cellular cholesterol deposition in atherogenesis. Eur. Heart J. 14(Suppl. K),105-117
  22. Kokkonen, J. O., Kovanen, P. T. (1987) Stimulation of mast cells leads to cholesterol accumulation in macrophages in vitro by a mast cell granule-mediated uptake of low density lipoprotein. Proc. Natl. Acad. Sci. USA 84,2287-2291[Abstract/Free Full Text]
  23. Wang, Y., Lindstedt, K. A., Kovanen, P. T. (1995) Mast cell granule remnants carry LDL into smooth muscle cells of the synthetic phenotype and induce their conversion into foam cells. Arterioscler. Thromb. Vasc. Biol. 15,801-810[Abstract/Free Full Text]
  24. Dvorak, A. M., Schleimer, R. P., Schulman, E. S., Lichtenstein, L. M. (1986) Human mast cells use conservation and condensation mechanisms during recovery from degranulation. Lab. Invest. 54,663-678[Medline]
  25. Taipale, J., Lohi, J., Saarinen, J., Kovanen, P. T., Keski-Oja, J. (1995) Human mast cell chymase and leukocyte elastase release latent transforming growth factor-ß1 from extracellular matrix of cultured human epithelial and endothelial cells. J. Biol. Chem. 270,4689-4696[Abstract/Free Full Text]
  26. Lützelschwab, C., Pejler, G., Aveskogh, M., Hellman, L. (1997) Secretory granule proteases in rat mast cells. Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations. J. Exp. Med. 185,13-29[Abstract/Free Full Text]
  27. Pinxteren, J. A., O’Sullivan, A. J., Tatham, P. E., Gomperts, B. D. (1998) Regulation of exocytosis from rat peritoneal mast cells by G-protein beta gamma-subunits. EMBO J 17,6210-6218[Medline]
  28. Kokkonen, J. O., Kovanen, P. T. (1989) Accumulation of low-density lipoproteins in stimulated rat serosal mast cells during recovery from degranulation. J. Lipid Res. 30,1341-1348[Abstract]
  29. Kawamoto, K., Okada, T., Kannan, Y., Ushio, H., Matsumoto, M., Matsuda, H. (1995) Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor. Blood 86,4638-4644[Abstract/Free Full Text]
  30. Lindstedt, L., Lee, M., Castro, G. R., Fruchart, J., Kovanen, P. T. (1996) Chymase in exocytosed rat mast cell granules effectively proteolyses apolipoprotein A1-containing lipoproteins, so reducing the cholesterol efflux-inducing ability of serum and aortic intimal fluid. J. Clin. Invest. 97,2174-2182[Medline]
  31. Lindstedt, K. A., Kokkonen, J. O., Kovanen, P. T. (1992) Soluble heparin proteoglycans released from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor-mediated phagocytosis. J. Lipid Res. 33,66-75
  32. Tsai, M., Miyamoto, M., Tam, S-.Y., Wang, Z.-S., Galli, S. J. (1994) Detection of mouse mast cell-associated mRNA. Heparinase treatment greatly improves RT-PCR of tissues containing mast cell heparin. Am. J. Pathol. 146,335-343[Abstract]
  33. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 3rd Ed Cold Spring Harbor Laboratory Cold Spring Harbor, New York.
  34. Taipale, J., Miyazono, K., Heldin, C.-H., Keski-Oja, J. (1994) Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via a latent TGF-ß binding protein. J. Cell Biol. 124,171-181[Abstract/Free Full Text]
  35. Taipale, J., Koli, K., Keski-Oja, J. (1992) Release of transforming growth factor-ß1 from the pericellular matrix of cultured fibroblasts and fibrosarcoma cells by plasmin and thrombin. J. Biol. Chem. 267,25378-25384[Abstract/Free Full Text]
  36. Hedman, K., Kurkinen, M., Alitalo, K., Vaheri, A., Johansson, S., Höök, M. (1979) Isolation of the pericellular matrix of human fibroblast cultures. J. Cell Biol. 81,83-91[Abstract/Free Full Text]
  37. Qian, S. W., Kondaiah, P., Casscells, W., Roberts, A. B., Sporn, M. B. (1991) A second messenger RNA species of transforming growth factor ß1 in infarcted rat heart. Cell Regul 2,241-249[Medline]
  38. Kokkonen, J. O., Kovanen, P. T. (1985) Low density lipoprotein degradation by rat mast cells. Demonstration of extracellular proteolysis by mast cell granules. J. Biol. Chem. 260,14756-14763[Abstract/Free Full Text]
  39. Urata, H., Kinoshita, A., Misono, K. S., Bumbus, F. M., Husain, A. (1990) Identification of a highly specific chymase as the major angiotensin-forming enzyme in the human heart. J. Biol. Chem. 265,22348-22357[Abstract/Free Full Text]
  40. Lyons, R. M., Keski-Oja, J., Moses, H. L. (1988) Proteolytic activation of latent transforming growth factor-ß from fibroblast-conditioned medium. J. Cell Biol. 106,1597-1605
  41. Shull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold, R. J., Yin, M., Allen, R., Sidman, C., Proetzel, G., Calvin, D., Annunziata, N., Doetschman, T. (1992) Targeted disruption of the mouse transforming growth factor-ß1 gene results in multifocal inflammatory disease. Nature (London) 359,693-699[Medline]
  42. Carmeliet, P., Schoonjans, L., Kieckens, L., Ream, B., Degen, J., Bronson, R., De Vos, R., van den Oord, J. J., Collen, D., Mulligan, R. C. (1994) Physiological consequences of loss of plasminogen activator gene function in mice. Nature (London) 368,419-424[Medline]
  43. Yu, Q., Stamenkovic, I. (2000) Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis. Genes Dev 14,163-176[Abstract/Free Full Text]
  44. D’Angelo, M., Billings, P. B., Pacifici, M., Leboy, P. S., Kirsch, T. (2001) Authentic matrix vesicles contain active metalloproteases: a role for matrix vesicle-associated MMP-13 in activation of TGFß. J. Biol. Chem. 276,11347-11353[Abstract/Free Full Text]
  45. Chandrasekharan, U. M., Sanker, S., Glynias, M. J., Karnik, S. S., Husain, A. (1996) Angiotensin II-forming activity in a reconstructed ancestral chymase. Science 271,502-505[Abstract]
  46. Sanker, S., Chandrasekharan, U. M., Wilk, D., Glynias, M. J., Karnik, S. S., Husain, A. (1997) Distinct multisite synergistic interactions determine substrate specificities of human chymase and rat chymase-1 for angiotensin II formation and degradation. J. Biol. Chem. 272,2963-2968[Abstract/Free Full Text]
  47. Caughey, G. H., Raymond, W. W., Wolters, P. J. (2000) Angiotensin II generation by mast cell alpha- and beta-chymases. Biochim. Biophys. Acta 1480,245-257[Medline]
  48. Lyon, M., Rushton, G., Gallagher, T. (1997) The interaction of the transforming growth factor-ßs with heparin and heparan sulfate is isoform-specific. J. Biol. Chem. 272,18000-18006[Abstract/Free Full Text]
  49. Chen, H., Centola, M., Altschul, S. F., Metzger, H. (1998) Characterization of gene expression in resting and activated mast cells. J. Exp. Med. 188,1657-1668[Abstract/Free Full Text]
  50. Gordon, J. R., Galli, S. J. (1990) Mast cells as a source of both preformed and immunologically inducible TNF-{alpha}/cachectin. Nature (London) 346,274-276[Medline]
  51. Kanbe, N., Kurosawa, M., Nagata, H., Saitoh, H., Miyachi, Y. (1999) Cord blood-derived human cultured mast cells produce transforming growth factor ß1. Clin. Exp. Allergy 29,105-113[Medline]
  52. Bissonnette, E. Y., Enciso, J. A., Befus, A. D. (1997) TGF-ß1 inhibits the release of histamine and tumor necrosis factor-{alpha} from mast cells through an autocrine pathway. Am. J. Resp. Cell Mol. Biol. 16,275-282[Abstract]
  53. Sillaber, C., Baghestanian, M., Bevec, D., Willheim, M., Agis, H., Kapiotis, S., Fureder, W., Bankl, H. C., Kiener, H. P., Speiser, W., Binder, B. R., Lechner, K., Valent, P. (1999) The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis. J. Immunol. 162,1032-1041[Abstract/Free Full Text]
  54. Sato, Y., Tsuboi, R., Lyons, R., Moses, H., Rifkin, D. B. (1990) Characterization of the activation of latent TGF-ß by cocultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system. J. Cell Biol. 111,757-763[Abstract/Free Full Text]
  55. Yang, L., Qiu, C. X., Ludlow, A., Ferguson, M. W. J., Brunner, G. (1999) Active transforming growth factor-ß in wound repair: determination using a new assay. Am. J. Pathol. 154,105-111[Abstract/Free Full Text]



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