Mast Cell Regulation of Human Endometrial Matrix Metalloproteinases: A Mechanism Underlying Menstruation¹

^a Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia ^b Department of Medicine, University of Manchester, Manchester Royal Infirmary, Manchester M13 9WL, United Kingdom

	ABSTRACT

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Endometrial matrix metalloproteinases (MMPs), which increase dramatically at menstruation, are purported to cause the focal tissue breakdown at menstruation, but how their expression or activation is locally regulated is unknown. Mast cell activation occurs within perimenstrual endometrium, and we postulated that mast cell products would regulate endometrial MMPs. We have examined the interaction between human mast cells and endometrial stromal cells with regard to MMP production and activation. The human mast cell line (HMC-1) in coculture with stromal cells stimulated stromal cell proMMP-1 and proMMP-3, and to a lesser extent proMMP-2 production, with increasing stimulation as mast cell number increased. Mast cell-conditioned medium also increased both protein and mRNA for stromal proMMP-1 and proMMP-3, this being abrogated by preadsorption of mast cell-conditioned medium with antisera to interleukin-1 and tumor necrosis factor . Mast cell-conditioned medium added to stromal cell culture medium in vitro along with added heparin (which stabilizes tryptase activity) resulted in the appearance of molecular weight forms indicative of active MMP-3 and MMP-1. Thus activated mast cells within the endometrium prior to menstruation have the potential to stimulate MMP production by endometrial stromal cells and to initiate precursor activation, and are likely to account for the local nature of endometrial MMP action resulting in foci of tissue breakdown at menstruation.

	INTRODUCTION

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The human endometrium undergoes monthly cycles of proliferation, differentiation, and secretory activity. In the absence of a blastocyst, the functionalis layer breaks down and is discharged at menstruation. After publication of the classic work of Markee [1], it was assumed that menstruation resulted from tissue necrosis following vasoconstriction of the spiral arterioles. Our understanding of the mechanisms underlying menstruation underwent a paradigm shift in the early 1990s after demonstrations of a dramatic increase in the endometrial expression of a number of matrix metalloproteinases (MMPs) immediately prior to and during menstruation [2–5]. We still know little of how this increased expression is brought about or how the enzymes are activated. Progesterone withdrawal clearly contributes to the increased production of endometrial MMPs [5–9]. However, given the low levels of MMP expression during the proliferative phase of the cycle and the focal nature both of menstrual shedding [10] and of MMP distribution in perimenstrual and menstrual endometrium [11], local rather than endocrine regulators must be the major stimuli for MMP production and activation at menstruation.

Menstruation has many features of an inflammatory process [12], including an influx of eosinophils, neutrophils, macrophages, and granulated lymphocytes (reviewed in [13]). Moreover, resident mast cells, which are present in endometrial stroma in fairly constant numbers throughout the cycle, undergo extensive activation prior to menstruation, as demonstrated by immunolocalization of the mast cell protease, tryptase, at extracellular sites [14]. Thus it is likely that mast cells assume important functional roles at menstruation [14]. Mast cells have the potential to produce a plethora of potentially important regulatory molecules including lipid-derived mediators, histamine and heparin, pleiotropic cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), and the mast cell-specific proteases, tryptase and chymase. There is substantial structural and functional heterogeneity in different mast cell populations with respect to both protease and cytokine production [15, 16]. In the endometrium, the mast cells in the functionalis layer are predominantly of the tryptase-only phenotype whereas those in the basalis layer express both chymase and tryptase [14]. The cytokines and other mediators produced by endometrial mast cells are currently unknown.

MMPs degrade almost all components of the extracellular matrix. They are grouped into four main subclasses: collagenases (including MMP-1 [interstitial collagenase]), gelatinases (MMP-2 and -9), stromelysins (including MMP-3) [17], and membrane-bound MMPs [18]. Regulation of MMP expression varies between enzymes and with cellular origin, but in general, expression of MMP-1, -3, and -9 is inducible by many cytokines and growth factors whereas that of MMP-2 is predominantly constitutive. Secreted MMPs are released as precursor forms requiring extracellular activation. While activation has been demonstrated in vitro by proteolytic cleavage of the propeptide domain [19], in vivo mechanisms are still largely unclear. Experiments in vitro using pure enzymes have shown that mast cell tryptase activates proMMP-3 [20], and this is a key enzyme in the proteolytic activation cascade of other MMPs [13]. The activators of endometrial MMPs at menstruation are not yet known.

Endometrial stromal cells in culture produce proMMP-1, proMMP-2, and proMMP-3 [21]; their production can be regulated in part by the presence or withdrawal of progesterone [7–9]. Production of proMMP-1 and -3 but not proMMP-2 may also be stimulated by both interleukin (IL)-1 and tumor necrosis factor (TNF) [21], but the local regulators controlling production and activation of the enzymes in vivo have not been identified. This study examined the potential role for mast cells in regulating both the expression of mRNA and the production of proMMPs from endometrial stromal cells, as well as in initiating activation of the MMP precursors.

	MATERIALS AND METHODS

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Mast Cell Line

The immature human leukemic mast cell line (HMC-1, generously provided by Dr. J. Butterfield, Mayo Clinic, Rochester, MN) was cultured in Dulbecco's modified Eagle's medium (DME) with 10% (v:v) fetal calf serum and penicillin, streptomycin, and fungizone (all from Trace Biosciences, Sydney, Australia) until of appropriate density. The cells were washed three times in PBS, counted, resuspended in DME, and either added to stromal cell cultures at defined densities or further cultured at defined density in DME with antibiotics for 24 h; at this time the medium was harvested, centrifuged to remove cellular debris, and snap frozen for subsequent studies. Some cells were cytocentrifuged onto glass slides while others were grown in drops of medium on glass coverslips for 24 h: both were fixed with 70% ethanol, air dried, and stored for immunohistochemistry. As controls, the Jurkat human leukemic T cell line and the BHK 21 (Syrian hamster kidney) cell line were cultured under conditions similar to those for the HMC-1 cell line, and medium was harvested.

Preparation of Mast Cell Products

Mast cells were prepared from histologically confirmed dog mastocytomas as described previously [20]. These preparations contained more than 98% mast cells as judged by metachromatic staining with toluidine blue. Soluble mast cell products were prepared by sonication and extraction with 1 M NaCl overnight at 2°C; granular debris was removed by centrifugation and filtration through a 45-µm filter, and mast cell products to the equivalent of 5 x 10⁶ cells/ml were stored in aliquots at -70°C until use.

Collection of Endometrial Tissue

Endometrial tissue was obtained at dilatation and curettage from women who had given informed consent who were undergoing assessment of tubal patency and had no evidence of endometrial dysfunction. Tissue was dated initially from the patient's testimony and confirmed histologically. All tissues used were from between cycle Days 8 and 24. Protocols were approved by appropriate Institutional Human Ethics Committees.

Stromal Cell Isolation and Culture

Stromal cells were prepared from human endometrial tissue as described previously [8, 21]. Briefly, chopped tissue was digested with bacterial collagenase type III (Worthington Biochemical Corporation, Freehold, NJ) at a concentration of 45 U/ml in the presence of 3.5 µg/ml deoxyribonuclease (Boehringer Mannheim Biochemica, Mannheim, Germany) for 40 min at 37°C and filtered sequentially through 45- and 10-µm nylon filters to remove glands; erythrocytes were removed by centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). The resulting cells were resuspended in a 1:1 mixture of DME and Ham's F-12 medium (Trace Biosciences) with 10% charcoal-stripped fetal calf serum and antibiotics (penicillin, streptomycin, and fungizone) and plated in 48-well dishes (2.5 x 10⁵ cells in 0.5 ml) or at a similar density in 35-mm dishes for subsequent RNA preparation. After 48 h, when the cells were nearly confluent, they were washed, and medium was replaced with serum-free medium. All experiments were performed in the presence of estradiol-17ß (E₂; 10 nM; Sigma), with or without the synthetic progestin ORG2058 (P; 100 nM; Organon Laboratories Ltd., Oss, The Netherlands). Mast cells (2 x 10⁵ cells unless otherwise stated) or mast cell-conditioned medium (67 µl, equivalent to 2 x 10⁵ cells) was added to wells as required, and final volumes were adjusted appropriately. Media from similar numbers of Jurkat or BHK 21 cells were similarly tested for effects on stromal cells. In some experiments, mast cell-conditioned medium was preadsorbed for 24 h at 4°C with a mixture of anti-human IL-1ß (300 µg/ml final concentration; Serotec Ltd., Oxford, UK; clone MCA 744) and anti-human TNF (380 µg/ml final concentration; Peptide Technology Ltd., Dee Why, NSW, Australia; clone 047) prior to addition to stromal cells. Preimmune IgG at the same concentration was used in a control preadsorption. All experiments were performed in triplicate or quadruplicate wells for 48 h, after which medium was collected, centrifuged to remove cellular debris, and stored at -20°C for subsequent analysis. Viability of cells at the end of the experiment was shown by their ability to exclude trypan blue. In some cases cells counts were performed at the end of the experiment, and in others, cells from selected wells were taken for RNA preparation. Each experiment was replicated at least three times using different primary cultures.

Zymography

Proteinase activity in samples of culture medium was analyzed by zymography on 10% SDS-polyacrylamide gels containing 1 mg/ml gelatin (all reagents from Bio-Rad, North Ryde, Australia) or 1 mg/ml casein (Sigma) under nonreducing conditions [8, 21]. Loading of samples of cell culture medium was normalized according to the DNA content of culture wells. Gelatinase or caseinase activity was visualized by negative staining, and bands were identified by comparison with standard preparations of pure human MMPs (a gift from Dr. Hideaki Nagase, Kansas City, KS) and with molecular weight markers (Bio-Rad; [21]). MMP identity of all bands was confirmed by incubation of parallel gels in the presence of EDTA (5 mM). For testing caseinase activities in mast cell-conditioned medium, gels were also incubated in chymostatin (100 µg/ml; Sigma), antipain (100 µg/ml; Sigma), and aprotinin (1 µg/ml; Sigma). Relative activities of MMPs were semiquantitated by densitometric analysis of zymograms [22] using the Hewlett-Packard Scanjet IIp with Deskscan software (Hewlett-Packard, Palo Alto, CA) set on a black-and-white photo with 256 gray shades. The area of the bands was analyzed, using the NIH (Bethesda, MD) Image Version 1.54 equipped with gel-plotting macros, by measuring the area beneath the peaks plotted through the lane profile for the appropriate enzyme. Comparisons were made only between samples run on the same gel. Previous analyses of doubling dilutions of samples on each of the types of zymograms verified the semiquantitative nature of these analyses, while analysis of the same samples for MMP-1 by casein zymography and ELISA substantiated changes in MMP-1 measured by this method [8].

Immunocytochemistry

Mast cells, grown or centrifuged onto glass coverslips, were subjected to immunocytochemistry using primary monoclonal antibody (mAb) against tryptase (MAB1222; Chemicon Int., Temecula, CA), chymase (MAB1254; Chemicon Int.), IL-1ß (clone MCA 744; Serotec, Oxford, UK), and TNF (clone 047; Peptide Technology Ltd.). The immunostaining procedure for tryptase and chymase was as described previously [14] and used a biotinylated goat anti-mouse IgG and the StreptABC complex (both from Dako A/S, Glostrup, Denmark) with New Fuchsin (Dako) as chromogen. The cytokine immunostaining used the antisera at 1:30 dilution and biotinylated horse anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA) with the same detection system. Human endometrium was used as a positive control tissue in all cases. Negative controls utilized an irrelevant -lactalbumin mAb at the same concentration to replace the primary antibody. Positive and negative controls were included in every staining run. Harris hematoxylin (1:10) was used as counterstain.

Dot Blots

Doubling dilutions of samples of mast cell-conditioned medium were spotted onto polyvinylidene difluoride (PVDF) membranes (Amersham Int., Buckinghamshire, UK). After blocking of nonspecific binding sites with 10% skim milk powder in Tris-buffered saline (TBS) with 0.1% Tween 20 for 30 min, the membranes were reacted overnight at 4°C with antisera to IL-1ß and TNF and then with goat anti-mouse IgG as above and visualized using the enhanced chemiluminescence detection kit (ECL plus; Amersham). Blots were exposed to Kodak X-OMAT AR scientific imaging film (Eastman Kodak, Rochester, NY) for 1 min and developed.

Electron Microscopy

HMC-1 cells were fixed in 2.5% glutaraldehyde in 0.12 M Millonig's phosphate buffer [23], washed three times in Millonig's phosphate buffer, and centrifuged at 200 x g 10 min to produce a pellet. After a 0.1 M cacodylate buffer wash, the cells were postfixed for 1 h in 1% (w:v) osmium tetroxide, rewashed, dehydrated in a graded series of ethanols, and embedded in agar 100 resin. Ultrathin sections were cut on a Reichert Jung (Nossloch, Germany) Ultracut ultramicrotome, mounted on copper grids, and stained with uranyl acetate and lead citrate. Grids were examined in an Associated Electrical Industries (Manchester, UK) EM 801 electron microscope.

Enzyme and Cytokine Assays

Tryptase activity in conditioned medium from HMC-1 cells was measured by RIA (Pharmacia & Upjohn Diagnostics, Uppsala, Sweden). This assay has a limit of detection of 0.5 U/L. Granulocyte-macrophage-colony stimulating factor (GM-CSF), granulocyte (G)-CSF, and IL-6 in the same conditioned medium were measured by ELISA assays with sensitivities of 4.1 pg/ml, 3 pg/ml, and 1.56 ng/ml, respectively.

RNA Extraction from Cultured Endometrial Stromal Cells

After culture and treatment, endometrial stromal cells were washed twice with PBS and either frozen in their culture dishes at -20°C or immediately processed. Total cellular RNA was extracted by a modified miniprep method [24]. Briefly, cells in the wells were incubated for 5 min at room temperature with 200 µl of 3 M LiCl/6 M urea. The solution was transferred to a microcentrifuge tube, vortexed vigorously, heated at 50°C for 3 min, and incubated for a further 5 min at room temperature. Extracted RNA was treated with DNase because substantial amounts of DNA were often detectable. RNA was quantitated by its absorbance at 260 nm.

Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Messenger RNA levels for MMPs 1, 2, and 3 and tissue inhibitor of metalloproteinases (TIMPs) 1, 2, and 3, along with the mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined by quantitative RT-PCR [25, 26] using a multispecific heterologous competitor that shares the same primer-binding sequences as the cellular mRNA of all 7 genes but yields different-sized PCR products; this competitor has been fully validated (unpublished results). In brief, a set of 7 competitive RT reactions (master RTs) was set up for each sample RNA; in each of these a constant amount (300-500 ng) of total DNA-free sample RNA was combined with a serial dilution of an exact amount of synthesized competitor RNA (range, 0.35-350 pg). The RNA mixture was then reverse transcribed at 46°C for 1-1.5 h in 20-µl reaction mixture using 100 ng random hexanucleotide primers and avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim, Nunawading, VIC, Australia) with the cDNA synthesis buffer supplied by the company for this enzyme. The resultant cDNA mixtures were then heated at 95°C for 3 min before storage at -20°C or immediately used for PCR amplification. This master RT set was used in PCR for different specific genes using individual primer pairs (Table 1); for each specific gene, PCR reactions corresponding to the whole series of RT reactions were performed. Each PCR (40 µl) contained 1-1.5 µl of the RT mixture, single-strength PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3), 20 µM dNTPs, 10 pmol forward primer, 10 pmol reverse primer, and 2.5 units of Taq DNA polymerase (Boehringer Mannheim). The PCR was performed in three stages as follows: the first stage was one cycle of an incubation for 5 min at 95°C, 1 min at 60°C, and 2 min at 72°C; the second stage involved 28 cycles, each cycle consisting of 2 steps—45 sec at 95°C and 2 min at 69°C; finally, the reaction mixture was incubated for 5 min at 72°C. The PCR products were separated on a 1.8% TBE agarose gel, stained with ethidium bromide, and photographed using a Polaroid (Cambridge, MA) system. The picture was scanned using the Hewlett-Packard Scanjet IIp with Deskscan software (Hewlett-Packard, Palo Alto, CA), and the band intensities were determined using NIH Image Version 1.54. To determine the competition equivalence point, the logarithm of the band intensity ratio of competitor product to the target product was graphed as a function of the logarithm of the initial amount (in mRNA copy numbers) of the competitor added. When the target and the competitor products are equal, the initial amount of the target sequence present in the RNA sample is equal to the initial amount of the competitor added to RNA sample in the RT reaction. The exact initial amount of the target sequence in the sample was calculated by linear regression analysis of the graph. To more directly compare the differences of each mRNA species between the treatments, the expression levels of MMPs and TIMPs in each treatment were normalized against GAPDH within that treatment, and the data were expressed as the percentage of the control (stromal cells alone, 100%).

In Vitro Activation Studies

Samples (100 µl) of conditioned medium from endometrial stromal cells and of pure standard solutions of proMMP-3 and proMMP-1 (in DME/Ham's F-12 medium) were incubated at 37°C for 16 h with mast cell-conditioned medium, mast cell products, or mast cells with or without heparin (0.5 mg/ml; Faulding and Co. Ltd., Mulgrave, Australia). Subsequent analysis was by casein zymography and Western blots.

Western Blots

Samples together with enzyme standards were subjected to SDS-PAGE on 12% gels under reducing conditions, and the proteins were transferred to PVDF membranes. After blocking of nonspecific binding sites with 10% skim milk powder in TBS with 0.1% Tween 20 for 30 min, blots were incubated overnight with affinity-purified rabbit antisera to MMP-1 (1:1000) or MMP-3 (1:2500). These antisera detect both latent and active forms of the enzymes. The blot was then incubated with donkey anti-rabbit IgG conjugated to horseradish peroxidase and the ECL plus kit (Amersham). Blots were exposed to Kodak X-OMAT film for 10 min and developed.

Statistical Analyses

Data from cell culture studies were expressed as mean percentage of control ± SEM (stromal cells alone, 100%); all treatments were in triplicate or quadruplicate wells. Data were analyzed either by unpaired Student's t-test or by ANOVA and Tukey's post hoc multiple comparison test (least significant difference procedure). Differences were considered significant at the 0.05 level.

	RESULTS

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Analysis of Mast Cell Products

Immunocytochemistry performed on HMC-1 cells after culture demonstrated the presence of tryptase, IL-1ß, and TNF but not chymase (Fig. 1). Tryptase was also detected in mast cell-conditioned medium by RIA, 2.3 mU being secreted by 10⁶ cells in 72 h. IL-1 and TNF were detected in mast cell-conditioned medium by dot blot (data not shown). No GM-CSF, G-CSF, or IL-6 were detected in the mast cell-conditioned medium by ELISA assays. Gelatin and casein zymograms of the mast cell-conditioned medium showed no detectable gelatinase activity, while bands of caseinase activity, of molecular mass approximately 30 kDa, were inhibitable by antipain and aprotinin but not by chymostatin or EDTA (data not shown), demonstrating that they represented activity of tryptase or another serine or cysteine protease, not chymase or MMP activities [20].

View larger version (119K): [in this window] [in a new window]   FIG. 1. HMC-1 cells subjected to immunocytochemistry for a) IL-1ß, b) TNF, c) tryptase, d) chymase. a and b) Cells grown on glass coverslips; c and d) cytocentrifuged cells. Negative controls in which an irrelevant mAb replaced the primary antibody were included for each staining and gave results comparable to those seen in d. x800.

Electron Microscopy of HMC-1 Cells

Ultrastructurally, the HMC-1 cells were approximately 10-16 µM in diameter; the ellipsoidal nucleus showed marginated chromatin, but nucleoli were not prominent (Fig. 2). The cytoplasm contained membrane-bound vesicles in addition to mitochondria, dictosomes, and centrioles. The vesicles were approximately 0.5 µM in diameter and contained either medium electron-dense granular contents or lucent areas with more condensed areas. Classical mast cell granules were not seen.

View larger version (171K): [in this window] [in a new window]   FIG. 2. Electron microscopy of HMC-1 cells after culture. Top) Low magnification. Note the absence of "classical" mast cell granules. x1500. Bottom left) Membrane-bound cytoplasmic vesicles with amorphous medium electron-dense granular contents. x9000. Bottom right) Vesicles showing more condensed contents within an electron-lucent matrix. x8000.

Steroid Hormones Regulated Stromal Cell Production of MMPs

Gelatin and casein zymography demonstrated that as previously described, the endometrial stromal cells released proMMP-1, proMMP-2, and proMMP-3 into the culture medium. Production of proMMP-1 and proMMP-3 by endometrial stromal cells was significantly lower when the cells were incubated in the presence of E₂+P compared to culture with E₂ alone (Table 2). This was not the case for proMMP-2: its secretion was not different with the two steroid treatments of the cells.

Coculture with Mast Cells Stimulated MMP Production by Endometrial Stromal Cells

When mast cells were cocultured with the endometrial stromal cells, stimulation of proMMP-1, proMMP-2, and proMMP-3 production was seen; this was dose dependent and reached a maximum in each case when 2 x 10⁵ mast cells were cultured with 2.5 x 10⁵ stromal cells (Fig. 3). In all cases, these effects were greater in the presence of E₂+P than with E₂ alone, although the total amounts of proMMP-1 and proMMP-3 released were higher in the absence of P (Table 2). The maximal increase in proMMP-1 and proMMP-3 with mast cells in the progesterone-treated cultures (8.2- and 5.8-fold, respectively, in the experiment shown) was substantially greater than the increase in proMMP-2 (3-fold). Mast cells cultured alone or in the presence of stromal cell-conditioned medium did not produce MMPs.

View larger version (34K): [in this window] [in a new window]   FIG. 3. Effect of coculture with increasing numbers of mast cells on endometrial stromal cell release of proMMP-1, proMMP-2, and proMMP-3: (lighter bars) in presence of E and (darker bars) in presence of E2+P. Culture medium was analyzed by zymography and densitometry, and data are expressed as percentage of control. Error bars represent SEM from triplicate or quadruplicate wells. Representative one of three separate experiments. *Represents significant increase over wells without mast cells (*p < 0.01, **p < 0.05).

Mast Cell-Conditioned Medium Stimulated MMP Production by Endometrial Stromal Cells Similarly to Coculture with Mast Cells

The result of the coculture experiment indicated an interaction between stromal cells and mast cells leading to increased MMP synthesis. To determine whether these effects were due to soluble mediators, stromal cells were cultured in the presence of E₂+P, with either mast cells (2 x 10⁵ cells) or mast cell-conditioned medium (at equivalent concentration). Both mast cell-conditioned medium and coculture with mast cells significantly stimulated the production of proMMP-1, proMMP-2, and proMMP-3, but there was no difference between the response to the two treatments (Fig. 4). The increase of proMMP-1 (12-fold) was greater than that of proMMP-3 (6-fold), which in turn was greater than that of proMMP-2 (2-fold), these data being in accord with those of the dose-dependency experiments (Fig. 3). Similar equality of response to mast cells and mast cell-conditioned medium was seen also in the presence of E₂ alone (data not shown). When mast cell-conditioned medium was substituted for by medium from culture of equivalent numbers of either Jurkat or BHK 21 cells (chosen because like mast cells, they do not produce proMMP-1 or -3), there was no increase in either proMMP-1 or proMMP-3 released by the stromal cells.

View larger version (42K): [in this window] [in a new window]   FIG. 4. Comparison of coculture with either mast cells (medium gray bars) or conditioned medium from an equivalent number of mast cells (dark bars) on production of proMMP-1, proMMP-2, and proMMP-3 from endometrial stromal cells, cultured in the presence of E2 (10 nM) + P (ORG2058, 100 nM). Light bars) Medium from control wells. Data are expressed as percentage of control (SC alone) ± SEM (n = 4 wells per treatment). Representative one of three experiments. Significant differences compared with control are represented by ** (p < 0.01), * (p < 0.02), (p < 0.05).

Mast Cell Products Regulated proMMP Gene Expression

Analysis of stromal cell mRNA at the end of culture demonstrated that mast cell-conditioned medium stimulated the expression of mRNA for proMMP-1 and proMMP-3 but not proMMP-2 (Fig. 5). This increase was apparent in both the absence and presence of P, although the effects were greater when P was present, and the production of MMPs from the control cells was lower. Interestingly, in contrast to the protein levels (Fig. 4), the increase in mRNA for proMMP-1 (4-fold) was less than that for proMMP-3 (7-fold), while no increase was seen for proMMP-2; these relative changes were consistent between individual stromal cell cultures although the absolute fold changes differed. No changes were seen in mRNA for TIMP-1, -2, or -3.

View larger version (61K): [in this window] [in a new window]   FIG. 5. Effect of mast cell-conditioned medium (MCCM) on stromal cell (SC) mRNA for proMMP-1, -2, and -3 and TIMP-1, -2, and -3. A) Agarose gel electrophoresis of the PCR products after quantitative RT-PCR reactions for GAPDH, MMP-1, MMP-2, and MMP-3. In each lane the upper band was from the wild-type target RNA and the lower band from the competitor RNA. Lane S, DNA 50-bp standard (the arrows indicate the sizes of the PCR products from the target RNA); lane 1, 350 pg of competitor RNA alone and lane 7, 230 ng of total RNA alone; lanes 2-6, 350, 21.88, 5.47, 1.37, 0.34 pg of competitor RNA, respectively, along with 230 ng of total cellular RNA. Gels for TIMP analyses are not shown. B) After electrophoresis of the PCR products, the band intensity of the competitor and target was determined; the amount of the wild-type target RNA copy numbers of each gene in each treatment was calculated; the data were normalized against GADH expression within each treatment; and the coculture effect of the SC together with MCCM (bars with squares) on the expression of MMPs and TIMPs in SC was expressed as percentage of control (SC alone, bars with dots). This experiment was performed twice with similar results.

Immunoneutralization of IL-1ß and TNF Abrogated Stromal Cell Response to Mast Cell-Conditioned Medium

Stromal cells (in the presence of E₂+P) were incubated either with mast cell-conditioned medium, with mast cell-conditioned medium preadsorbed with combined antisera to IL-1ß and TNF, or with mast cell-conditioned medium treated similarly with an irrelevant IgG at the same concentration; the cells were then examined for expression of mRNA for proMMP-1 and proMMP-3. Stromal cell expression of mRNA for both enzymes was increased by mast cell-conditioned medium alone, but this effect was abrogated by preadsorption of the mast cell-conditioned medium with antisera against IL-1ß and TNF: abolition of the response was complete in the case of proMMP-1 but incomplete for proMMP-3, raising the possibility that some additional factor that selectively stimulates expression of proMMP-3 may be present in the mast cell-conditioned medium (Fig. 6). Preincubation with nonimmune IgG resulted in a only a very small reduction in stromal cell mRNA (data not shown).

View larger version (80K): [in this window] [in a new window]   FIG. 6. Inhibition of effects of mast cell-conditioned medium (MCCM) on stromal cell mRNA for proMMP-1 and proMMP-3 by pretreatment of MCCM with anti-IL-1ß plus anti-TNF. A) Agarose gel electrophoresis of the PCR products for GAPDH, MMP-1, MMP-2, and MMP-3. In each lane the upper band was from the target RNA and the lower band from the competitor RNA. Lane S, DNA 50-bp standard (the arrows indicate the 250-bp position); lane 1, 87.5 pg of competitor RNA alone and lane 6, 296 ng of total RNA alone; lanes 2-5, 21.88, 5.47, 1.37, 0.34 pg of competitor RNA, respectively, along with 296 ng of total cellular RNA. SC are the data for stromal cells alone; SC+MCCM, stromal cells with mast cell-conditioned medium; and SC+MCCM (spreads), stromal cells with mast cell-conditioned medium that had been preadsorbed with antisera to IL-1ß and TNF. B) After electrophoresis of the PCR products, the band intensity of the competitor and target was determined; the amount of the wild-type target RNA copy numbers of each gene in each treatment was calculated; the data were normalized against GAPDH expression within each treatment; and the coculture effect of stromal cells together with MCCM, which was or was not preabsorbed with antibodies, on the expression of MMP-1 and MMP-3 mRNA was expressed as percentage of control (SC alone). SC alone (bars with dots), coculture of SC with MCCM (bars with squares) or after preadsorption with anti-IL-1ß plus anti-TNF (solid bars). Representative one of two experiments.

Mast Cell Products Could Activate proMMPs of Endometrial Stromal Cell Origin

In cocultures of stromal cells with mast cells, or when mast cell-conditioned medium was added to stromal cell cultures, only small amounts of the active forms of MMP-1, -2, and -3 were detected, although the mast cells produced substantial quantities of immunoreactive tryptase, a known activator of proMMP-3. Given the stringent requirements for stabilization of tryptase activity [27], we incubated pure proMMP-1, proMMP-3, and stromal cell-conditioned medium in vitro with mast cells in the presence of the tryptase stabilizer, heparin, and also with mast cell products that had previously been shown to contain active tryptase and chymase [20]. The products were examined by Western blot analysis using antisera that detect both latent and active forms of MMP-3 and MMP-1 (Fig. 7, A and B). Incubation of proMMP-1 and proMMP-3 with mast cell products resulted in production of several lower molecular weight forms, indicative of activation of the enzymes. Similar products were obtained when stromal cell-conditioned medium was incubated with mast cell products. When stromal cell-conditioned medium was incubated with HMC-1 in the presence of heparin, these lower molecular weight forms of MMP-3 and MMP-1 were also produced, although the extent of conversion was less than with mast cell products, particularly in the case of MMP-1. HMC-1 cells without added exogenous heparin did not have this effect, strongly suggesting that tryptase was the active product and that its stabilization by heparin was necessary. ProMMP-1 in the stromal cell-conditioned medium was converted to lower molecular weight active forms to a lesser extent with the HMC-1 cells plus heparin than with mast cell products (which contains chymase as well as tryptase), consistent with this activation resulting secondarily from the action of active MMP-3. No MMPs were detected in culture medium containing HMC-1 cells plus heparin alone.

View larger version (53K): [in this window] [in a new window]   FIG. 7. Activation of proMMP -1 and -3 by dog mastocytoma products (MCP) or by products of MC. Western blot analysis using antibodies against human MMP-3 (A) and human MMP-1 (B) was performed on purified enzymes, MMP-3 (P3) and MMP-1 (P1), or SCCM alone or following incubation overnight at 37°C with either dog mastocytoma extract (MCP) or MC with added heparin (HMC). The largest molecular weight forms represent the glycosylated and nonglycosylated forms of the MMP zymogens: doublets of decreasing size represent partially and fully activated enzymes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study was an investigation of a likely role for mast cells in stimulating the local production of MMPs by endometrial stromal and decidual cells and in setting up a cascade of MMP activation within the endometrium, which could result in the focal breakdown of extracellular matrix and lead to menstruation. The results indicate that soluble mast cell products can stimulate expression of mRNA for MMP-1 and -3 and production of latent MMP-1, -2, and -3 through signaling pathways that do not depend upon cell-cell contact and that are largely inhibited by preadsorption of the mast cell products with combined antisera against IL-1 and TNF. Further, the mast cell enzyme tryptase resulted in appearance of molecular weight forms indicative of active MMP-3, and via its action, active MMP-1.

No changes were seen in mRNA for TIMP-1, TIMP-2, or TIMP-3 in response to mast cell products. Protein levels of TIMPs were not measured in these studies: these are unresponsive to withdrawal of P from such cell cultures [8], and immunohistochemical analysis of human endometrium across the menstrual cycle has indicated little major cyclical variation [28]. As TIMPs inhibit MMP activity by binding active MMPs with a 1:1 stoichiometry [29], tissue degradation will result only from an increase in active MMPs without a concomitant increase in TIMPs. Hence, the selective effect of mast cell products on production and activation of MMPs, but not TIMPs, would favor degradation of the extracellular matrix.

The concentration-dependent stimulatory effects of mast cell products on endometrial cell MMP production are demonstrable in both the presence and absence of progesterone, although they are more marked when the basal MMP production has been inhibited by progesterone. IL-1 and TNF have previously been shown to stimulate MMP-1 and -3 but not MMP-2 production by endometrial stromal cells [21], while IL-1ß elevated MMP-3 in the presence of E₂ plus medroxyprogesterone acetate [30]. These observations are extended here to include stimulation of MMP-1 in the presence of P and by a combination of IL-1 and TNF produced by mast cells. Such data suggest that different intracellular mediators may be involved in the inhibitory effects of progesterone and the stimulatory effects of the cytokines on MMP expression. Moreover, the lack of complete abrogation of the stimulatory effect on MMP-3 mRNA suggests that the HMC-1 cells may produce an additional factor with a selective action on MMP-3 but not MMP-1 or -2 expression. This factor has not been identified. Anti-TNF and anti-IL-1 were not tested separately, as they were both present in substantial quantities in the mast cell-conditioned medium and as they separately and equally stimulated MMP-1 and -3 production by endometrial stromal cells [21].

Some disparity appears in the data for MMP mRNA and protein levels. Small (2- to 3-fold) increases in proMMP-2 protein but not mRNA are observed in response to mast cell-conditioned medium, while the greater stimulation of proMMP-1 in comparison to proMMP-3 protein is not in accord with the greater increase in mRNA for proMMP-3 than for proMMP-1. Variability in the stability of the mRNA for different MMPs [31] and variability in their rate of translation are likely explanations for these discrepancies.

Considerable heterogeneity exists between mast cells in different tissue locations [32], and mast cell phenotype is probably dependent on the anatomical microenvironment [16, 33]. In the endometrium, the mast cells in the functionalis layer are predominantly of the mast cell T (MC_T) phenotype (produce tryptase but not chymase) while those in the basalis layer are MC_TC (produce both tryptase and chymase) [14]. The cytokines produced by endometrial mast cells have not been determined. As it was not possible to obtain human endometrial mast cells for this study due to the difficulties of isolation and propagation in sufficient numbers, the HMC-1 cell line was chosen. This cell line, derived from a patient with mast cell leukemia [34], is the only established cell line exhibiting a phenotype similar to that of human mast cells [35]. However, it has the limitation of being a very immature mast cell, as evidenced by the lack of characteristic electron-dense granules seen by electron microscopy. Nevertheless, these cells produce a range of products similar to those synthesized by mature mast cells [36], albeit probably in much lower concentrations. This would account for the relatively large number of mast cells (a 1:1 ratio with stromal cells) required for maximal effects in our coculture system. In vivo, where mature mast cells are present, the high local concentrations of their products (for example, the concentration of active tryptase in secretory granules is estimated to be at least 100 µg/ml [37]) would be expected to maximally stimulate a number of stromal cells in the region. The HMC-1 cell line has previously been usefully applied to the study of mast cell effects on fibroblast activation [38].

Since mast cells are commonly associated with sites of connective tissue degradation, a potential role for this cell in matrix degradation has been proposed [39]. Mast cell tryptase has a remarkably restricted substrate specificity for proteins: substrates include proMMP-3 [20, 40], urokinase type plasminogen activator [41], collagen VI [42], and 1 macroglobulin [43], while chymase is able to activate proMMP-1 [20]. Neither apparently activates proMMP-2 or proMMP-9 [20]. As MMP-3 itself activates a number of other latent MMPs, including MMP-1, -7, and -9, all of which are produced in human endometrium at menstruation, at sites where active tryptase is also produced, the potential exists for a cascade of MMP activations mediated via mast cell serine proteases to occur [13].

The lack of active forms of MMPs, particularly MMP-3, in the cocultures was initially of some concern, given that tryptase had been detected in the mast cells and mast cell-conditioned medium by both immunohistochemistry and RIA. However, human tryptase binds tightly to heparin, which stabilizes its enzymatic activity. Once dissociated from heparin, the enzyme converts from a tetramer to a monomer and irreversibly loses activity [15, 44]. Since electron microscopy of the HMC-1 cells showed little evidence of the characteristic heparin-containing granules, it appeared likely that there was insufficient heparin produced to stabilize the active tryptase: this was substantiated by the active tryptase that resulted after addition of heparin to the mast cell culture.

Tryptase has additional roles of potential importance in the endometrium: these include mitogenic actions on epithelial [45], smooth muscle, and mesenchymal cells [46] and hence a possible role in endometrial repair. The mitogenic signals generated by tryptase can synergize with those generated by both tyrosine kinase-coupled and G protein-coupled growth factor receptors [47]. Mast cells can also play a critical role in leukocyte recruitment to sites of inflammation by stimulating IL-8 [45, 48] and intracellular adhesion molecule 1 [45] release.

The mechanism of endometrial mast cell activation is currently unknown. Candidates for this action include corticotropin-releasing hormone, which is present in endometrial epithelial cells [49] and has a proinflammatory role [50] with activation of mast cells as one target, and endothelin-1, which is produced maximally by endometrial epithelial and decidual cells perimenstrually [51] and is a potent inducer of histamine release from mouse peritoneal mast cells [52].

The studies described here, along with our previous observations that endometrial mast cells become activated just prior to menstruation [14], support an important contributory role for mast cells in regulating menstruation. While withdrawal of progesterone directly stimulates MMP production by endometrial explants or stromal cells in culture [5–9], such a mechanism cannot explain the very focal nature of tissue breakdown at menstruation [13]. Furthermore, immunolocalization of MMPs in human endometrium at menstruation [11, 53] supports the concept of microenvironmental rather than widespread regulation. An important contributory role for the mast cell in mediating matrix degradation within microfoci via stimulation of MMP production and activation, has been established in pathological situations including the rheumatoid lesion [54] and tumor invasion [55, 56], and can now be extended to a role for mast cells at menstruation. Whether or not these cells are also involved in situations of abnormal uterine bleeding is now being explored.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Joseph Butterfield for providing the HMC-1 cell line, Dr. Hideaki Nagase for antisera and MMP standards, Ms. Sonia Garcia for performing the tryptase and chymase immunolocalization, Dr. Sue Blackwell and Professor John Hamilton (Melbourne University) for performing the ELISA assays for M-CSF, GM-CSF, and IL-6, Dr. Allan Curry (Withington Hospital, Manchester, UK) for the electron microscopy, Sue Panckridge for assistance in preparation of figures, Professor Gabor Kovacs and Dr. Jason Clark for providing the tissue, and Sr. Catherine Canny for its collection.

	FOOTNOTES

 
¹ This work was supported by the National Health and Medical Research Council of Australia (Grant 971292) and the NIH (Grant HD-33233-02). J.W. was a recipient of a training fellowship from the Human Reproduction Program of WHO.

² Correspondence: L.A. Salamonsen, Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia. FAX: 61 3 9550 6125; lois.salamonsen{at}med.monash.edu.au

Accepted: May 5, 1998.

Received: February 13, 1998.

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