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Am J Physiol Lung Cell Mol Physiol 296: L236-L247, 2009. First published December 5, 2008; doi:10.1152/ajplung.90462.2008
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MMP dependence of fibroblast contraction and collagen production induced by human mast cell activation in a three-dimensional collagen lattice

Alexander Margulis, Karl H. Nocka, Nancy L. Wood, Stanley F. Wolf, Samuel J. Goldman, and Marion T. Kasaian

Department of Inflammation, Wyeth Research, Cambridge, Massachusetts

Submitted 28 August 2008 ; accepted in final form 29 November 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cell-fibroblast interactions may contribute to fibrosis in asthma and other disease states. Fibroblast contraction is known to be stimulated by coculture with the human mast cell line, HMC-1, or by mast cell-derived agents. Matrix metalloproteinases (MMPs) can also mediate contraction, but the MMP-dependence of mast cell-induced fibroblast contractility is not established, and the consequences of mast cell activation within the coculture system have not been fully explored. We demonstrate that activation of primary human mast cells (pHMC) with IgE receptor cross-linking, or activation of HMC-1 with C5a, enhanced contractility of human lung fibroblasts in a three-dimensional collagen lattice system. This enhanced contractility was inhibited by the pan-MMP antagonist, batimastat, and was transferrable in the conditioned medium of activated mast cells. Exogenously added MMPs promoted gel contraction by mediating the proteolytic activation of latent transforming growth factor-β (TGF-β). Consistent with this, fibroblast contraction induced by mast cell activation was enhanced by addition of excess latent TGF-β to the cultures. Batimastat inhibited this response, suggesting that MMPs capable of activating latent TGF-β were released following mast cell activation in coculture with fibroblasts. Collagen production was also stimulated by activated mast cells in an MMP-dependent manner. MMP-2 and MMP-3 content of the gels increased in the presence of activated mast cells, and inhibition of these enzymes blocked the contractile response. These findings demonstrate the MMP dependence of mast cell-induced fibroblast contraction and collagen production.

transforming growth factor-β; C5a; immunoglobulin E receptor

SUBEPITHELIAL FIBROSIS is a hallmark of human asthma characterized by increased thickness and density of the airway wall due to the fibroblast-mediated deposition of extracellular proteins (51). These changes may be associated with reduced airway elasticity, increased contractile responses, and progressive loss of lung function (51). Lung mast cell numbers are increased in asthma and correlate with increased thickness of the extracellular matrix in asthmatic airways (3). Lung mast cells are found in close proximity to fibroblasts (26), which are an important source of the essential mast cell growth and survival agent, stem cell factor (SCF) (49). Direct cell-cell contacts can be maintained through interaction of fibroblast surface SCF with mast cell surface c-kit and through homotypical interactions of spermatogenic immunoglobulin superfamily (SgIGSF) on each cell type (33). In an animal model of asthma, mast cell-deficient W/Wv mice have reduced subepithelial fibrosis, and the fibrotic response can be restored by reconstitution of mast cells (39).

Mast cells are found at sites of tissue fibrosis not only in asthma, but also in other fibrotic diseases and in healing wounds (17, 25). A role for mast cells in contributing to fibrosis is supported by data from in vitro coculture systems in which coincubation of human mast cells with fibroblasts enhances mast cell production of proinflammatory cytokines, increases fibroblast proliferation, and drives fibroblast synthesis of collagen (7, 23, 24). To assay contractility, fibroblasts can be embedded in a three-dimensional collagen gel lattice. Fibroblast contraction shrinks the gel, leading to formation of a dense fibrous matrix (6). Embedding mast cells along with the fibroblasts increases fibroblast contractility (5, 46, 59). Although a number of mast cell-derived mediators have been proposed to contribute to this response, including cytokines, histamine, and the proteolytic enzymes, tryptase and chymase (2, 52, 56), the specific mechanisms by which mast cells enhance fibrotic responses are not completely understood. In some reports, the presence of mast cells was sufficient to elicit a response, and neither mast cell degranulation nor the supernatants of degranulated mast cells resulted in further enhancement of fibroblast contraction (43). Thus the requirement for specific mast cell activation in these coculture models remains unclear.

Transforming growth factor-β (TGF-β) is a key profibrotic agent, expressed by multiple structural and inflammatory cells in the inflamed airway, including epithelial cells, macrophages, mast cells, and fibroblasts (27). Secreted and sequestered in the extracellular matrix in latent (precursor) form, TGF-β may be proteolytically processed by a variety of extracellular enzymes. Once activated, TGF-β is a potent inducer of fibroblast contractility in the collagen gel matrix (14). Enzymes with known TGF-β processing activity include the gelatinases, matrix metalloproteinase (MMP)-9 and MMP-2 (13, 60), the stromelysin, MMP-3 (37), the collagenase MMP-13 (11), and membrane type 1 (MT1)-MMP (30). These enzymes may be expressed by fibroblasts (18, 55) or mast cells (16, 29, 50). Increased levels of activated MMP-9 and MMP-2 are found in lung and sputum of asthmatics and have been associated with severity of the asthmatic response (10, 40, 57). MMP-3 has been localized to sites of mast cell infiltration in the asthmatic lung (12), and its expression can be induced in fibroblasts under inflammatory conditions (55). Thus MMPs derived from mast cells or fibroblasts have the potential to regulate fibrotic responses.

Although mast cells have been shown to influence the activation of fibroblast-derived MMPs (1, 7, 23), the MMP-dependence of mast cell-induced fibroblast contraction has not been clearly demonstrated, and the potential contribution of MMP-dependent cleavage of TGF-β in promoting contraction has not been examined. We addressed these questions using two model systems. HMC-1 is a widely used mast cell line that has been shown to promote the contractility of primary human lung and dermal fibroblasts (22, 24, 46, 59), but the role of MMPs in mediating this response is unknown. HMC-1 cells lack the high-affinity IgE receptor but can be activated by exposure to the anaphylatoxin, C5a (58). To confirm that the response of the cell line was similar to that of primary cells, we derived primary human mast cells (pHMC) from progenitors in peripheral blood and activated them by IgE receptor cross-linking. Mast cells were cocultured with human lung fibroblasts (HFL-1), and effects on fibroblast contractility and collagen synthesis were investigated. Our findings show that soluble mediators secreted from activated mast cells induce MMP-dependent fibroblast contractility. Contraction was enhanced in the presence of latent TGF-β and was accompanied by increased de novo synthesis of collagen. These findings provide a potential mechanism by which mast cells may contribute to fibrosis in asthma and other disease states.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. HFL-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were grown in DMEM containing 10% fetal bovine serum (HyClone, Logan, UT). HMC-1 (gift from Dr. J. Butterfield, Mayo Clinic, Rochester, MN) was cultured in Iscove's medium with 10% defined iron-supplemented calf serum (HyClone) and 1.2 mM {alpha}-thioglycerol (Sigma, St. Louis, MO). All cells were maintained at 37°C in 5% CO2.

pHMC generation. To generate primary mast cells, CD34+ progenitors were purified from heparinized human blood (Massachusetts General Hospital, Boston, MA) using CD34-coated magnetic beads (Miltenyi Biotec, Auburn, CA). Cells were seeded in Iscove's medium containing L-glutamine (2 mM), 30% charcoal-treated fetal bovine serum (Sigma), 50 µg/ml iron-saturated holo-transferrin (Sigma), 1x penicillin-streptomycin (Sigma), 10–5 M 2-mercaptoethanol (Sigma), 100 ng/ml SCF (Invitrogen, Carlsbad, CA), 3% 20x concentrated conditioned medium generated from HCC2175 BL lymphoblastoid cell line (ATCC), and 10 pg/ml GM-CSF (R&D Systems, Minneapolis, MN) at 104 cells per milliliter. After the first 2 wk of culture, cells were maintained at a density of 1.5 x 105 cells per milliliter, and half of the medium was replaced weekly. At 6 wk, the culture was depleted of macrophages by magnetic bead separation using CD14 antibody (Invitrogen). Mast cell phenotype and purity were evaluated by Wright stain and by flow cytometry using antibodies to CD14 and CD117 (BD Pharmingen, San Jose, CA) with mast cells identified as CD117+/CD14–. Cultures used in these experiments contained >95% mast cells.

Type I collagen gel contraction. Collagen lattices were prepared by mixing neutralized bovine type I collagen (Organogenesis, Canton, MA) with HFL-1 at 2.5 x 105 cells per milliliter in 24-well plates. HMC-1 (2.5 x 105 cells per milliliter) or pHMC (1 x 105 cells per milliliter) were added to the mixture as indicated, and gels were solidified overnight at 37°C 10% CO2. Polymerized gels were gently released into a 6-well plate that contained 3 ml of serum-free DMEM and treated with 10 nM C5a (Sigma), 5 ng/ml latent or activated TGF-β (R&D Systems), 400 ng/ml catalytic domain of MMP-2 or MMP-3 (Wyeth Research), 5 or 0.67 µM batimastat (Wyeth Research), 2 µM MMP-2 inhibitor 2-[isopropoxy-(1,1'-biphenyl-4-ylsulfonyl)-amino]-N-hydroxyacetamide (EMD Biosciences, Gibbstown, NJ), or 0.67 µM MMP-3 inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH; EMD Biosciences) as indicated. For coculture with primary mast cells, the incubation medium contained 50 ng/ml SCF (R&D Systems). To assay the role of IgE-dependent mast cell activation, cells were sensitized with 0.1 µg/ml human IgE (Millipore, Billerica, MA) for 1 h before the addition of 10 µg/ml anti-IgE (KPL, Gaithersburg, MD). Gels were photographed 48 h prorelease using a digital camera. The degree of collagen gel contraction was quantified by measuring the gel area using LaserPix software (Bio-Rad, Hercules, CA). Approximately 40% of the original area was found to be the maximum contraction induced under these experimental conditions. Data are shown on a scale of 40–100%.

In some cases, collagen gels were incubated with conditioned medium from activated mast cells. To generate conditioned medium, HMC-1 cells were seeded in 24-well plates at 1 x 106 cells per well in 1 ml of serum-free Iscove's medium containing {alpha}-thioglycerol and treated for 24 h with activation agents as indicated. pHMC were seeded in a round-bottom 96-well plate at 5 x 104 cells per well in 200 µl of serum-free Iscove's medium containing 50 ng/ml SCF and treated for 24 h with activation agents as indicated.

Collagen assembly analysis. To measure hydroxyproline content, collagen gels were cultured for 72 h and subsequently lysed in a buffer containing Nonidet P-40 and acetic acid for 72 h. Hydrochloric acid was added to the lysate, and gels were hydrolyzed overnight at 110°C. The resulting hydrolyzates were dried by speed vacuum overnight, reconstituted in distilled water, and then oxidized by shaking with a mixture of isopropanol, sodium acetate, citric acid, and chloramine T. Color was detected by adding p-dimethylaminobenzaldehyde in perchloric acid, diluted in isopropanol, and incubated at 70°C. All reagents were obtained from Sigma. Absorbance was read at 550 nm.

Mast cell activation. Calcium mobilization was assayed in HMC-1 cells loaded for 30 min at 30°C with fluo-3 (Invitrogen). The cells were washed in PBS and warmed to 37°C, and time-based data acquisition was initiated by flow cytometry (FACScan, Becton-Dickinson, San Jose, CA). At the indicated time point, acquisition was interrupted, and C5a (10 nM; Sigma) was added. Intracellular free calcium content was monitored as an increase in fluorescence. For analysis of IL-3 production, HMC-1 cells were incubated for 24 h at 37°C in media containing 10 nM C5a. Supernatants were assayed for IL-3 content by ELISA (Invitrogen). For pHMC activation studies, cells were incubated overnight with 0.1 µg/ml human IgE, washed into serum-free Iscove's medium with 0.005% human serum albumin (Sigma), and then treated with 10 µg/ml anti-human IgE (KPL). Supernatants were collected after 30 min for quantitation of histamine by ELISA (Coulter Immunotech, Dardilly, France). Alternatively, IgE-loaded pHMC were challenged with 10 µg/ml anti-human IgE in serum-free media with 50 ng/ml SCF for 24 h, and supernatants were assayed for IL-8 by ELISA (limit of detection was 8 pg/ml; R&D Systems).

Gelatin zymography. Collagen gels embedded with HFL-1 alone or cocultures of HFL-1 and HMC-1 were incubated in serum-free DMEM with C5a (10 nM; Sigma) and latent TGF-β (5 ng/ml; R&D Systems), as indicated, in a 37°C incubator for 48 h. Media surrounding the gels was concentrated 10-fold using Nanosep Omega 10K concentrators (Pall, Ann Arbor, MI). An equal volume of concentrated media from each sample was subjected to electrophoresis under reducing conditions in 10% precast polyacrylamide gels containing gelatin (Bio-Rad). After electrophoresis, gels were incubated in renaturing buffer containing Triton X-100 (Invitrogen) for 30 min at room temperature. The gels were then incubated in Novex Zymogram Developing Buffer (Invitrogen) for 30 min at room temperature and for 48 h at 37°C. After incubation, gels were stained in SimplyBlue SafeStain (Invitrogen).

Fluorokine assay. Collagen gels embedded with cell types as described above were treated as indicated in a 37°C incubator for 48 h. Media surrounding the gels was concentrated 10-fold using Nanosep Omega 10K concentrators (Pall). Equal volumes of the concentrated samples were subjected to Multiplex microbead-based immunoassay using the Fluorokine human MMP Multianalyte profiling kit (R&D Systems) and analyzed on a Luminex 100 instrument (Luminex, Austin, TX) using StarStation software (Applied Cytometry Systems, Sacramento, CA). Limits of detection for MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, and MMP-13 were 6, 38, 3, 30, 12, 11, 2, and 30 pg/ml, respectively.

TGF-β assay. Collagen gels were embedded with HFL-1 in the presence or absence of mast cells and cultured with the indicated agents 48 h at 37°C. Media surrounding the gels was collected, concentrated 10-fold using Nanosep Omega 10K (Pall), acidified to activate TGF-β, and assayed for TGF-β content using a specific ELISA (R&D Systems). In some cases, the assay was performed without acidification to quantify active TGF-β. The limit of assay sensitivity was 4 pg/ml.

Statistical analysis. Collagen gel contraction data are presented as the means ± SD of 3–10 experiments, each containing 3–4 replicate gels. ELISA data are presented as the means ± SD of triplicate cultures per treatment. Student's t-test was used to analyze differences between two groups. Time-course curves were compared by ANOVA. A difference of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mast cell activation models. The human mast cell line, HMC-1, can be activated in an IgE-independent manner in response to the anaphylatoxin, C5a (58). HMC-1 undergo calcium mobilization (Fig. 1A) and IL-3 production (Fig. 1B) in response to C5a. To study IgE-dependent mast cell activation responses, pHMC were derived from CD34+ progenitors in peripheral blood. These cells displayed mast cell morphology, with dense intracellular granules (Fig. 1C), and bound to human IgE (Fig. 1D). In response to IgE receptor cross-linking, the pHMC underwent degranulation (Fig. 1E) and produced cytokines (Fig. 1F).


Figure 1
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  		Fig. 1. Mast cell activation responses. A: human mast HMC-1 cells were loaded with the calcium indicator dye, fluo-3, and fluorescence was analyzed by flow cytometry. At the time point indicated by the arrow, analysis was interrupted, and C5a was added to the cells. Intracellular free calcium levels were monitored as green fluorescence. B: HMC-1 cells were incubated overnight in the presence or absence of C5a (10 nM), and supernatants were assayed for IL-3 by ELISA. n.d., not detectable. C: morphology of primary human mast cells (pHMC), as determined by Wright-Giemsa staining. D: flow cytometric analysis of IgE binding by pHMC. Cells were cultured overnight in the absence (left) or presence (right) of human IgE (0.1 µg/ml) and then stained with FITC-labeled antibody to human IgE (open) or isotype control antibody (filled). E: pHMC were incubated overnight with IgE (0.1 µg/ml) and then challenged with anti-human IgE (10 µg/ml) for 30 min. Supernatants were analyzed for histamine content. F: supernatants of pHMC activated in response to IgE + anti-IgE were collected 24 h postactivation and assayed for concentration of IL-8 by ELISA.

 
Activated mast cells promote contractility of fibroblasts in collagen gels. The effects of mast cell activation on fibroblast contractile responses were studied by coembedding either HMC-1 cells or pHMC with HFL-1 fibroblasts in a three-dimensional collagen lattice. Fibroblast contraction resulted in shrinkage of the gel, quantitated as a decrease in total gel surface area after 48 h. Gels containing HFL-1 alone did not contract in response to the mast cell activating agents, C5a (Fig. 2A), or IgE and anti-IgE (Fig. 2B). When gels coembedded with HFL-1 and HMC-1 were treated with C5a (Fig. 2A) or those containing HFL-1 and pHMC were treated with IgE and anti-IgE (Fig. 2B), however, significant gel shrinkage was seen (P < 0.005), suggesting that mast cell activation resulted in contraction of HFL-1. IgE alone produced a low level of contraction in the presence of pHMC (P < 0.005), and IgE receptor cross-linking produced a greater response (Fig. 2B). Gels embedded with mast cells in the absence of HFL-1 did not contract under these conditions (data not shown).


Figure 2
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  		Fig. 2. Activated mast cells release soluble factors that contribute to human lung fibroblast (HFL-1) contractility. Collagen lattices were seeded with HFL-1 in the absence or presence of HMC-1 cells (A) or pHMC (B). Gels were treated with media alone or with the appropriate mast cell activating agent for 48 h at 37°C. A 3rd group of gels was seeded with HFL-1 only and treated for 48 h at 37°C with conditioned medium (c.m.) harvested from resting or activated mast cells. Gel surface area was quantitated and expressed as a percentage of the area of a lattice that contained no cells. Data shown are the means ± SD across 3–5 experiments, each of which included triplicate gels per condition. *P < 0.005 for treatment with mast cell activating agents compared with media.

 
Contraction-inducing activity can be transferred in conditioned medium of activated mast cells. To address whether direct cell-cell contact between mast cells and fibroblasts was required for the contractile response, conditioned medium was collected after 24 h from HMC-1 cells activated with C5a or from pHMC activated by IgE receptor cross-linking. Treatment of lattices containing HFL-1 with conditioned medium from C5a-activated HMC-1 increased contraction over that seen for gels exposed to the conditioned medium from resting HMC-1 (Fig. 2A; P < 0.005), indicating the contribution of secreted agent(s) to the fibroblast contractile response. Treatment of gels containing HFL-1 with conditioned medium from pHMC activated with IgE + anti-IgE also produced a significant increase in contraction over that seen with conditioned medium from resting pHMC (Fig. 2B; P < 0.005).

MMPs contribute to the induction of fibroblast contraction by activated mast cells. TGF-β is a well-characterized profibrotic agent that can be produced by multiple cell types including mast cells (27). TGF-β is produced in latent form and processed extracellularly by a variety of enzymes including MMPs. To address whether mast cells promote fibroblast contraction through an MMP-dependent mechanism, excess latent TGF-β was added to collagen gels seeded with HFL-1 and HMC-1, and the MMP dependence of contractile responses was assessed. Either C5a alone or latent TGF-β alone produced a significant contractile response (P < 0.01). The combination of C5a and latent TGF-β resulted in more pronounced contraction than was seen with either agent alone (P < 0.0005; Fig. 3A). Similarly, anti-IgE or latent TGF-β induced significant contraction in gels coembedded with HFL-1 and pHMC (P < 0.01), whereas the combination induced a stronger response than either agent alone (P < 0.0005; Fig. 3C). In all cases, the pan-selective MMP antagonist, batimastat, blocked contraction, such that gel area in the presence of mast cells no longer differed significantly from the area of gels embedded with HFL-1 alone (Fig. 3, A and C).


Figure 3
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  		Fig. 3. Contractile responses of HFL-1 fibroblasts to mast cell activation can be enhanced by matrix metalloproteinase (MMP)-dependent cleavage of latent transforming growth factor-β (TGF-β). HFL-1-embedded collagen gels were seeded with: HMC-1 cells treated with the indicated agents (A); conditioned medium harvested from resting or activated HMC-1 cells (B); pHMC treated with the indicated agents (C); and conditioned medium harvested from resting or activated pHMC (D). Batimastat was used at 5 µg/ml. After 48-h incubation at 37°C, gel surface area was quantitated and expressed as a percentage of the area of a lattice that contained no cells. Data shown are the means ± SD across 3–6 experiments, each of which included triplicate gels per condition. *P < 0.01 for gels with mast cells or mast cell conditioned media vs. HFL-1 only. aIgE, anti-IgE.

 
MMPs can exist in cell-associated or secreted form (44, 60). To determine whether a secreted MMP was contributing to contractile activity in this model, supernatant (conditioned medium) was collected from HMC-1 cells treated for 24 h with C5a and latent TGF-β or from pHMC treated for 24 h with IgE receptor cross-linking agents and latent TGF-β. Conditioned medium was transferred to collagen gels embedded with HFL-1 cells and incubated for 48 h in the presence or absence of batimastat. Increased contraction was seen in the presence of conditioned medium, and the most potent effect was produced by supernatants of cells treated with the combination of mast cell activating agent and latent TGF-β (Fig. 3, B and D; P < 0.01). In all cases, activity was blocked with batimastat.

Activation of latent TGF-β promotes HFL-1 contraction. To determine whether TGF-β were being endogenously produced in the coculture system, supernatants of collagen gels embedded with HFL-1 and human mast cells were examined. To be detected, the TGF-β measured in these samples was activated by acid treatment before analysis and consisted of the latent form. Gels embedded with HFL-1 alone did not produce detectable TGF-β under resting conditions and produced low levels following 48 h treatment with C5a (Fig. 4A; P < 0.05). In gels coembedded with HFL-1 and HMC-1, C5a treatment induced the release of higher concentrations of TGF-β into the supernatant (Fig. 4A; P < 0.05 compared with media treatment or HFL-1 alone). Gels coembedded with HFL-1 and pHMC released high concentrations of TGF-β even under resting conditions, and TGF-β release was further enhanced following IgE receptor cross-linking (Fig. 4B; P < 0.05 compared with media treatment or HFL-1 alone). No TGF-β was detectable in cultures of HMC-1 or pHMC alone, either under resting or activated conditions (data not shown). These findings suggest that in cocultures of HFL-1 with HMC-1 or pHMC, TGF-β was produced.


Figure 4
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  		Fig. 4. TGF-β is produced in coculture and promotes HFL-1 contraction. Collagen gels were embedded with HFL-1 alone or HFL-1 and HMC-1 (A) or embedded with HFL-1 alone or with HFL-1 and pHMC (B). Gels were incubated for 48 h with media, C5a (10 nM), or IgE (0.1 µg/ml) and anti-IgE (10 µg/ml), as indicated. Media surrounding the gels were acidified to activate latent TGF-β and assayed by ELISA. *P < 0.05 for C5a or anti-IgE treatment compared with media; ^P < 0.05 for HFL-1 + HMC-1 or pHMC compared with HFL-1 alone. Collagen gels embedded with HFL-1 and HMC-1 (C) or HFL-1 and pHMC (D) were treated with C5a or IgE and anti-IgE in the presence or absence of latent TGF-β (5 ng/ml) for 48 h. Media surrounding the gels were assayed without acidification for active TGF-β. *P < 0.005 compared with latent TGF-β alone; ^P 0.05 for treatment with batimastat (5 µM) compared with no inhibitor. Dashed line indicates the limit of assay sensitivity (4 pg/ml). E: collagen gels containing HFL-1 alone were treated with active TGF-β (5 ng/ml) as positive control, latent TGF-β (5 ng/ml), MMP catalytic domain (400 ng/ml), or the combination of latent-TGF-β and MMP. After 48-h incubation at 37°C, gel surface area was quantitated and expressed as a percentage of the area of a lattice that contained no cells. *P < 0.005 compared with latent TGF-β alone or MMP alone. ^P < 0.005 compared with media control. F: the media surrounding collagen gels treated as in E was assayed without acidification for active TGF-β. *P < 0.005 compared with latent TGF-β alone or MMP alone. Data shown are means ± SD of 3 determinations.

 
Active TGF-β was also assayed, by omitting acidification before analysis. C5a treatment of gels embedded with HMC-1 and HFL-1, or IgE receptor cross-linking agents added to gels embedded with pHMC and HFL-1, did not result in detectable release of active TGF-β to the media (Fig. 4, C and D). This may have been because concentrations produced were undetectable or because the cells used any active TGF-β that was produced. On addition of excess latent TGF-β, however, the active form could be detected. Active TGF-β concentrations were increased following HMC-1 treatment with C5a and latent TGF-β over levels seen with latent TGF-β alone (Fig. 4C; P < 0.005) in accordance with the increased collagen gel contraction seen under these conditions (Fig. 3A). There was also a trend toward increased release of active TGF-β following IgE receptor cross-linking of pHMC in the presence of latent TGF-β, but this did not reach statistical significance over concentrations seen with latent TGF-β alone (Fig. 4D). To evaluate whether MMPs contributed to production of active TGF-β under these conditions, batimastat was added. Batimastat inhibited the release of active TGF-β in HMC-1 containing gels treated with C5a and latent TGF-β (Fig. 4C; P = 0.05) and showed a trend toward inhibition of active TGF-β release with pHMC treated with IgE receptor cross-linking and latent TGF-β (Fig. 4D).

Collagen gels embedded with HFL-1 alone had a strong contractile response to active TGF-β and a greatly attenuated, but significant, response to latent TGF-β. (Fig. 4E; P < 0.005). To confirm that MMP activation of latent TGF-β could promote contraction in this system, collagen gels containing HFL-1 alone were treated with the active catalytic domains of MMP-2 or MMP-3. The MMPs themselves induced marginal but significant contraction (P < 0.005 vs. media). In the presence of latent TGF-β, however, both MMP-2 and MMP-3 produced a strong contractile response (Fig. 4E; P < 0.005 compared with latent TGF-β alone or MMP alone). Analysis of conditioned media from these gels confirmed accumulation of active TGF-β on exposure to the combination of latent TGF-β and catalytically active MMP-2 or MMP-3 (Fig. 4F).

Time course of collagen gel contraction in response to activating agents. Mast cell activation involves short-term mediator release, including degranulation and eicosanoid production responses, which occur within minutes of exposure to the activating agent. This is followed by production of cytokines, chemokines, and other mediators over several hours. We compared the activity of conditioned media collected from HMC-1 cells 2 h after activation with that of media collected at 24 h. The 2-h conditioned medium did not mediate fibroblast contraction in collagen gels (data not shown). This indicates that the agent(s) responsible was not released on short-term activation of the mast cells.

To further explore this response, we assayed the time course of collagen gel contraction. Gels embedded with HFL-1 alone demonstrated robust contraction in response to active TGF-β with the majority of the response occurring within the first 24 h (Fig. 5, A and C). Collagen gels coembedded with HFL-1 and HMC-1 treated with either C5a alone or latent TGF-β alone displayed a low-level contractile response between 3 and 24 h with only a marginal further increase at 48 h postactivation. In response to the combination of C5a and latent TGF-β, however, contraction continued to increase between the 24- and 48-h time points (Fig. 5B). A similar time course was observed for gels coembedded with HFL-1 and pHMC and treated the combination of anti-IgE and latent TGF-β (Fig. 5D).


Figure 5
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  		Fig. 5. Time course of collagen gel contraction in the presence or absence of mast cells. Collagen gels embedded with HFL-1 alone (A), HFL-1 + HMC-1 (B), HFL-1 alone (C), or HFL-1 + pHMC (D) were treated with the indicated agents for 3, 24, or 48 h. HFL-1 alone were treated with active TGF-β as a control. Gel surface area was quantitated and expressed as a percentage of the area of a lattice that contained no cells. Data shown are means ± SD for 3–4 gels assayed per time point. *P < 0.01 for variation from media-only time course by 2-way repeated-measures ANOVA.

 
MMPs contribute to the induction of fibroblast collagen production by activated mast cells. In addition to contractile responses, HFL-1 cells produce collagen on treatment with TGF-β (4). To characterize the influence of mast cells on extracellular matrix production by fibroblasts, collagen gels were embedded with HFL-1 in the presence of HMC-1 or pHMC and then treated for 24 h with mast cell activating agents and assayed for hydroxyproline content. No change in collagen content was seen when lattices embedded with HFL-1 alone were treated with C5a, IgE receptor cross-linking agents, or latent TGF-β, compared with treatment with media alone (Fig. 6, A and B). Under coculture conditions, the presence of HMC-1 did not stimulate de novo collagen synthesis by HFL-1 in the absence of mast cell activators (Fig. 6A), whereas pHMC resulted in a significant increase in collagen content (Fig. 6B; P < 0.001). In response to the combination of latent TGF-β and mast cell activating agents, hydroxyproline content of the lattices was further increased (Fig. 6A and B; P < 0.01 compared with single agents alone). For both HMC-1 and pHMC, batimastat blocked the increase in collagen synthesis in response to latent TGF-β in combination with mast cell activating agents (Fig. 6A and B; P < 0.05), suggesting that MMP-dependent cleavage of latent TGF-β leads to increased matrix deposition by HFL-1 in response to mast cell activation.


Figure 6
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  		Fig. 6. Activated mast cells increase MMP-dependent collagen synthesis by HFL-1 fibroblasts. Collagen gels containing HFL-1 alone or cocultured with HMC-1 (A) or pHMC (B) were treated as indicated. Gels containing HFL-1 were treated with conditioned media collected from HMC-1 (C) or pHMC (D) were activated as indicated. Gels were incubated for 48 h in the presence or absence of batimastat (5 µM) and then assayed for hydroxyproline content (micrograms per gel). Data shown are the means ± SD across 2–3 experiments, each of which included triplicate gels per condition. *P < 0.001 compared with the corresponding media control. ^P < 0.05 for batimastat-treated compared with untreated.

 
Conditioned media of HMC-1 activated by C5a and latent TGF-β also increased the hydroxyproline content of the gel lattice (Fig. 6C; P < 0.001). Inhibition by batimastat (P < 0.05) suggested a role for MMP-dependent cleavage of latent TGF-β in this response. Conditioned media of pHMC activated by IgE receptor cross-linking in the presence of latent TGF-β also induced collagen production by HFL-1 cells (P < 0.001), and this response was significantly reduced by batimastat (Fig. 6D; P < 0.05).

MMP-2 and MMP-3 are induced under conditions supporting HFL-1 contraction. To determine which MMPs were present in cultures of HFL-1 and mast cells under the experimental conditions examined, collagen gels embedded with HFL-1 alone, HFL-1 and HMC-1, or HFL-1 and pHMC were cultured for 48 h under resting conditions or in the presence of mast cell activating agents. Media surrounding the gels were concentrated 10-fold, and MMP content was evaluated by fluorokine assay. MMP-2 content was increased in supernatants of gels embedded with HFL-1 and HMC-1 following treatment with C5a and latent TGF-β (Fig. 7A; P < 0.05) and in supernatants of gels embedded with HFL-1 and pHMC-1 following treatment with anti-IgE and latent TGF-β (Fig. 7B; P < 0.05). MMP-3 was also detectable under all conditions, increased on treatment of HMC-1 embedded gels with C5a (P < 0.05), and further increased with C5a and latent TGF-β (Fig. 7C; P < 0.05). MMP-3 was also induced on treatment of pHMC-embedded gels with anti-IgE (P < 0.05) and further increased with anti-IgE and latent TGF-β (Fig. 7D; P < 0.05). A fluorokine multiplex assay format revealed strong expression of MMP-1 associated with the HFL-1 cells, but this was not modulated by the presence of HMC-1 or treatment with C5a and latent TGF-β (data not shown). Trace amounts of MMP-9 were released by pHMC activated by IgE receptor cross-linking under coculture conditions but were not further modulated by latent TGF-β (data not shown). MMP-7, MMP-8, MMP-12, and MMP-13 were undetectable under all treatment conditions (data not shown).


Figure 7
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  		Fig. 7. Expression of MMPs in coculture systems. HFL-1 cells were cultured in the presence or absence of HMC-1 or pHMC for 48 h with the indicated agents. Concentrated samples were tested for content of MMP-2 and MMP-3 by fluorokine assay. A: MMP-2 release from gels embedded with HFL-1 ± HMC-1. B: MMP-2 release from gels embedded with HFL-1 ± pHMC. C: MMP-3 release from gels embedded with HFL-1 ± HMC-1. D: MMP-3 release from gels embedded with HFL-1 ± pHMC. *P < 0.05 compared with media control. E: HFL-1 cells were cultured in the presence of HMC-1 cells or pHMC for 48 h with the indicated agents. Media surrounding the gels were concentrated 10-fold and assayed for gelatinase activity by zymography. Numbers indicate the relative quantitation of band intensities for pro-MMP-2 and the processed form, with intensity of pro-MMP-2 in the media-treated HFL-1-only control sample set at 100. lat TGF-β, latent TGF-β.

 
To confirm the expression of MMP-2, media surrounding the gels were concentrated 10-fold and assayed for gelatinase activity by zymography. The unprocessed, full-length (pro-) form of MMP-2 was detected under all conditions and increased on treatment of HFL-1-HMC-1 cocultures with C5a and latent TGF-β (Fig. 7E). Trace amounts of processed MMP-2 were also detectable, most prominently on treatment with C5a and latent TGF-β, and were reduced with batimastat (Fig. 7E). No bands corresponding to MMP-9 were seen under these conditions (Fig. 7E). Media from cultures of HMC-1 alone did not contain detectable MMP-2 or MMP-9 by zymography (data not shown). In contrast, MMP-9, but not MMP-2, was readily detectable in media from cultures of pHMC alone (Fig. 7E) as has been previously reported for pHMC (16, 32). On coculture of pHMC with HFL-1, the MMP-9 was reduced to barely detectable levels (Fig. 7E). In contrast, expression of both pro- and processed MMP-2 increased over that seen with HFL-1 alone and increased further on addition of C5a and latent TGF-β (Fig. 7E).

MMP-2 and MMP-3 antagonists block mast cell-stimulated HFL-1 contraction. To further explore the requirement for MMP-2 or MMP-3 in mast cell-mediated HFL-1 contraction, inhibitors targeted to these MMPs were added to the collagen gel system. As described above, catalytically active MMP-2 or MMP-3 could induce contraction of collagen gels embedded with HFL-1 alone (Fig. 4E). Contraction induced by MMP-2 was inhibitable by the MMP-2 selective antagonist, 2-[isopropoxy-(1,1'-biphenyl-4-ylsulfonyl)-amino]-N-hydroxyacetamide (MMP-2 inhibitor; Fig. 8A; P < 0.005). Contraction induced by MMP-3 was not affected by the MMP-2 inhibitor but was blocked by NNGH (Fig. 8A; P < 0.005). Although NNGH has been described to be MMP-3-selective (21, 31, 47), it may also have activity against the gelatinase MMP-2 (36), and, in our studies, NNGH antagonized the activity of recombinant, catalytically active MMP-2 (Fig. 8A; P < 0.005). To evaluate the role of MMP-2 or MMP-3 in mast cell-induced HFL-1 contraction, collagen gels embedded with HFL-1 and HMC-1 were treated with batimastat, MMP-2 inhibitor, or NNGH (Fig. 8B). All three agents significantly blocked the contraction (P < 0.005), with the MMP-2 inhibitor producing a partial inhibitory response. The activity of the MMP-2 inhibitor indicates a role for MMP-2 in the mast cell activation model. Because NNGH inhibited the activity of both MMP-2 and MMP-3, a functional role for MMP-3 in mast cell-mediated HFL-1 contraction is suggested but not definitively demonstrated by these studies.


Figure 8
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  		Fig. 8. MMP-2 and MMP-3 antagonists block mast cell-stimulated HFL-1 contraction. A: collagen gels embedded with HFL-1 cells were treated with catalytically active MMP-2 or MMP-3 in the absence or presence of limiting concentrations of batimastat (0.67 µM), MMP-2 inhibitor (2 µM), or N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH; 0.67 µM). B: collagen gels embedded with HFL-1 and HMC-1 cells were treated with C5a (10 nM), latent TGF-β (5 ng/ml), or C5a + latent TGF-β, and MMP inhibitors as described above. After 48 h, gel surface area was quantitated and expressed as a percentage of the area of a lattice that contained no cells. *P < 0.005 compared with no inhibitor.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Mast cell activation contributes to the pathology of asthma (8). In the lung, mast cells are localized in close apposition to fibroblasts (26) and are spatially associated with regions of fibrosis in fibrotic lung disorders (45). Previous reports demonstrated that mast cells can support contraction and collagen production by fibroblasts in coculture systems and suggested the involvement of cytokines, histamine, and proteases (7, 9, 23, 24, 46). Separate studies have demonstrated that MMPs could promote fibroblast contractile responses (15, 53). Despite these observations, a direct role for MMPs in mediating mast cell-induced fibroblast contraction has not been previously demonstrated. Using a coculture model with pHMC or HMC-1 and HFL-1 embedded in a collagen gel matrix, we have demonstrated that MMPs contribute to the contraction-stimulating activity of both HMC-1 and pHMC. These data definitively establish the primary and critical role of MMPs in mast cell-dependent fibroblast gel contraction and collagen production.

MMPs are able to mediate the proteolytic activation of latent TGF-β (11, 60), providing one potential mechanism whereby MMP release could lead to fibrotic responses. In support of this model, neither latent TGF-β alone nor MMPs alone induced a strong contractile response in HFL-1, but the combination of latent TGF-β with MMP-2 or MMP-3 resulted in potent gel contraction. Furthermore, exogenously added latent TGF-β promoted fibroblast contraction in the presence of activated mast cells or their conditioned media, and this activity was inhibited by batimastat. Active TGF-β could be detected in media surrounding collagen gels under conditions that led to a contractile response. Although active MMPs can exist in a cell-associated form (44, 60), the finding of batimastat-sensitive contractile activity in mast cell conditioned medium is consistent with a primary role for secreted MMPs. These findings demonstrate that MMP-dependent mechanisms, such as activation of latent TGF-β, could support profibrotic changes triggered by mast cell activation.

Zymography revealed constitutive expression of the pro- form of MMP-2 (gelatinase) in cultures of HFL-1 cells, as has been previously reported for this cell type (19, 20, 34). Trace levels of processed MMP-2 were also detectable in the presence of HMC-1 cells, and higher levels in the presence of pHMC. In the presence of latent TGF-β, mast cell activation enhanced expression of pro-MMP-2, processed MMP-2, and MMP-3, in association with collagen gel contraction. Latent TGF-β processing activity has been described for several MMPs, including MMP-2 (13), MMP-3 (37), MMP-9 (13, 60), MMP-13 (11), or MT1-MMP (30) in various in vitro systems, and was supported by our findings of collagen gel contraction and TGF-β activation in the presence of MMP-2 or MMP-3 in combination with latent TGF-β. These findings suggest that the generation and activation of MMP-2 and/or MMP-3 under mast cell-fibroblast coculture conditions contributes to the contractile phenotype.

MMPs are released from the cell as latent proenzymes, and mast cell derived serine proteases are among the enzymes able to process pro-MMP-1, MMP-3, MMP-9, and MMP-2 to the catalytically active forms (35, 48, 50, 52). Because granule-associated serine proteases are released immediately on degranulation, the time course of contractile responses to mast cell-derived agents was investigated. Gel contraction was initiated between 3 and 24 h after mast cell activation and continued through 48 h. The lack of appreciable contractile activity at 3 h indicates that agents released on mast cell degranulation do not directly promote contraction. This was supported by observations that mast cell supernatants collected 2 h postactivation did not support HFL-1 contraction (data not shown), whereas those collected 24 h postactivation did promote contraction.

In addition to driving gel contraction and collagen production, TGF-β is a potent inducer of fibroblast differentiation to myofibroblasts (27). Myofibroblast differentiation can lead to increased contractile responses in collagen gels (28, 54), can be induced by HMC-1 (22), and can involve MMP induction (28). Therefore, myofibroblast differentiation may contribute to the mast cell-induced contractile responses described here. Although HFL-1 cells express {alpha}-smooth muscle actin and other myofibroblast-associated proteins in response to TGF-β exposure (38), it is apparent that TGF-β-induced contractility (42) and collagen production (41) responses may precede differentiation of human fibroblasts to myofibroblasts. This appears to be the case in the current model, as no induction of {alpha}-smooth muscle actin expression was noted in response to mast cell activation over the 48-h time course of these experiments (data not shown).

A histopathological association between activated mast cells and fibrosis has been recognized in numerous studies of asthma, autoimmune disease, and wound healing (17, 25). Collagen gel lattice systems, coembedded with human fibroblasts and HMC-1 or rodent mast cells, have provided an in vitro model to investigate potential mechanisms of interaction between mast cells and fibroblasts. The present study outlines a critical role for MMPs in mediating mast cell-induced fibroblast contractility and collagen production and implicates involvement of MMP-2 and MMP-3. Activation of latent TGF-β is one potential mechanism by which MMPs could promote fibrotic changes. This was seen with HMC-1 cells and confirmed using pHMC derived from progenitors in peripheral blood and activated in an IgE-dependent manner. These findings define a link between MMP-dependent and mast cell-dependent profibrotic mechanisms and support the potential utility of MMP antagonists in addressing airway remodeling changes in atopic asthma.


   ACKNOWLEDGMENTS
 
We thank Maria Lorenzo for providing catalytically active MMP, John Kubera for help with zymography, and Agnes Brennan for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. T. Kasaian, Wyeth Research, 200 CambridgePark Dr., Cambridge, MA 02140 (e-mail: mkasaian{at}wyeth.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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