The Differentiation and Function of Myofibroblasts is Regulated by Mast Cell Mediators
2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2001.15211.x
ISSN1523-1747
AutoresJames Gailit, Mary J. Marchese, Richard R. Kew, Barry L. Gruber,
Tópico(s)Fibroblast Growth Factor Research
ResumoMyofibroblasts are fibroblasts that express certain features of smooth muscle differentiation. Increased numbers of myofibroblasts and mast cells are frequently found together in a wide variety of settings, such as normal wound repair and scleroderma skin, which suggests that mediators produced by the mast cells could play a role in the regulation of myofibroblast differentiation and function. We used a human mast cell line, HMC-1, to determine if mast cells can induce normal human dermal fibroblasts to differentiate into functional myofibroblasts in vitro. We monitored the differentiation process by assaying two properties of the myofibroblast phenotype: expression of α-smooth muscle actin and functional capacity to contract a collagen matrix. In both a simple coculture system and in a skin-equivalent culture system, HMC-1 cells induced α-smooth muscle actin expression by fibroblasts. HMC-1 cells also stimulated fibroblast contraction of collagen gels, and the relative amount of contraction was dependent upon the number of HMC-1 cells present. To characterize the individual contributions made by specific mast cell products, we examined the effects of histamine, tumor necrosis factor α, and tryptase. Histamine induced a clear increase in α-smooth muscle actin expression, but it did not appear to stimulate fibroblast contraction. Tumor necrosis factor α had no effect in either assay. Purified human tryptase induced α-smooth muscle actin expression, and blocking the proteolytic activity of tryptase with specific inhibitors reduced that response. Tryptase inhibitors also eliminated the ability of HMC-1 cells to stimulate fibroblast contraction, suggesting that tryptase secreted by the HMC-1 cells may be one of the active mast cell mediators. Myofibroblasts are fibroblasts that express certain features of smooth muscle differentiation. Increased numbers of myofibroblasts and mast cells are frequently found together in a wide variety of settings, such as normal wound repair and scleroderma skin, which suggests that mediators produced by the mast cells could play a role in the regulation of myofibroblast differentiation and function. We used a human mast cell line, HMC-1, to determine if mast cells can induce normal human dermal fibroblasts to differentiate into functional myofibroblasts in vitro. We monitored the differentiation process by assaying two properties of the myofibroblast phenotype: expression of α-smooth muscle actin and functional capacity to contract a collagen matrix. In both a simple coculture system and in a skin-equivalent culture system, HMC-1 cells induced α-smooth muscle actin expression by fibroblasts. HMC-1 cells also stimulated fibroblast contraction of collagen gels, and the relative amount of contraction was dependent upon the number of HMC-1 cells present. To characterize the individual contributions made by specific mast cell products, we examined the effects of histamine, tumor necrosis factor α, and tryptase. Histamine induced a clear increase in α-smooth muscle actin expression, but it did not appear to stimulate fibroblast contraction. Tumor necrosis factor α had no effect in either assay. Purified human tryptase induced α-smooth muscle actin expression, and blocking the proteolytic activity of tryptase with specific inhibitors reduced that response. Tryptase inhibitors also eliminated the ability of HMC-1 cells to stimulate fibroblast contraction, suggesting that tryptase secreted by the HMC-1 cells may be one of the active mast cell mediators. 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride bis(5-amidino-2-benzimidazolyl)methane Iscove's modified Dulbecco's medium recombinant leech-derived tryptase inhibitor serum-free medium containing 2 mg per ml bovine serum albumin Although fibroblasts are abundant in connective tissues throughout the body, not all populations of fibroblasts are homogeneous (Sappino et al., 1990bSappino A.P. Schürch W. Gabbiani G. Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations.Lab Invest. 1990; 63: 144-161PubMed Google Scholar;Schmitt-Gräff et al., 1994Schmitt-Gräff A. Desmoulière A. Gabbiani G. Heterogeneity of myofibroblast phenotypic features: an example of fibroblastic cell plasticity.Virchows Arch. 1994; 425: 3-24Crossref PubMed Scopus (403) Google Scholar). Some fibroblasts express features of smooth muscle differentiation. These smooth muscle-like fibroblasts are referred to as myofibroblasts (Serini and Gabbiani, 1999Serini G. Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation.Exp Cell Res. 1999; 250: 273-283Crossref PubMed Scopus (501) Google Scholar) and they can be identified by certain characteristic features of the cytoskeleton, particularly by the expression of α-smooth muscle actin (Sappino et al., 1990bSappino A.P. Schürch W. Gabbiani G. Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations.Lab Invest. 1990; 63: 144-161PubMed Google Scholar). Myofibroblasts make important contributions to the growth and differentiation of tissues and organs through their interactions with epithelial cells (Gabbiani et al., 1971Gabbiani G. Ryan G.B. Majno G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction.Experientia. 1971; 27: 549-550Crossref PubMed Scopus (1170) Google Scholar;Gabbiani and Majno, 1972Gabbiani G. Majno G. Dupuytren's contracture: fibroblast contraction? An ultrastructural study.Am J Pathol. 1972; 66: 131-146PubMed Google Scholar;Schmitt-Gräff et al., 1994Schmitt-Gräff A. Desmoulière A. Gabbiani G. Heterogeneity of myofibroblast phenotypic features: an example of fibroblastic cell plasticity.Virchows Arch. 1994; 425: 3-24Crossref PubMed Scopus (403) Google Scholar;Gabbiani, 1998Gabbiani G. Evolution and clinical implications of the myofibroblast concept.Cardiovasc Res. 1998; 38: 545-548Crossref PubMed Scopus (132) Google Scholar;Powell et al., 1999Powell D.W. Mifflin R.C. Valentich J.D. Crowe S.E. Saada J.I. West A.B. Myofibroblasts. I. Paracrine cells important in health and disease.Am J Physiol Cell Physiol. 1999; 277: C1-C19Crossref PubMed Google Scholar;Serini and Gabbiani, 1999Serini G. Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation.Exp Cell Res. 1999; 250: 273-283Crossref PubMed Scopus (501) Google Scholar). They play a major role in inflammatory responses and in wound repair through their production of growth factors, cytokines, and other soluble mediators. Myofibroblasts also contribute to wound repair by contracting granulation tissue to reduce wound volume, and by repairing damaged extracellular matrix through the synthesis of collagen, fibronectin, and proteoglycans. When myofibroblast activity is not regulated properly, however, the result may be destructive tissue remodeling (Adler et al., 1989Adler K.B. Low R.B. Leslie K.O. Mitchell J. Evans J.N. Contractile cells in normal and fibrotic lung.Lab Invest. 1989; 60: 473-485PubMed Google Scholar;Hebda et al., 1993Hebda P.A. Collins M.A. Tharp M.D. Mast cell and myofibroblast in wound healing.Dermatol Clin. 1993; 11: 685-696Abstract Full Text PDF PubMed Google Scholar;Weber, 1997Weber K.T. Fibrosis, a common pathway to organ failure: angiotensin II and tissue repair.Semin Nephrol. 1997; 17: 467-491PubMed Google Scholar;Powell et al., 1999Powell D.W. Mifflin R.C. Valentich J.D. Crowe S.E. Saada J.I. West A.B. Myofibroblasts. I. Paracrine cells important in health and disease.Am J Physiol Cell Physiol. 1999; 277: C1-C19Crossref PubMed Google Scholar). Myofibroblasts appear to play a fundamental role in many diseases, including scleroderma (Sappino et al., 1990aSappino A.P. Masouye I. Saurat J.H. Gabbiani G. Smooth muscle differentiation in scleroderma fibroblastic cells.Am J Pathol. 1990; 137: 585-591PubMed Google Scholar;Kirk et al., 1995Kirk T.Z. Mark M.E. Chua C.C. Chua B.H. Mayes M.D. Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1.J Biol Chem. 1995; 270: 3423-3428Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), hepatic and pancreatic fibrosis (Bachem et al., 1998Bachem M.G. Schneider E. Gross H. et al.Identification, culture, and characterization of pancreatic stellate cells in rats and humans.Gastroenterology. 1998; 115: 421-432Abstract Full Text Full Text PDF PubMed Scopus (846) Google Scholar;Friedman, 2000Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to injury.J Biol Chem. 2000; 275: 2247-2250https://doi.org/10.1074/jbc.275.4.2247Crossref PubMed Scopus (1885) Google Scholar), and pulmonary fibrosis (Gauldie et al., 1999Gauldie J. Sime P.J. Xing Z. Marr B. Tremblay G.M. TGF-β gene transfer to the lung induces myofibroblast presence and pulmonary fibrosis.Curr Top Pathol. 1999; 93: 35-45Crossref PubMed Scopus (91) Google Scholar;Low, 1999Low R.B. Modulation of myofibroblast and smooth-muscle phenotypes in the lung.Curr Top Pathol. 1999; 93: 19-26Crossref PubMed Scopus (28) Google Scholar). Perhaps the most critical event in the normal process of wound repair and in the evolution of fibrosis is the appearance of activated myofibroblasts (Powell et al., 1999Powell D.W. Mifflin R.C. Valentich J.D. Crowe S.E. Saada J.I. West A.B. Myofibroblasts. I. Paracrine cells important in health and disease.Am J Physiol Cell Physiol. 1999; 277: C1-C19Crossref PubMed Google Scholar). These cells can apparently originate from several different sources, and a number of different activating factors may be involved. Mast cells are a potential source of some of those factors. One remarkably consistent finding is that myofibroblasts arise in a wide variety of settings concurrently with a local increase in the number of tissue mast cells (Choi and Claman, 1987Choi K.L. Claman H.N. Mast cells, fibroblasts, and fibrosis. New clues to the riddle of mast cells.Immunol Res. 1987; 6: 145-152Crossref PubMed Scopus (62) Google Scholar;Rothe and Kerdel, 1991Rothe M.J. Kerdel F.A. The mast cell in fibrosis.Int J Dermatol. 1991; 30: 13-16Crossref PubMed Scopus (33) Google Scholar;Hebda et al., 1993Hebda P.A. Collins M.A. Tharp M.D. Mast cell and myofibroblast in wound healing.Dermatol Clin. 1993; 11: 685-696Abstract Full Text PDF PubMed Google Scholar). The concept that biologically significant interactions occur between mast cells and fibroblasts is supported by the fact that numerous mediators produced by mast cells, such as histamine, tumor necrosis factor α (TNF-α), and tryptase, can influence fibroblast development, proliferation, and behavior. Tryptase, for example, is a serine protease unique to mast cells, and it can stimulate fibroblast proliferation (Ruoss et al., 1991Ruoss S.J. Hartmann T. Caughey G.H. Mast cell tryptase is a mitogen for cultured fibroblasts.J Clin Invest. 1991; 88: 493-499Crossref PubMed Scopus (344) Google Scholar;Hartmann et al., 1992Hartmann T. Ruoss S.J. Raymond W.W. Seuwen K. Caughey G.H. Human tryptase as a potent, cell-specific mitogen: role of signaling pathways in synergistic responses.Am J Physiol Lung Cell Mol Physiol. 1992; 262: L528-L534PubMed Google Scholar;Abe et al., 1998Abe M. Kurosawa M. Ishikawa O. Miyachi Y. Kido H. Mast cell tryptase stimulates both human dermal fibroblast proliferation and type I collagen production.Clin Exp Allergy. 1998; 28: 1509-1517Crossref PubMed Scopus (91) Google Scholar) and collagen synthesis (Cairns and Walls, 1997Cairns J.A. Walls A.F. Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts.J Clin Invest. 1997; 99: 1313-1321Crossref PubMed Scopus (268) Google Scholar;Gruber et al., 1997Gruber B.L. Kew R.R. Jelaska A. et al.Human mast cells activate fibroblasts. Tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis.J Immunol. 1997; 158: 2310-2317PubMed Google Scholar;Abe et al., 1998Abe M. Kurosawa M. Ishikawa O. Miyachi Y. Kido H. Mast cell tryptase stimulates both human dermal fibroblast proliferation and type I collagen production.Clin Exp Allergy. 1998; 28: 1509-1517Crossref PubMed Scopus (91) Google Scholar). As mast cells have such pleiotropic effects on fibroblasts, we explored the possibility that specific mast cell products might be able to induce normal human dermal fibroblasts to differentiate into functional myofibroblasts. Several different fibroblast cultures were used over the course of this work. One culture of normal human fibroblasts isolated from foreskin, AG01523C, was obtained from the National Institute on Aging, Aging Cell Culture Repository, Coriell Institute for Medical Research (Camden, NJ). These cells were fed minimum essential medium containing Earle's salts, and 26 mM sodium bicarbonate, 25 mM HEPES, pH 7.3 (catalog #42360, Life Technologies, Gaithersburg, MD), supplemented with nonessential amino acids (#11140, Life Technologies), 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 U per ml penicillin and 100 µg per ml streptomycin. Three other cultures of foreskin fibroblasts were prepared in this laboratory from enzyme-digested tissue. These cultures were fed Dulbecco's modified Eagle's medium (DMEM; #12100, Life Technologies), supplemented with 44 mM sodium bicarbonate, 1 mM sodium pyruvate, 10% heat-inactivated fetal bovine serum, 100 U per ml penicillin, 100 µg per ml streptomycin, and 250 ng per ml fungizone (Life Technologies). All fibroblast cultures were maintained in an atmosphere of 5% CO2 at 37°C. The human mast cell line, HMC-1 (Butterfield et al., 1988Butterfield J.H. Weiler D. Dewald G. Gleich G.J. Establishment of an immature mast cell line from a patient with mast cell leukemia.Leukemia Res. 1988; 12: 345-355Abstract Full Text PDF PubMed Scopus (635) Google Scholar), was obtained from J. H. Butterfield (Mayo Clinic, Rochester, MN). Cultures of HMC-1 cells were grown at 37°C, 5% CO2, in Iscove's modified Dulbecco's medium (IMDM; catalog #12200, Life Technologies), supplemented with 36 mM sodium bicarbonate, 1.2 mM monothioglycerol (Sigma-Aldrich, St. Louis, MO), 10% heat-inactivated fetal bovine serum, 100 U per ml penicillin, 100 µg per ml streptomycin, and 250 ng per ml fungizone. A freeze/thaw lysate of HMC-1 cells was prepared by the following procedure. HMC-1 cells were washed once with IMDM supplemented only with 36 mM sodium bicarbonate, resuspended in the same medium at a concentration of 5 × 106 cells per ml, and then sequentially frozen and thawed five times. Finally, the cell lysate was clarified by centrifugation to remove cell debris. The concentration of total protein in a typical lysate preparation was ≈0.5 µg per ml, as measured in the BCA protein assay (Pierce, Rockford, IL). Organotypic skin cultures were prepared as described previously (Garlick and Taichman, 1994bGarlick J.A. Taichman L.B. Fate of human keratinocytes during reepithelialization in an organotypic culture model.Lab Invest. 1994; 70: 916-924PubMed Google Scholar;Gruber et al., 1997Gruber B.L. Kew R.R. Jelaska A. et al.Human mast cells activate fibroblasts. Tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis.J Immunol. 1997; 158: 2310-2317PubMed Google Scholar). The culture system used here consisted of a collagen gel matrix, containing human foreskin fibroblasts and human dermal microvascular endothelial cells, supporting a stratified layer of human foreskin keratinocytes. In some cases, HMC-1 cells were also embedded in the collagen gel. Organotypic cultures, or rafts, were grown submerged for 5 d and then raised to the air interface for another 5 d to allow for complete stratification and differentiation of surface keratinocytes. We have used this skin-equivalent culture system previously to study interactions between mast cells and dermal fibroblasts (Meng et al., 1995Meng H. Marchese M.J. Garlick J.A. et al.Mast cells induce T-cell adhesion to human fibroblasts by regulating intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression.J Invest Dermatol. 1995; 105: 789-796Crossref PubMed Scopus (37) Google Scholar;Gruber et al., 1997Gruber B.L. Kew R.R. Jelaska A. et al.Human mast cells activate fibroblasts. Tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis.J Immunol. 1997; 158: 2310-2317PubMed Google Scholar) because it manifests epithelial stratification and other properties of living skin (Garlick and Taichman, 1994aGarlick J.A. Taichman L.B. Effect of TGF-β1 on re-epithelialization of human keratinocytes in vitro: an organotypic model.J Invest Dermatol. 1994; 103: 554-559Abstract Full Text PDF PubMed Scopus (64) Google Scholar). Transforming growth factor β1 (TGF-β1) purified from human platelets, recombinant human platelet-derived growth factor BB (PDGF-BB), and recombinant human TNF-α were purchased from R&D Systems. Histamine (#H-7375) was purchased from Sigma-Aldrich. Normal human foreskin fibroblasts (105 cells) were grown for 24 h on glass coverslips (18 mm × 18 mm) in complete medium containing serum, and then HMC-1 cells (106 cells) or TGF-β1 (5 ng per ml) was added to some of the coverslips and cultures were continued for an additional 48 h. The coverslips were washed once gently with phosphate-buffered saline (PBS) before the cells were fixed with ice-cold acetone for 15 min. The fixed cells were washed three times with PBS containing 0.05% Tween 20 (PBS/T) and then stained with a mouse monoclonal antibody specific for α-smooth muscle actin, clone 1A4 from Sigma-Aldrich, followed by fluorescein isthiocyanate conjugated goat antimouse IgG. To identify and locate mast cells, coverslips to which HMC-1 cells had been added were stained a second time using a monoclonal antibody against human tryptase (clone B2 from L.B. Schwartz, Virginia Commonwealth University, Richmond, VA) followed by a TRITC conjugate. Organotypic cultures containing fibroblasts and HMC-1 cells, or fibroblasts without HMC-1 cells, were prepared and grown in complete medium as described above. Paraffin-embedded sections from individual rafts were deparaffinized, rehydrated with PBS, fixed in acetone, blocked for 60 min with 1% nonfat milk in PBS, and then incubated with the 1A4 anti-α smooth muscle actin monoclonal antibody for 60 min at room temperature. After sections were washed three times with PBS, they were incubated with biotinylated horse antimouse IgG for 30 min. The slides were again washed before incubation with streptavidin-alkaline phosphatase conjugate for 30 min. After three more washes, the slides were developed with fast red substrate (BioGenex, San Ramon, CA), and then counterstained with hematoxylin. To quantitate the results of the staining, we counted the number of positive cells within a defined area (450 µm × 150 µm) on three different sections from each raft. This assay was adapted from a published protocol (Greiling and Clark, 1997Greiling D. Clark R.A. Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix.J Cell Sci. 1997; 110: 861-870Crossref PubMed Google Scholar). Fibroblast cultures at 80% confluence were harvested by treatment with 0.05% trypsin/0.02% ethylenediamine tetraacetic acid (Clonetics, Walkersville, MD). Trypsin was inactivated with trypsin neutralizing solution (Clonetics). The cells were resuspended in DMEM containing 2% bovine serum albumin (BSA) (DMEM/BSA) at a concentration of 1 × 106 cells per ml. The fibroblast suspension was mixed with neutralized collagen (Vitrogen 100, Celtrix Laboratories, Santa Clara, CA), 5 × concentrated DMEM, and sodium bicarbonate, and, in certain experiments, HMC-1 cells. The final volume was adjusted so that DMEM and sodium bicarbonate were present at normal strength and the concentration of collagen was 1.8 mg per ml. The number of fibroblasts in the final mixture was kept constant at 500,000 fibroblasts per ml. In certain experiments, the mixture also contained from 20,000 to 500,000 HMC-1 cells per ml. Aliquots (0.5 ml) of the cell mixture were added to wells of a 24-well tissue culture plate, which had been coated with 2% BSA, and the collagen was allowed to polymerize at 37°C. After a 2 h incubation, the round gels were gently detached from the plastic surface to allow contraction, 0.5 ml DMEM/BSA was added to each well, and the gels were incubated at 37°C for 1–72 h. The DMEM/BSA fed to the cells at this point, when specified, contained fetal bovine serum (10%), PDGF-BB (5 ng per ml), TGF-β1 (5 ng per ml), histamine (0.5–5 µg per ml), or TNF-α (10–100 ng per ml). It is important to emphasize that the DMEM/BSA medium used for the preparation and incubation of collagen gels did not routinely contain serum; serum was present, to stimulate contraction, only when specified. The amount of contraction was assessed by measuring collagen gel diameter after incubation for the indicated time, usually 72 h. The results presented here are expressed as a percentage of the contraction stimulated by PDGF-BB, which was used at the optimal dose established in preliminary experiments. Stimulation with PDGF-BB for 72 h typically caused the fibroblasts to contract the collagen gels to 50% or less of the size of control, unstimulated gels. Collagen gel contraction in all of these experiments was completely dependent upon the presence of fibroblasts. Gels prepared without cells, or with only HMC-1 cells, did not contract (data not shown). Tryptase inhibitors were tested by adding the inhibitors to the HMC-1 cell suspension (500,000 cells per ml) before the HMC-1 cells were mixed with the fibroblasts and collagen solution as described above. Bis(5-amidino-2-benzimidazolyl)methane (BABIM) was kindly provided by R. Tidwell (University of North Carolina, Chapel Hill, NC). Recombinant leech-derived tryptase inhibitor (rLDTI) was a generous gift from C. P. Sommerhof (Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, München, Germany). BABIM (Caughey et al., 1993Caughey G.H. Raymond W.W. Bacci E. Lombardy R.J. Tidwell R.R. Bis(5-amidino-2-benzimidazolyl)methane and related amidines are potent, reversible inhibitors of mast cell tryptases.J Pharmacol Exp Ther. 1993; 264: 676-682PubMed Google Scholar) and rLDTI (Sommerhoff et al., 1994Sommerhoff C.P. Söllner C. Mentele R. Piechottka G.P. Auerswald E.A. Fritz H. A Kazal-type inhibitor of human mast cell tryptase: isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis.Biol Chem Hoppe-Seyler. 1994; 375: 685-694Crossref PubMed Scopus (108) Google Scholar) are both specific inhibitors of tryptase proteolytic activity. The general protocol described above was modified slightly to measure the effect of HMC-1 cells on fibroblast contraction when the HMC-1 cells were cocultured with the fibroblast, but were not incorporated into the collagen gel. In those experiments, the porous membrane of a cell culture insert (catalog #3401, Corning Costar, Cambridge, MA) was used to separate the HMC-1 cells from the collagen gel, which contained only fibroblasts. The cup-shaped inserts were carefully positioned a few millimeters above the collagen gels, and 0.5 ml aliquots of the HMC-1 cell suspension (500,000 cells per ml) were dispensed directly into the inserts. Subsequent steps followed the standard protocol. Normal human dermal fibroblasts were seeded into six-well plates, 100,000 cells per well, and fed culture medium fully supplemented with serum and the other components listed above. After 24 h, cells were washed twice with PBS and fed serum-free medium containing 2 mg per ml BSA (SFA). After another 24 h, cells were washed twice with PBS, and then either treated as described below or fed SFA containing the specified additions and incubated for 72 h. Finally, the wells were washed three times with PBS and the cells in each well were lysed with 0.1 ml of Tris-buffered saline containing 1% sodium dodecyl sulfate (SDS) and protease inhibitors. The protein concentration in each sample was determined with the BCA protein assay using the supplied BSA as a standard. Equal amounts of protein from each sample, typically 1–10 µg, were separated under reducing conditions on a 10% Tris-HCl Ready Gel (Bio-Rad Laboratories, Hercules, CA) in SDS running buffer, and then the proteins were electrophoretically transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membrane blot was blocked with 1% nonfat milk in PBS/T and then incubated with the 1A4 anti-α smooth muscle actin monoclonal antibody, followed sequentially by biotinylated antimouse IgG (Vector Laboratories, Burlingame, CA) and alkaline phosphatase-streptavidin conjugate (Vector). After these incubations, the immunoblot was stained using BCIP/NBT substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and the stained immunoblot was quantitated using NIH Image. In certain experiments, fibroblast cultures were treated with tryptase immediately before the 72 h incubation in SFA medium. Tryptase solutions of 1–10 nM were prepared in SFA adjusted to pH 6.5 (Ren et al., 1997Ren S. Lawson A.E. Carr M. Baumgarten C.M. Schwartz L.B. Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12.J Immunol. 1997; 159: 3540-3548PubMed Google Scholar,Ren et al., 1998Ren S. Sakai K. Schwartz L.B. Regulation of human mast cell β-tryptase: conversion of inactive monomer to active tetramer at acid pH.J Immunol. 1998; 160: 4561-4569PubMed Google Scholar). Aliquots of these solutions were incubated at ambient temperature for 60 min with no additions, with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF Pefabloc SC; 2 mM), or with a combination of soybean trypsin inhibitor (10 µg per ml) and aprotinin (2 µg per ml). These three different tryptase solutions were then applied to fibroblast cultures prepared as described above. The cultures were incubated for 30 min at 37°C, washed twice with PBS, and finally fed SFA containing the specified additions and incubated for 72 h. Purified human lung tryptase, soybean trypsin inhibitor (#T-9003), and aprotinin (#A-4529) were obtained from Sigma-Aldrich. AEBSF was purchased from Roche Molecular Biochemicals (Indianapolis, IN). The goal of this research was to determine if HMC-1 mast cells can induce normal human dermal fibroblasts to differentiate into functional myofibroblasts. We monitored the differentiation process by assaying two important properties of the myofibroblast phenotype: α-smooth muscle actin expression and functional capacity to contract a collagen matrix. TGF-β1 was used as a positive control in many of these experiments because it potently induces α-smooth muscle actin expression (Desmoulière et al., 1993Desmoulière A. Geinoz A. Gabbiani F. Gabbiani G. Transforming growth factor-β1 induces α-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.J Cell Biol. 1993; 122: 103-111Crossref PubMed Scopus (1873) Google Scholar;Ronnov-Jessen and Peterson, 1993Ronnov-Jessen L. Peterson O.W. Induction of α-smooth muscle actin by transforming growth factor-β1 in quiescent human breast gland fibroblasts.Lab Invest. 1993; 68: 696-707PubMed Google Scholar) and markedly stimulates collagen gel contraction (Montesano and Orci, 1988Montesano R. Orci L. Transforming growth factor β stimulates collagen-matrix contraction by fibroblasts: implications for wound healing.Proc Natl Acad Sci USA. 1988; 85: 4894-4897Crossref PubMed Scopus (487) Google Scholar;Finesmith et al., 1990Finesmith T.H. Broadley K.N. Davidson J.M. Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors.J Cell Physiol. 1990; 144: 99-107Crossref PubMed Scopus (103) Google Scholar;Fukamizu and Grinnell, 1990Fukamizu H. Grinnell F. Spatial organization of extracellular matrix and fibroblast activity: effects of serum, transforming growth factor β, and fibronectin.Exp Cell Res. 1990; 190: 276-282Crossref PubMed Scopus (55) Google Scholar). In the first set of experiments normal human dermal fibroblasts were cultured on coverslips, either with or without HMC-1 cells, to determine if the presence of mast cells could induce fibroblasts to express α-smooth muscle actin. As shown in Figure 1, only a low level of α-smooth muscle actin was detected in unstimulated fibroblasts Figure 1a. The immunofluorescence is weak and comparatively diffuse. As expected, however, treating the fibroblasts with TGF-β1 strongly induced α-smooth muscle actin expression Figure 1c. The cytoskeleton in the spindle-shaped fibroblasts is much more brightly stained and many individual actin filaments are discernible. Notably, fibroblasts cocultured with HMC-1 cells were also strongly induced to express α-smooth muscle actin Figure 1b. The effect of HMC-1 cells on α-smooth muscle actin expression by fibroblasts was also studied using an organotypic (Garlick and Taichman, 1994bGarlick J.A. Taichman L.B. Fate of human keratinocytes during reepithelialization in an organotypic culture model.Lab Invest. 1994; 70: 916-924PubMed Google Scholar), or skin-equivalent, culture system. Using this culture system for an earlier study (Gruber et al., 1997Gruber B.L. Kew R.R. Jelaska A. et al.Human mast cells activate fibroblasts. Tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis.J Immunol. 1997; 158: 2310-2317PubMed Google Scholar), we demonstrated that fibroblast synthesis of type α1(I) procollagen mRNA was substantially increa
Referência(s)