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Am. J. Respir. Cell Mol. Biol., Volume 17, Number 5, November 1997 583-591

Increased Release of Matrix Metalloproteinase-9 in Bronchoalveolar Lavage Fluid and by Alveolar Macrophages of Asthmatics

Gisèle Mautino, Nicolas Oliver, Pascal Chanez, Jean Bousquet, and Françoise Capony

INSERM U 454 and Clinique des Maladies Respiratoires, Hôpital Arnaud de Villeneuve, Montpellier, France


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In order to determine whether matrix metalloproteinases (MMPs) contribute to inflammation in asthma, we have examined the release of MMPs in bronchoalveolar lavage (BAL) fluids and their production and regulation by alveolar macrophages (AM), in short-term culture. BAL was collected from 38 asthmatic subjects (24 untreated and 14 treated with inhaled corticosteroids), 26 healthy nonsmokers, and 18 patients with chronic bronchitis used as a control group for another inflammation. The profile of MMPs present in BAL fluid and AM supernatant, determined by zymographic analysis, was found to be similar in all populations. The main enzyme released was identified immunologically as MMP-9, a potent collagenolytic and elastolytic enzyme. Its release, measured using enzyme immunoassay, was significantly enhanced in fluids and in AM supernatants from untreated asthmatics compared with those from the other populations. Enhanced MMP-9 levels, in asthma, could not be explained by a different sensitivity of AM to interleukin-4, interferon-gamma , or dexamethasone, compounds that have been shown to inhibit MMP-9. The phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, significantly increased MMP-9 in AM from healthy control subjects but not in those from untreated asthmatics. Calphostin C and H7, PKC inhibitors, significantly reduced PMA-stimulated MMP-9 release in AM from healthy control subjects and spontaneous MMP-9 release in AM from untreated asthmatics. H8, a PKA inhibitor, was inactive in both populations. These data suggest that the stimulation of MMP-9 release in AM from untreated asthmatic patients occurs, at least partly, via signals activating PKC.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Matrix metalloproteinases (MMPs) are major proteolytic enzymes which are involved in extracellular matrix (ECM) turnover, due to their ability to cleave all the proteins constituting ECM (1, 2). During inflammation and tissue remodeling and repair, MMP gene expression is regulated by many factors including cytokines, growth factors, proinflammatory mediators, and hormones (1, 2). Dysregulation of MMP production has been implicated in chronic inflammatory diseases (3) and several lung diseases (4). The possible implication of MMPs in asthma is suggested by morphologic studies that have observed some features of matrix remodeling, lung tissue damage, alveolar structure alterations, or abnormal repair (5).

Macrophages are the most abundant defense cells present in both normal lung tissue and chronic inflammatory lung diseases with the capacity to degrade and remodel the ECM and the basement membrane through synthesis and secretion of proteinases, including MMPs. The expression and regulation of MMPs in human macrophages has been studied using monocytes/macrophages, purified from peripheral blood mononuclear cells or alveolar macrophages (AM) (9). AM expressed MMP-9, a 92 kD gelatinase (9, 12, 14) with collagenolytic and elastolytic activities (10). Activation of AM by inflammatory agents, such as LPS and phorbol esters (phorbol 12-myristate 13-acetate [PMA]), stimulates MMP-9 production and an induction of other MMPs (15) via protein kinase C (PKC) or PGE2-dependent protein kinase A (PKA) signaling pathways (13). In asthma, there is an increase of activated macrophages found in bronchial biopsies (18). Moreover, in this disease, AM recovered by bronchoalveolar lavage (BAL) were found to be hyperactive (19, 20) and less sensitive to in vitro modulation by cytokines such as interleukin (IL)-4 (21).

The purpose of this study was, therefore, to explore whether MMPs were differently expressed and regulated in asthmatic patients and in control populations. Asthmatic patients, untreated or treated with inhaled corticosteroids, were compared with healthy, nonsmoking control subjects and with patients with chronic bronchitis (CB) enrolled as controls for another pulmonary inflammatory disease. We studied first the release of MMPs in BAL fluid and in the supernatants of AM after short-term culture, and second, their regulation by IL-4, interferon-gamma (IFN-gamma ), dexamethasone, and the phorbol ester PMA, factors known to modulate MMP production in AM from control subjects.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents

Recombinant human IFN-gamma was kindly provided by Pr. G. Garotta (Hoffmann-Laroche, Basel, Switzerland). Recombinant human IL-4 was purchased from Immugenex Corp. (Los Angeles, CA). RPMI 1640 was purchased from Seromed, Biochrom KG (Berlin, Germany); penicillin, streptomycin, L-glutamine, phosphate-buffered saline (PBS), and fetal calf serum (FCS) were obtained from Gibco BRL (Cergy Pontoise, France). Triton X-100, phenylmethylsulfonyl-fluoride (PMSF), 1-10-phenanthroline, gelatin, PMA, lipopolysaccharide (LPS), dexamethasone, and Calphostin C were purchased from Sigma Chemical Co. (St. Louis, MO). H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] and H8 [N-2-(methylaminoethyl)-5-isoquinoline-sulfonamide] were obtained from Calbiochem Corp. (La Jolla, CA). Monoclonal antibodies and enzyme immunoassay (EIA) kits for MMP-9 were obtained from Fuji Chemical Industries (Toyama, Japan).

Subjects

Thirty-eight asthmatic patients (mean ± SD: 40 ± 13 yr, range: 15-69 yr) were studied. All patients had a reversible airways disease according to the criteria of the American Thoracic Society (22). The severity of asthma, assessed by the Aas score (23), varied from mild to severe with FEV1 values ranging from 40 to 100% of their predicted values (mean ± SD: 73 ± 18%). None of these subjects was a smoker and none had any bronchial infection during the month preceding the study. Twenty-four patients had not been treated with corticosteroids, nedocromil, or cromoglycate for a period of at least 3 mo prior to the study. Fourteen patients were currently being treated with a daily dose of 1,000 µg of beclomethasone dipropionate or 800 µg budesonide and had been receiving the treatment for at least 3 mo. Their disease state was diagnosed as steady at the start of the study.

Eighteen patients with CB and/or COPD (chronic obstructive pulmonary disease) (mean ± SD: 56 ± 11 yr, range: 41-73 yr) were studied. CB and COPD were defined according to the criteria of the American Thoracic Society (22) as previously described in detail (24). Patients were excluded if they showed a history of allergic diseases or wheezing, or if they had a bronchial infection during the month preceding the study. Patients diagnosed as COPD had a FEV1 level under 70% of predicted value and displayed a 10% or less increase in their FEV1 at the time of the procedure after an inhaled dose of 200 µg of albuterol. They were all smokers (>=  30 annual packs) and had not received corticosteroids of any form during the 3 mo prior to the study.

Twenty-six healthy subjects (mean ± SD: 40 ± 14 yr, range: 23-63 yr) were used as a control group. None of these subjects was a smoker or had any bronchial infection during the previous month. None suffered from a lung disease.

Informed consent was obtained from all the participants prior to the study. The design of the study fulfilled all the criteria of the Ethics Committee of our hospital.

Recovery of BAL Fluid and AM

BAL was carried out, using fiberoptic bronchoscopy as described previously (25), in one of the subsegmental bronchi of the middle lobe by the injection of 5 aliquots of 50 ml of saline at room temperature, reaspirated by gentle syringe suction. The different BAL fractions retrieved were pooled in order to obtain enough cells.

Cells were separated from the fluid phase by centrifugation at 400 × g for 10 min. An aliquot of cells resuspended in PBS was taken to perform cell counting, to assess cellular viability using the trypan blue exclusion dye test, and to characterize the different cell populations by cytocentrifugation and May Giemsa Grünwald staining. Total cell counts and percentages of different cell types are reported in Table 1.

                              
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TABLE 1
BAL cell content

AM were isolated by adherence onto plastic wells for 1 h in RPMI containing L-glutamine (2 mM) and antibiotics (penicillin, 100 U/ml; and streptomycin, 100 µg/ml) at 37°C in a moist atmosphere of 95% air and 5% CO2. After removal of the nonadherent cells by 3 washes in RPMI with L-glutamine and antibiotics, the remaining cells were checked microscopically and found to consist of > 90% AM. Few epithelial cells were visible. Viability of AM was between 75 and 96% in all groups (mean ± SD = 86 ± 6) and cells were used immediately after separation.

Processing of BAL Fluid

The fluid phase was centrifuged at 5,000 × g for 20 min at 4°C to remove debris and stored as aliquots in plastic vials at -80°C. For zymographic analysis, Triton X100 was added at a final concentration of 0.05% (wt/vol) to fresh aliquots and the samples were concentrated using centricon filters with a membrane of 10,000 molecular weight cutoff (Amicon Inc., Beverly, MA), according to manufacturer's instructions, and kept at -20°C for subsequent analysis.

Cell Culture and Collection of Supernatants

Adherent AM were incubated in RPMI in the absence of serum, at a density of 106 cells/ml (14). Cultures were generally performed without serum as described (26, 27) or, when specified, with 10% heat-inactivated FCS. Since the viability of AM in culture in the absence of serum declines after 24 h, we selected a 24-h culture time. Supernatants were collected; centrifuged at 400 × g for 10 min, then at 13,000 × g for 30 min; and stored at -20°C for MMP measurements. The cells were counted or fixed with methanol and saved to measure their DNA content (28).

IL-4, IFN-gamma , dexamethasone, PMA, and protein kinase inhibitors were used at concentrations and for periods of time indicated in the figures or in the legends of figures.

The human monocytic-like cell line U937 (American Type Culture Collection, CRL 1593, Rockville, MD), stimulated with PMA and LPS, was used as a reference for the secretion of MMP-9 (17). Cells were maintained in RPMI containing 10% heat-inactivated FCS, L-glutamine, and antibiotics at 37°C with 5% CO2 in a humidified atmosphere.

Zymographic Analysis of MMPs

Using zymography, MMPs were detected by their capacity to degrade gelatin (29). Aliquots of concentrated BAL fluids or unconcentrated cell supernatants were run on 10% polyacrylamide gels copolymerized with gelatin under nonreducing conditions. After electrophoresis and renaturation, gels were incubated in activation buffer (30) for 18-20 h at 37°C. For inhibition studies, gel slices were incubated in activation buffer with 1,10-phenanthroline (5 mM) or EDTA (10 mM), inhibitors of metalloproteinases, and with PMSF (2 mM), an inhibitor of serine proteases. The degree of gelatin digestion on the zymographs was estimated by densitometric scanning. Results were expressed as relative gelatinolytic activities as compared with the gelatinolytic activity of samples not exposed to cytokines.

Molecular weight of the gelatinolytic bands was estimated relative to the U937 MMP-9 reference and to prestained molecular-weight markers from Novex (Novex, San Diego, CA).

Measurement of MMP-9

The absolute value of MMP-9 in unconcentrated BAL fluids and supernatants of unstimulated AM was measured by a solid-phase EIA based on the recognition of the pro and intermediate forms of MMP-9, as well as their complexed forms with tissue inhibitor of metalloproteinase (TIMP)-1 (31). The sensitivity of the assay was 3 ng/ml. The data on MMP-9 concentration in BAL fluids were normalized to the amount of albumin present and expressed in ng/µg albumin. Levels of albumin were measured using an EIA (32). The limit of detection of the assay was 10 ng/ml. In AM supernatants, results were normalized to the same number of secreting AM and MMP-9 was expressed as ng/106 AM.

Immunoprecipitation

A mouse monoclonal antihuman MMP-9 was used to immunoprecipitate MMP-9 from BAL fluids and AM supernatants. After addition of Triton X-100 at 0.5% final concentration, the samples were centrifuged at 13,000 × g to eliminate debris and then incubated with anti-MMP-9 antibody at a final concentration of 2 µg/ml in a 1.5-ml Eppendorf tube. After overnight incubation at 4°C, 10 µl of packed protein G-Sepharose was added to isolate the antigen-antibody complex and a further incubation was performed for 90 min on a rotating device. The immunoprecipitate was collected after centrifugation at 10,000 × g for 2 min and the supernatant discarded. The immunoprecipitates were washed 4 times with 1 ml PBS containing 0.5% Triton X-100. Pellets were resuspended in electrophoresis sample buffer and analyzed by zymography.

Design of the Study

Due to the limited number of cells recovered, it was not always possible to perform all tests on samples from every patient. The numbers of subjects tested are indicated in the figure legends.

Statistical Analysis of the Data

Data were analyzed using nonparametric tests. The Mann-Whitney U test was applied for unpaired comparisons and paired comparisons were analyzed using the Wilcoxon rank test. Correlations were performed using the Spearmann rank test.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Zymographic Analysis of MMPs in BAL Fluids

The profile of proteinases released into BAL fluid from untreated asthmatic subjects, healthy control subjects, and CB patients was analyzed using zymography. Representative examples of BAL fluids from 4 patients in each group are shown in Figure 1a. Five gelatinolytic activities, varying widely in intensity, were detectable in all subjects. In all samples, the more abundant gelatinolytic activity was observed in the 92 kD area, relative to molecular-weight standards. When analyzed by densitometry scanning, no significant differences were found between the three groups of subjects studied in terms of qualitative or semiquantitative analysis. When BAL fluid was submitted to immunoprecipitation with a monoclonal antibody anti-MMP-9 and analyzed by zymography, the main gelatinase activity of 92 kD was immunoprecipitated along with the two upper bands migrating over 98 kD (Figure 1b, lane 1). These two bands represent MMP-9 homodimers or heterodimers probably associated with microglobulin (29). The minor bands migrating faster than MMP-9 were not characterized immunologically. Gelatin-specific degradation by metalloproteinases was confirmed by the disappearance of all gelatin digestion bands in the presence of protease inhibitors, 1,10-phenanthroline (Figure 1b, lane 2) and EDTA (Figure 1b, lane 3), but not in the presence of PMSF (Figure 1b, lane 4).


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  		Figure 1.   Analysis of MMPs in BAL fluid. BAL fluids were analyzed by zymography. For each sample, 1% of the original volume of BAL fluid, concentrated as specified in MATERIALS AND METHODS, was loaded onto the gels. The positions of molecular weight standards are marked on the right. Arrowheads indicate the position of MMP-9. (a) Fluids harvested from 4 healthy, 4 asthmatic, and 4 CB patients. (b) Four aliquots of the same BAL fluid were analyzed, after immunoprecipitation as described in MATERIALS AND METHODS (lane 1) or without immunoprecipitation (lanes 2-4). Enzymatic activity was revealed in the absence of inhibitors (lane 1) or in the presence of 1-10 phenanthroline (1,10-Phe; lane 2), EDTA (lane 3), or PMSF (lane 4). In lane 1 the volume of fluid submitted to immunoprecipitation was 3-fold larger than that of the others.

Zymographic Analysis of MMPs Released by AM

Macrophages from 6 healthy controls secreted a major gelatinase species (Figure 2a). Its molecular weight was identical to the MMP-9/92 kD gelatinase B released by U937 cells stimulated with PMA and LPS and used as a standard (Figure 2a, lane 1, arrowhead). The pattern of products secreted by AM from asthmatics (Figure 2b, lanes 1 and 2) and CB patients was similar (Figure 2b, lanes 3 and 4). Again, gelatin digestion bands disappeared in the presence of 1,10-phenanthroline (Figure 2b, lanes 5 and 6) and EDTA, but not in the presence of PMSF (Figure 2b, lanes 7 and 8). Gelatinolytic activity was immunoprecipitated with a monoclonal antibody to MMP-9, confirming its identity with MMP-9 (not shown). These data show that the pattern of proteinases released in BAL fluids and by AM are similar in all populations studied.


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  		Figure 2.   Analysis of MMPs released by AM in culture. Macrophages and U937 cells stimulated by PMA and LPS (17) were cultured in serum-free medium for 24 h. Culture supernatants were analyzed by zymography as described in MATERIALS AND METHODS. (a) U937 (lane 1). The upper band (arrowhead) is the MMP-9/92 kD reference enzyme (see MATERIALS AND METHODS); macrophages harvested from 6 healthy patients (lanes 2-7). (b) Macrophages harvested from 2 asthmatic (lanes 1-2) and 2 CB (lanes 3-8) subjects. Enzymatic activity was revealed in the absence of inhibitors (lanes 1-4) or in the presence of 1-10 phenanthroline (1,10-Phe; lanes 5 and 6) or PMSF (lanes 7 and 8). For each sample, a volume of unconcentrated cell supernatant corresponding to 0.4 µg DNA of secreting cells was loaded onto the gels. The positions of molecular weight standards are marked on the right.

Measurement of MMP-9 in BAL Fluids and AM Supernatants

Since MMP-9 represents the major MMP present in BAL fluid and in AM supernatants, it was quantitated using EIA in the different groups of subjects. The mean MMP-9 concentration, normalized to BAL fluid albumin content, was > 4 times higher in BAL fluids obtained from untreated asthmatics than in those of healthy controls (P = 0.03) and was also higher than in BAL fluid from corticosteroid-treated asthmatics or from CB patients (Figure 3a). The concentration of MMP-9 in BAL fluids recovered from CB subjects was 2.5 times greater than that detected in BAL fluid from healthy control subjects (P = 0.03). There was, however, no correlation between the amount of MMP-9 in BAL fluids and the severity of asthma (Figure 3; Aas scores < 3 [closed circles] or >=  3 [open circles]) or between the amount of MMP-9 and the presence of bronchial obstruction in the CB population (Figure 3 with [open circles] or without [closed circles] bronchial obstruction). However, in a few patients, high MMP-9 concentrations (Figure 3a, open circles and *) were found to be associated with an elevated percentage of neutrophils (>=  10%) among the cells recovered in BAL fluid.


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  		Figure 3.   Quantitative analysis of MMP-9 in BAL fluids and AM culture supernatants. Unconcentrated BAL fluids (a) or AM culture supernatants (b) were assayed for MMP-9 by EIA as described in MATERIALS AND METHODS. (a) Fluids from 11 healthy control subjects and 17 untreated and 11 corticosteroid-treated (+Cs) asthmatics with Aas scores < 3 ( closed circle) or >=  3 (open circle) and 10 CB subjects with (open circle) or without ( closed circle) bronchial obstruction. (*) presence of number of neutrophils > 10% of total cells in BAL fluid. (b) Supernatants from AM, cultured in serum-free medium for 24 h, harvested from 18 healthy control subjects and 21 untreated and 12 corticosteroid-treated (+Cs) asthmatics with Aas scores < 3 ( closed circle) or >=  3 (open circle) and 14 CB subjects, with (open circle) or without ( closed circle) bronchial obstruction.

Similarly, the level of MMP-9 released by AM was the highest in the untreated asthmatic population (Figure 3b). The mean production was significantly >=  3 times greater than that of healthy control subjects (P = 0.008), CB population (P = 0.004), and treated asthmatics (P = 0.011). Again, no correlation was observed between the amount of MMP-9 released and the severity of the disease for both asthma and CB.

Effect of IL-4 and IFN-gamma on AM MMP-9 Secretion

Since IL-4 and IFN-gamma were shown previously to downregulate the biosynthesis of MMP-1 and MMP-9 by human monocytes and AM (12, 13, 16), we studied their effects in order to determine whether they would modulate differently MMP-9 in AM from untreated asthmatics.

The ability of IL-4 and IFN-gamma to inhibit MMP-9 release in AM from untreated asthmatics, healthy control subjects, or CB subjects was evaluated using zymography. The addition of either cytokine induced a slight but nonsignificant decrease in the release of MMP-9 (Figure 4). No significant difference was observed between the three groups of subjects.


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  		Figure 4.   Modulation of MMP-9 release by IL-4 and IFN-gamma . Airway macrophages from 7 healthy control subjects, 8 untreated asthmatics, and 7 CB patients were stimulated or not with IL-4 or with IFN-gamma at the indicated concentrations in serum-free medium for 24 h. The harvested culture supernatants corresponding to 0.4 µg DNA of secreting cells from unstimulated and stimulated samples from the same patient were run on the same gel. They were analyzed for gelatinolytic activities by zymography followed by scanning densitometry as described in MATERIALS AND METHODS. Results were expressed as relative gelatinolytic activities ± SD; 100% is the gelatinolytic activity of samples not exposed to cytokines.

Effect of Dexamethasone on AM MMP-9 Secretion

As dexamethasone was shown to be a potent inhibitor of MMP-9 synthesis (33), its ability to inhibit MMP-9 release was compared in AM from asthmatic patients and healthy control subjects. AM were incubated in the presence or absence of 10-6 M dexamethasone for 48 h, according to conditions previously described (33). Dexamethasone decreased MMP-9 release in AM supernatants from healthy subjects and from asthmatic patients, whether they were under steroid treatment or not, by approximately 80% in all cases (Figure 5).


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  		Figure 5.   Modulation of MMP-9 release by dexamethasone. AM from 4 healthy control subjects and 4 untreated and 3 corticosteroid-treated (+Cs) asthmatics were stimulated or not with dexamethasone at the indicated concentration in heat-inactivated 10% FCS containing medium for 48 h (33). The harvested culture supernatants were assayed for MMP-9 using EIA.

Effect of PMA and PKC Inhibitors on AM MMP-9 Secretion

We then tested the effects of PMA, an inducer of PKC and MMP-9 expression (15) in this system, as well as the effects of protein kinase inhibitors on PMA-stimulated and spontaneous MMP-9 release.

PMA stimulated MMP-9 release in AM supernatants from healthy subjects (P = 0.007) and to a lesser extent in those from untreated asthmatic patients (Figure 6).


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  		Figure 6.   Effects of PMA on MMP-9 release by AM from healthy subjects and untreated asthmatics. AM from 8 healthy and 7 untreated asthmatic subjects were incubated in 10% heat-inactivated FCS containing medium for 24 h in the presence or absence of PMA (50 ng/ml). The harvested culture supernatants were assayed for MMP-9 by EIA.

In the presence of the PKC inhibitors Calphostin C and H7, PMA-dependent MMP-9 release was significantly inhibited in AM from healthy subjects (P = 0.008), but not in those from untreated asthmatic patients (Figure 7a). The PKA inhibitor H8 had no inhibitory effect on MMP-9 release in AM from both populations (Figure 7a).


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  		Figure 7.   Effects of protein kinase inhibitors on PMA-dependent and spontaneous MMP-9 release by AM from healthy subjects and untreated asthmatics. AM from 6 healthy control subjects and 6 untreated asthmatics were incubated in the presence (a) or absence (b) of PMA plus Calphostin C (250 nM), H7 (20 µM), and H8 (2.5 µM) (3) added 1 h prior to PMA. PMA-dependent MMP-9 values are the difference between MMP-9 values obtained from AM incubated in the presence of PMA and in its absence. Results are presented as percentage of MMP-9 value (± SD) obtained from AM incubated in the absence of inhibitor.

Spontaneous MMP-9 release by AM from control subjects was not inhibited by Calphostin C or H8, while it was downregulated by H7 (Figure 7b). In contrast, MMP-9 release by AM from untreated asthmatics was significantly decreased by H7 and to a lesser extent by Calphostin C (P  = 0.03), but not by H8 (Figure 7b).

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, MMP-9 was found to be increased in BAL fluids and in supernatants from AM recovered by BAL from untreated asthmatic patients, in comparison with control healthy subjects, patients suffering from chronic bronchitis, and asthmatic patients treated with inhaled corticosteroids. Our data, based on the differential effects of protein kinase inhibitors on MMP-9 release by AM, suggest that this increase is at least partly dependent on signals activating PKC pathway.

The production and the regulation of MMPs by AM has been largely investigated in vitro. First, they can degrade ECM components and connective tissues via their own production and secretion of MMPs. Second, they are easily accessible by BAL and provide a model to study the production and regulation of MMPs by inflammatory mediators. AM have been demonstrated to spontaneously secrete MMP-9 (14, 15). Stimulation of MMP-9 and induction of other collagenases such as MMP-1, MMP-2, and MMP-3 occur with differentiation and with activation by PMA and/or LPS (15). In asthma, AM are present in the airways inflammatory infiltrate and have been detected in biopsies in the submucosa and among epithelial cells (18, 34, 35). Several studies revealed an increased activation of AM recovered by BAL (19, 36) which, in some cases, was significantly correlated with the severity of asthma (40, 41). In our study, we found that MMP-9 was the major MMP detected in both BAL fluid and AM supernatants from all populations. Two additional MMPs, not reacting with anti-MMP-9 antibody, were detected in BAL fluids, but they were present in all populations. They could possibly be MMP-2 and MMP-1 or -3 according to their molecular weight, but we could not identify them by Western blot and immunodetection. They were not detected in AM supernatants, indicating that activation of AM in asthma is not accompanied by induction of any other MMP. If such an induction exists, it is not detectable by zymography under our culture conditions. These MMPs may originate from other cell types, such as epithelial or stromal cells (42, 43).

In AM from untreated asthmatics, the amplitude of MMP-9 increase is highly variable, possibly due to the heterogeneity of the disease and of the cells, which are in different states of activation and differentiation (44, 45). But the increase was statistically significant in comparison with the other populations studied. This increase was not observed in patients suffering from chronic bronchitis, enrolled as control subjects with another inflammatory disease of the airways, in which inflammation is a continuous process (46) leading to fibrosis of the airway wall with ECM deposition (47, 48). MMP-9 content was also higher in BAL fluid of untreated asthmatics than in control populations, indicating that MMP-9 released by AM contributes mainly to MMP-9 levels found in BAL fluid. However, we observed that the highest MMP-9 levels in BAL fluids correlated with a high percentage of neutrophils among total cells. The release of MMP-9 by neutrophils (14) may explain this observation.

Corticosteroids are potent inhibitors of MMPs in vitro (33), but the in vivo effect of inhaled corticosteroids have not yet been reported. They also inhibit inflammatory mediators and cytokines, and were found to decrease AM activation in vivo (38). We found here that the levels of MMP-9 released by AM from corticosteroid-treated asthmatic patients was similar to that of AM from normal subjects. This result suggests that inhaled corticosteroids, in vivo, either directly inhibit MMP-9 transcription or indirectly inhibit the release of inflammatory mediators stimulating MMP production.

We have attempted to provide some insight into the mechanism of MMP-9 increase by investigating the regulation of MMP-9 release in AM from untreated asthmatic patients by agents known to modulate MMP in monocytes and AM. First, spontaneous and stimulated release of MMP-9, -1, and -3 have been demonstrated to be suppressed by IL-4 and IFN-gamma in AM from smokers (12, 16). Moreover, stimulated---but not spontaneous---MMP-9 release by monocytes/macrophages was inhibited by IL-4 and IFN-gamma via a PGE2-cAMP-dependent pathway (13). In our study, IL-4 and IFN-gamma did not display any significant inhibitory effect on MMP-9 release by AM from all populations studied. In fact, these compounds were found to inhibit MMP-9 release with a highly variable efficiency according to the subject tested. These variations are probably related to the heterogeneity of AM and/or subjects. However, IL-4 and IFN-gamma effects were not different in AM from untreated asthmatic patients, indicating that the stimulus responsible for increased MMP-9 release in these subjects is unlikely to proceed via the PGE2-cAMP-dependent pathway.

Second, dexamethasone, a potent inhibitor of all MMPs in AM (33), was found to suppress to the same extent MMP-9 release in AM obtained from healthy controls as well as from untreated and corticosteroid-treated asthmatic patients.

Third, it has been shown that MMP-9, which is expressed by mononuclear phagocytes at an early stage of differentiation, increases as monocytes differentiate into macrophages in vitro and in vivo (9, 49, 50). PMA, an inducer of cell differentiation and of MMP synthesis via a pathway involving PKC activation (15), significantly increased MMP-9 release in AM from healthy controls (Figure 6). In our study, PMA-dependent MMP-9 release was inhibited by PKC inhibitors Calphostin C and H7 (51, 52) (Figure 7b). These results are in agreement with those found using peritoneal mouse macrophages (27) and human fibroblasts (3). In untreated asthmatics, Calphostin C and H7 had no inhibitory effect, or it may be undetectable since PMA-dependent MMP-9 release was low. These results suggest that, in asthma, either activation of PKC has already occurred in vivo or AM are refractory to PMA. H8, a PKA inhibitor (53), had no inhibitory effects in AM from healthy controls and asthmatic patients. On the other hand, spontaneous MMP-9 release was significantly decreased by both PKC inhibitors in asthmatics. Spontaneous MMP-9 release in healthy control subjects was not inhibited by Calphostin C but was inhibited by H7, although to a lesser extent than in asthmatics. Calphostin C is highly specific for PKC, whereas H7 has less specificity for PKC, inhibiting also serine/threonine protein kinases (51, 53). The fact that in our experiments H7 was more potent than Calphostin C suggests that MMP-9 release is mediated by both PKC and serine/threonine protein kinases. Thus our findings suggest that MMP-9 stimulation in AM from untreated asthmatic patients occurred via signals activating, at least partially, PKC. The absence of any significant inhibitory effect of H8 suggested that spontaneous MMP-9 release in asthma does not proceed through a cAMP-dependent protein kinase pathway.

MMPs have been implicated in several pulmonary diseases characterized by lung tissue damage, alveolar structure alterations, or abnormal repair. Neutrophil collagenase has been detected in BAL fluids in acute respiratory distress syndrome, in interstitial fibrosis diseases (review by O'Connor and FitzGerald [4]) and in the sputum of cystic fibrosis subjects (54). In asthma, the mean production of MMP-9 by AM was increased by 3- to 4-fold on a per-cell basis and over in BAL fluids, but can reach > 10-fold in some individuals. If one considers that the number of AM are locally increased in inflammatory conditions and that the inflammation is chronic, then the actual in vivo concentration of MMP-9 may be more important and its effect potentially more deleterious. MMP-9 is able to degrade type IV collagen, native type V collagen, denatured collagens, and fibronectin; also has elastolytic capacity (55, 56); and may be considered in ECM breakdown and elastolysis observed in the biopsies of asthmatic patients (8). MMPs are secreted as inactive zymogens and require in vitro chemical activators, other enzymes, or in vivo pathophysiologic conditions to digest their substrates (1). In vitro contact between cells and specific matrices has also been shown to activate MMPs (57, 58). Our results show that MMP-9 is secreted by AM in its 92 kD inactive zymogen form. This is probably due to the fact that, in our experiments, the synthesis and release of MMP-9 occurs in vitro on a plastic surface and not under physiologic conditions or on matrices. However, in asthma there are potential activators of MMP-9 such as highly reactive oxygen species, generated in excess by various cells, including AM. In addition, latent MMP-9 has been shown to be sensitive to oxidative activation (59).

In conclusion, our findings demonstrate a significant increased release of MMP-9 in AM from asthmatics via PKC activation, suggesting its implication in inflammatory processes in asthma. Its significance requires further studies, however, since MMPs are produced by a large variety of other lung cells and macrophage-mediated connective tissue alterations would depend on the balance between MMP-9 and its inhibitor TIMP-1. TIMP-1 production and regulation in AM is currently under study.

    Footnotes

Address correspondence to: Dr. Françoise Capony, INSERM U454, Hôpital Arnaud de Villeneuve, Avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France. E-mail: caponi{at}montp.inserm.fr

(Received in original form March 1, 1996 and in revised form December 10, 1996).

Acknowledgments: Gisèle Mautino is supported by AIR 2 CT94 1166 contract from the European Union. The authors thank Drs. D. Jaffuel, M. Mathieu, I. Vachier, and H. Yssel for their helpful suggestions in the preparation of the manuscript.

Abbreviations AM, alveolar macrophages; BAL, bronchoalveolar lavage; CB, chronic bronchitis; ECM, extracellular matrix; EIA, enzyme immunoassay; FCS, fetal calf serum; IFN-gamma , interferon-gamma; IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; C, protein kinase A; PKC, PKA; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl-fluoride; TIMP-1, tissue inhibitor of metalloproteinase.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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