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*
Department of Pediatrics, School of Medicine, University of Tokushima,
Tokushima, Japan
Institute for Enzyme Research, University of Tokushima, Tokushima,
Japan
Department of Nephrology and Rheumatology, Georg August University Medical
Center, Göttingen, Germany.
Correspondence to Dr. Shoji Kagami, Department of Pediatrics, School of Medicine, University of Tokushima, Kuramoto-cho-3-chome, Tokushima 770-8503, Japan. Phone: +81-886-31-3111; Fax: +81-886-31-8697; E-mail: kagami{at}medclin.clin.med.tokushima-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, several lines of evidence have suggested the participation of mast cells in fibroblast proliferation in a number of organs. Increased numbers of mast cells are observed in close proximity to proliferating fibroblasts in healing wounds and in fibrotic lesions of lungs, vessel walls, and skin in human subjects (6,7,8,9). The most abundant product of human mast cells is the serine protease tryptase. Tryptase is a tetrameric serine protease with a molecular size of 134 kD; it is composed of four monomers of 32 to 34 kD, each with one catalytic site (10). Its presence is restricted almost exclusively to mast cells, where tryptase exists within the secretary granules in a complex with heparin proteoglycan (11). Tryptase has various biologic activities, such as fibrinogenolysis, augmentation of histamine-mediated contractility of airway smooth muscle, and degradation of vasoactive intestinal peptide (12,13,14). Importantly, tryptase has been demonstrated to be mitogenic for fibroblasts, smooth muscle cells, and bronchial epithelial cells (15,16,17). Recent studies have indicated that tryptase-positive mast cells may be involved in renal interstitial injury (18,19,20). However, the significance of mast cell tryptase in human renal interstitial fibrosis has not been investigated. To evaluate the role of mast cell tryptase, we first examined the localization of tryptase-positive mast cells in various types of human renal diseases. In addition, we investigated the possible role of tryptase in renal fibroblast proliferation and matrix protein synthesis in vitro.
We report for the first time that mast cell tryptase may have a role in the development of renal interstitial fibrosis, because the number of infiltrating tryptase-positive mast cells was well correlated with the degree of interstitial scarring. Furthermore, we observed that mast cell tryptase is a mitogen and fibrogenic factor for human renal fibroblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemical Studies
Immunohistochemical studies were performed as described previously
(21). Formalin-fixed tissue
sections were deparaffinized with xylene and rehydrated with graded ethanols.
Endogenous peroxidase was blocked with hydrogen peroxide, and the samples were
then rinsed in phosphate-buffered saline (PBS). To yield adequate signals with
the respective antibodies, the slides were trypsinized for 30 min at 37°C.
For reduction of background labeling, the sections were blocked for 30 min in
normal horse serum. The sections were incubated for 24 h at 4°C with mouse
anti-human mast cell tryptase monoclonal antibody (Chemicon International,
Temecula, CA) diluted in PBS containing 1% bovine serum albumin (1:1000). It
has been demonstrated that tryptase serves as a selective marker that
distinguishes mast cells from other cell types
(11). After washing with PBS,
the tissue was incubated with biotinylated secondary antibody,
avidin-biotin-peroxidase complex (ABC Elite; Vector Laboratories, Burlingame,
CA), and then 3,3'-diaminobenzidine (Dojindo, Kumamoto, Japan). Each
section was counterstained with Mayer's hematoxylin (Wako, Tokyo, Japan),
dehydrated, and coverslipped. A total of 10 consecutive nonoverlapping
interstitial areas (area, 0.625 mm2) were analyzed in each section
of the biopsy (18).
Tubulointerstitial damage was estimated on the basis of the degree of
tubulointerstitial fibrosis, as primarily judged using Azan-Mallory staining.
The degree of tubulointerstitial fibrosis was semiquantitatively graded by two
independent observers and was expressed as a ratio of the area of fibrosis to
that of the renal interstitium in biopsy sections.
Cell Culture
Human renal interstitial fibroblast cell lines were established as
described previously (22).
Briefly, human renal interstitial fibroblasts were obtained from the medullary
part of a kidney biopsy with histologically proven GN and interstitial
fibrosis. Fibroblast clones, as identified by morphologic criteria, were
subcultured in Dulbecco's modified Eagle's medium (Sigma Chemical Co., St.
Louis, MO) with 10% fetal calf serum (FCS) (HyClone, Logan, UT). Clones of
these early-passage human renal fibroblasts were immortalized by transfection
with the SV40 plasmid pSVgpt
(23). Stable cell lines of
these SV40-immortalized fibroblasts were obtained by repeated subcloning of
transfected cells (24). The
renal fibroblast cell lines were comparable to the parental nontransformed
interstitial fibroblasts, as indicated by similar morphologic features,
unchanged patterns of expression of COL I, III, and V, fibronectin (FN),
laminin, vimentin, tenascin, and intercellular adhesion molecule-1, and
negative immunostaining for cytokeratin, desmin, and factor VIII
(24). In addition, these
fibroblasts were positive for CD90, a marker for cultured renal fibroblasts
(25). The renal fibroblast
cell lines (fibrosis-derived renal fibroblasts) were maintained in RPMI 1640
medium (Life Technologies/BRL, Grand Island, NY) containing 2 mM L-glutamine
(Sigma), 25 mM Hepes (Sigma), 100 U/ml penicillin, 100 µU/ml streptomycin
(Life Technologies/BRL), and 10% FCS. All experiments were performed after
24-h incubations in serum-free RPMI 1640 medium, to achieve fibroblast
quiescence.
Proteinase Catalytic Assays
Human tryptase was purchased from Biogenesis (Poole, Dorset, UK). Tryptase
activity was assayed using the peptide substrate
N-tert-butoxycarbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide (Peptide
Institute, Japan). Tryptase activity was expressed in milliunits per
milliliter, where 1 U of enzyme activity was defined as the amount degrading 1
µmol of substrate per minute at 25°C
(26). The specific activity of
the tryptase preparations used in this study was 1.64 mU/µg.
Cell Proliferation Studies
Confluent human renal fibroblasts were detached from culture flasks with
trypsin-ethylenediaminetetraacetate (Life Technologies/BRL) and were seeded
into 24-well plates (Costar Corp., Cambridge, MA) at a density of 4 x
104 cells/ml, in RPMI 1640 medium containing 10% FCS. At
subconfluence (60%), the medium was replaced with serum-free medium for 24 h.
The cells were treated with tryptase in the presence of 1 U/ml heparin (Sigma)
for 24 h and were radiolabeled with 0.5 µCi/well
[methyl-3H]thymidine (Dupont/New England Nuclear, Boston,
MA) for the last 12 h. The cells were collected and counted in a liquid
scintillation counter (27). In
experiments with protease inhibitors, tryptase was preincubated with 50
µg/ml leupeptin (a nonselective serine protease inhibitor; Sigma) or 50
µM benzamidine hydrochloride (a relatively selective tryptase inhibitor;
Sigma) for 1 h at 4°C
(28). Similar experiments
studying DNA synthesis by fibroblasts were performed by incubating cells with
either trypsin or tryptase in the presence or absence of heparin or with
heparin alone. Cell proliferation in response to incubation with tryptase was
also monitored by cell counting with a hemocytometer.
[35S]Methionine-Labeling of Cells and Measurement of ECM
Protein Synthesis
Growth-arrested renal fibroblasts in six-well culture plates (Costar Corp.)
were incubated for 24 h in methionine-free medium containing either various
concentrations of tryptase with 1 U/ml heparin or heparin alone and were
metabolically labeled with 100 µCi/ml [35S]methionine (Dupont)
for the last 18 h. For immunoprecipitation and gelatin-Sepharose absorption
assays, the medium was collected and centrifuged for removal of cellular
debris. Radiolabeled protein in the supernatant was quantified by counting,
and a standard amount of radioactivity was used in the immunoprecipitation
assays. Aliquots (150 µl) of the radiolabeled medium were diluted with 800
µl of RIPA buffer [25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl
sulfate (SDS), 0.5% Triton X-100, 0.5% deoxycholate]. For removal of
nonspecific binding, 10 µl of normal rabbit serum and 50 µl of protein
A-Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden) were added, and
tubes were incubated and centrifuged. Then, 10 µl of a rabbit antibody to
human COL I (Chemicon International) was added to the supernatant, and the
mixture was incubated for 16 h at 4°C. Immune complexes were recovered by
binding to protein A-Sepharose beads (Pharmacia LKB Biotechnology). Beads were
washed three times with RIPA buffer, and the immunoprecipitates were analyzed
by electrophoresis on 4 to 12% SDS-polyacrylamide gels under reducing
conditions, as described previously
(29,30).
Gelatin-Sepharose was used to isolate FN from the radiolabeled medium, as
described previously (31).
Briefly, 50 µl of the gelatin-Sepharose suspension was added to each
sample, followed by incubation for 16 h at 4°C. The gelatin-Sepharose
beads were recovered by centrifugation and washed three times with RIPA
buffer. FN was released by resuspension of the beads in electrophoresis sample
buffer for 3 min at 100°C, under heating conditions, and was analyzed by
electrophoresis, as described above.
Northern Blot Analyses
Growth-arrested fibroblasts were treated for 24 h with either various
concentrations of tryptase with 1 U/ml heparin or heparin alone. Total
cellular RNA was isolated from the fibroblasts using RNAzol (Biotex
Laboratories, Houston, TX). For Northern blot analysis, RNA was denatured and
fractionated by electrophoresis through a 1.2% agarose gel (20 µg/lane) and
was then transferred to a Hybond nylon membrane (Amersham, Buckinghamshire,
UK). Nucleic acids were immobilized by ultraviolet irradiation (Stratagene, La
Jolla, CA) and hybridized with 1 x 106 cpm/ml
32P-labeled cDNA probes in hybridization buffer (50% formamide, 19%
Denhardt's solution, 0.1% SDS, 5x SSC, 200 µg/ml denatured salmon
sperm DNA). The cDNA probes were radiolabeled with [32P]dCTP by
random oligonucleotide priming (Boehringer Mannheim Biochemicals,
Indianapolis, IN). The cDNA probes used in this study included a rat 
1
COL I cDNA and, for normalization of the signals obtained with the cDNA
probes, a rat glyceraldehyde-3-phosphate dehydrogenase cDNA; the probes
specifically bind to human 1 COL I mRNA and human
glyceraldehyde-3-phosphate dehydrogenase cDNA, respectively
(32). The hybridization
products were observed by autoradiography. Densitometric scans were obtained
from autoradiographs, for quantification of hybridization intensity.
Statistical Analyses
Statistical significance in the immunohistochemical studies (defined as
P < 0.01) was evaluated using Pearson's correlation coefficients.
In vitro findings were presented as mean ± SD, and a value of
P < 0.05 was used to establish statistical significance
(t test). Triplicate wells were analyzed for cell proliferation
studies, and each experiment was performed independently at least twice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mitogenic Response of Human Renal Fibroblasts to Tryptase
To investigate whether mast cell tryptase has mitogenic potential against
renal fibroblasts, we examined the effect of purified tryptase on cultured
human renal interstitial fibroblasts in [3H]thymidine uptake
experiments. It was demonstrated that tryptase was stabilized as an
enzymatically active tetramer by association with heparin and dissociated to
inactive monomers without heparin
(11). A dose-dependent
increase in DNA synthesis by renal fibroblasts was observed after treatment
with tryptase in the presence of heparin (0.5 to 5.0 nM)
(Figure 3A). In addition,
[3H]thymidine uptake of fibroblasts stimulated by tryptase, with or
without heparin, was examined. The mitogenic activity of fibroblasts treated
with tryptase without heparin was decreased, compared with that of cells
coincubated with tryptase and heparin, indicating the substantial role of
heparin in maintaining tryptase activity
(Figure 4). Because heparin is
well known to inhibit the growth of various types of cells in vivo
and in vitro, we examined the effect of heparin alone on human renal
fibroblasts. Heparin suppressed [3H]thymidine uptake by
fibroblasts, suggesting that the mitogenic action in renal fibroblasts in this
experiment is induced not by heparin but rather by tryptase with heparin
(Figure 5). The proteolytic
activity of tryptase was similar to that of trypsin, and this action was
reported to play a role in the induction of cell signaling events by tryptase
(33). Therefore, we performed
an experiment to clarify whether trypsin exhibits any mitogenic effect on
cultured fibroblasts. A similar dose-dependent increase in DNA synthesis by
fibroblasts was observed with incubation with trypsin (10 to 50 ng/ml), but
treatment of fibroblasts with higher concentrations of trypsin resulted in
cell rounding and detachment from the plates
(Figure 6). Finally, we
examined whether the proteolytic cell signaling event induced by tryptase and
trypsin at the catalytic site was directly involved in their mitogenic effects
on fibroblasts. Preincubation of tryptase- or trypsin-stimulated fibroblasts
with the protease inhibitor leupeptin (50 µg/ml) or benzamidine
hydrochloride (50 µM) resulted in a significant decrease in
[3H]thymidine uptake (Figures
3 and
6), indicating that the
proteolytic action of tryptase is critical for induction of the mitogenic
response in fibroblasts. No significant change in mitogenesis was observed in
cells incubated with either leupeptin or benzamidine hydrochloride, compared
with vehicletreated control cells (Figures
3 and
6). Similar to
[3H]thymidine uptake experiments, cell-counting experiments
indicated that treatment of renal fibroblasts with tryptase and heparin
significantly increased cell numbers at tryptase doses of 2 and 5 nM
(Figure 3B). This increased
response to tryptase stimulation was suppressed by coincubation with either
leupeptin or benzamidine hydrochloride.
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Effects of Tryptase on ECM Protein Synthesis and COL I mRNA Level in
Human Renal Fibroblasts
To clarify whether tryptase regulates ECM protein synthesis by renal
fibroblasts, we performed gelatin-absorption assays for FN and
immunoprecipitation assays for COL I, using [35S]methionine-labeled
renal fibroblasts. As shown in Figure
7, incubation of renal fibroblasts with tryptase and heparin for
24 h resulted in increased synthesis of FN and COL I, in a dose-dependent
manner. The synthesis of FN and COL I by tryptase- and heparin-treated
fibroblasts was approximately 1.9- and 2.3-fold greater, respectively, than
that of control cells in response to tryptase at a dose of 2 nM. Incubation of
fibroblasts with concentrations of heparin identical to those used to
stabilize tryptase activity increased synthesis of COL I protein 1.6-fold,
compared with control values (Figure
7). Furthermore, Northern blot analysis revealed that 24-h
incubations with tryptase and heparin increased COL I mRNA levels in cultured
renal fibroblasts, whereas heparin alone had no significant effect on COL I
mRNA levels (Figure 8),
suggesting that the increases in COL I protein levels induced by heparin may
be attributable to post-translational events in heparin-treated fibroblasts
(34).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These findings from immunohistochemical assays demonstrated significant
correlations between the degree of renal interstitial fibrosis and the number
of infiltrating tryptase-positive mast cells, suggesting that mast cells play
an important role in renal interstitial fibrotic processes regardless of the
type of renal disease. These findings extended other studies that focused on
the involvement of mast cells in tubulointerstitial injuries accompanied by
GN, to determine the general role of mast cells in the development of
tubulointerstitial fibrosis not only in GN but also in acute and chronic TIN
(18,19,20).
Of note, mast cells were mainly detected in fibrotic lesions of TIN, rather
than in edematous areas. Furthermore, mast cells could not be detected in any
damaged glomeruli examined. These findings strongly suggested that mast cells
are unique contributors to the development of renal interstitial fibrosis.
Activation of mast cells can usually be induced by multiple mechanisms,
including IgE- and complement-mediated pathways. Stem cell factor (SCF) has
also been identified as a differentiation, chemoattraction, and activation
factor for mast cells in human subjects
(35,36,37).
Recently, Hogaboam et al.
(38) reported that SCF was
constitutively produced by pulmonary fibroblasts and could be induced by tumor
necrosis factor-, which is contained in mast cells. Therefore, it was
suggested that activated renal interstitial fibroblasts produce SCF after
injuries, to attract mast cells to injured sites.
Mast cells synthesize and secrete histamine, heparin, and various proteases and chemical mediators, such as prostaglandins, leukotrienes, and platelet-activating factors (39). Mast cell tryptase is a major protease and produces mitogenic effects on various types of cultured cells, such as smooth muscle cells and bronchial epithelial cells (16,17). Its mitogenic potential may be dependent on its proteolytic activity. The mechanism of the signaling event that mediates the fibrogenic effects of tryptase remains unclear. Like the thrombin receptor, proteinase-activated receptor-2 (PAR-2), a prototypic G protein-coupled receptor, was recently considered to be activated by proteolytic cleavage and synthetic peptides corresponding to the newly generated amino terminus after receptor cleavage (40). PAR-2 is activated by not only trypsin but also tryptase in vitro (33). Consistent with these findings, our [3H]thymidine uptake experiments demonstrated that mast cell tryptase and trypsin have mitogenic activity for human renal fibroblasts and that tryptase-mediated cellular activation requires a proteolytic event similar to thrombin-mediated signal transduction. The development of selective PAR-2 antagonists is necessary for determination of the precise mechanisms by which tryptase induces mitogenic responses in renal fibroblasts.
It is interesting that heparin is required for induction of the full activity of tryptase in fibroblasts, because the effects of tryptase without heparin on fibroblast proliferation are less potent than those observed with heparin. Heparin is generally accepted to suppress cell proliferation in vivo and in vitro (41,42). Floege et al. (43) demonstrated that heparin suppressed mesangial cell proliferation and matrix expansion in experimental GN in rats. Castellot et al. (44) reported that heparin inhibited mitogenesis of cultured rat mesangial cells. The findings presented here also demonstrated that heparin suppressed proliferation of renal fibroblasts in vitro. Tryptase was recently demonstrated to be stabilized as an enzymatically active tetramer by association with heparin and dissociated to inactive monomers in the absence of heparin, suggesting that heparin is an essential component for the induction of tryptase action on cultured renal fibroblasts (11).
Mast cells are a major source of basic fibroblast growth factor (bFGF) in chronic inflammation (45). bFGF is mitogenic for a variety of cell types, including fibroblasts, smooth muscle cells, and endothelial cells, and is known to play an important role in wound healing and fibrosis (46). Ehara and Shigematu (19) suggested that mast cell-derived bFGF may be a key mediator in renal fibrosis. In addition, it was demonstrated that bFGF had very robust proliferative effects on renal interstitial fibroblasts, without significant effects on matrix synthesis (47). bFGF seems to be an important factor, similar to tryptase, in the development of renal fibrosis.
ECM protein deposition in the interstitium is a very important step leading to renal interstitial fibrosis. ECM deposition occurs when the balance between matrix synthesis and degradation is disturbed (48). Our findings indicated that tryptase, when coincubated with heparin, stimulated FN and COL I protein synthesis by renal fibroblasts. The increased COL I protein synthesis seemed to result from the increased COL I mRNA levels induced by tryptase. The amounts of both FN and COL I were demonstrated to increase in interstitial fibrosis of the kidney (49). Because tryptase does not alter matrix metalloproteinase expression in human dermal fibroblasts (50), this study strongly indicates that tryptase is a fibrogenic factor. Interestingly, heparin itself stimulated COL I protein synthesis but did not affect COL I mRNA expression in these experiments. This response may be attributable to the inhibitory effect of heparin on gene expression of matrix metalloproteinase-degrading ECM proteins (34).
Although it is possible that SV40-transformed human renal fibroblasts may have increased proliferative capacity, compared with primary fibroblasts, we think that our cell lines were suitable for these experiments, because our transformed fibroblasts maintained the same characteristics and produced the same ECM proteins as their primary counterparts (24).
In conclusion, we presented data indicating that mast cell tryptase may play a role in the proliferation of and ECM protein production in renal interstitial fibroblasts and may thus contribute to the development of renal interstitial fibrosis. These findings also indicated that the proteolytic activity of tryptase may be involved in the fibrogenic action of renal interstitial fibroblasts. Further studies using either administration of a neutralizing antibody against tryptase or application of a mast cell tryptase-knockout mouse strategy in experimental models of kidney fibrosis are required for more definitive determination of the role of tryptase in the development of kidney scarring.
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Acknowledgments |
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- and ß-tryptases.
Blood 90:3914
-3922, 1997-smooth muscle actin expression in cultured fibroblasts and in
granulation tissue myofibroblasts. Lab Invest67
: 716-726,1992[Medline]
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), non-IgA GN (n = 5) (
), minor glomerular abnormalities
(n = 5) (
), membranous GN (n = 5) (x), focal
segmental glomerulosclerosis (n = 4) (
), acute TIN (n = 9) ([UNK]), and chronic TIN
(n = 9) (
). The degree of renal interstitial fibrosis was
significantly correlated with the number of infiltrating mast cells
(r = 0.834, P < 0.01).
) on DNA synthesis by human renal fibroblasts. Values
for [3H]thymidine uptake by renal fibroblasts treated for 24 h with
tryptase (0.5 to 5 nM) in the absence or presence of 1 U/ml heparin were
compared. The cells were radiolabeled with 0.5 µCi of
[3H]thymidine during the last 12 h. *, P, <
0.05, significant difference between untreated fibroblasts and
tryptase-treated fibroblasts with heparin; **, P <
0.05, significant difference between untreated fibroblasts and
tryptase-treated fibroblasts without heparin. Values are mean ± SD.
