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From the Department of Respiratory Diseases*and Laboratory of Biology of Tumours and Development,University of Liege, Liege; and the Department of Respiratory Diseases,Ghent University Hospital, Ghent, Belgium
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Abstract |
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Matrix metalloproteinases (MMPs) are a family of calcium- and zinc-dependent enzymes involved in many physiological and pathological processes. Most MMPs are secreted from the cells as inactive zymogens requiring the cleavage of an amino terminal peptide of 
10 kd for activation. The mechanisms leading to activation of MMPs in vivo are poorly known but the plasminogen/plasmin system is likely to be involved.4
MMPs are selectively inhibited by the tissue inhibitor of metalloproteinases (TIMPs). MMP-9 is produced in vivo by many inflammatory cells (eosinophils, lymphocytes, neutrophils, macrophages, and so forth) and resident cells of the lungs such as bronchial epithelial cells and alveolar epithelium.5-8
MMP-9 is considered to play a key role in inflammatory cell trafficking and inflammation through the degradation of type IV collagen, the major component of basement membranes.9,10
Several observations suggest a potential role of MMP-9 in the pathogenesis of asthma. Alveolar macrophages of patients with asthma spontaneously release an increased amount of MMP-9.7
MMP-9 concentration is elevated in induced sputum from patients with asthma.11,12
Allergen challenge results in an increase of MMP-9 in the bronchoalveolar lavage fluid (BALF) in asthmatics13
and the inhibition of MMPs by intranasally administered TIMP-2 decreased the airway responsiveness in a mouse model of asthma.14
However, no experimental data are available on whether the absence of endogenous MMP-9 influences allergen-induced airway changes. To address this question, we applied a murine model of allergen-induced asthma to MMP-9-/- knockout mice and their corresponding WT littermates. In this model, we also studied the link between MMP-9 expression during the allergic pulmonary inflammation and production of other MMPs, urokinase-type plasminogen activator (uPA) and their inhibitors, TIMP-1 and plasminogen activator inhibitor-1 (PAI-1), respectively.
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Materials and Methods |
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MMP-9-/- mice and matched wild-type (WT) control (MMP-9+/+) littermates were generated as described previously15 and kindly provided by Professor Z. Werb (University of California at San Francisco, San Francisco, CA). The mice used were littermates produced by mating of heterozygous brothers and sisters. The mice were between 6 and 8 weeks old at the beginning of the experimental protocol. All in vivo manipulations were approved by the local Ethics Committee.
Sensitization and Allergen Exposure
On the first day of the experiments (day 0), all mice (n = 6 to 7 per group) were actively immunized by intraperitoneal injection of 10 µg ovalbumin (OVA) (Sigma, St Louis, MO), adsorbed to 1 mg of aluminum hydroxide. From day 14 to day 21, the mice were exposed daily to phosphate-buffered saline (PBS) or OVA aerosols (1%) for 30 minutes in a Plexiglas exposure chamber (30 x 20 x 15 cm). The aerosol was produced by an ultrasonic nebulizer (Ultraschallvernebler Sirius Nova; Heyer Medizintechnologie, Bad Ems, Germany) as described elsewhere.16 According to the specifications of the manufacturer, the output of the nebulizer was 3 ml/min and the mean particle size of the aerosol was 3.2 µm.
Airway Responsiveness Measurements
Airway responsiveness to carbachol was measured 24 hours after the final allergen exposure as previously described.16 The mice were anesthetized with pentobarbital (100 mg/kg intraperitoneally) and a tracheal cannula was inserted. The femoral artery and the jugular vein were catheterized. The animals, placed on a 37°C heated blanket, were ventilated with a Palmer respirator (Bioscience, Sheerness, UK) at 145 strokes/minute (stroke volume of 0.5 ml). To prevent spontaneous respiration, neuromuscular blockade was induced by injecting pancuronium bromide (1 mg/kg i.v.) (Organon Teknika N.V., Turnhout, Belgium). The lung resistance (RL) was calculated from the differential pressure between the airways and the pleural cavity, tidal volume, and flow. Measurements were performed with a computerized pulmonary mechanics analyzer (Mumed PMS800 system; Mumed Ltd., London, UK). Increasing doses of carbachol were administered (microinfusion pump) intravenously (20, 40, 80, and 160 µg/kg). Between each dose, the airway resistance was allowed to return to the baseline level.
Bronchoalveolar Lavage (BAL)
Twenty-four hours after the final allergen exposure and immediately after the assessment of airway responsiveness, 1 ml of Hanks’ balanced salt solution free of ionized calcium and magnesium but supplemented with 0.05 mmol/L of sodium ethylenediaminetetraacetic acid was instilled four times via the tracheal cannula and recovered by gentle manual aspiration. The recovered BALF was centrifuged (1800 rpm for 10 minutes at 4°C). The supernatant was processed for protein assessments whereas the cell pellet was washed twice and finally resuspended in 1 ml of Hanks’ balanced salt solution. A total cell count was performed in a Bürker chamber and the differential cell counts on at least 400 cells were performed on cytocentrifuged preparations (Cytospin 2; Cytospin, Shandon Ltd., Runcorn, Cheshire, UK) using standard morphological criteria after staining with May-Grünwald-Giemsa. The fluid phase of the BALF was stored at -80°C until analyzed in zymography.
Pulmonary Tissue Processing
After BAL, the thorax was opened and the left main bronchus was clamped. The left lung was excised and frozen immediately in liquid N2 for protein chemistry and mRNA extraction while the right lung was processed for histology.
Histological Analysis of Pulmonary Inflammation
As previously described,16 the right lung was infused with 4% paraformaldehyde and embedded in paraffin. Sections of 2.5-µm thickness from all lobes were stained with hematoxylin and eosin. The extent of peribronchial infiltrates was estimated by an inflammation score and by quantifying the peribronchial mononuclear cells and eosinophils. Slides were coded and the peribronchial inflammation was graded in a blinded manner using a reproducible scoring system described elsewhere.16 A value from 0 to 3 per criteria was adjudged to each tissue section scored. A value of 0 was adjudged when no inflammation was detectable, a value of 1 for occasional cuffing with inflammatory cells, a value of 2 when most bronchi were surrounded by a thin layer (one to five cells) of inflammatory cells, and a value of 3 when most bronchi were surrounded by a thick layer (more than five cells) of inflammatory cells. Because five to seven randomly selected tissue sections per mouse were scored, inflammation scores could be expressed as a mean value per animal and could be compared between groups.
In addition, the cells in the peribronchial area were counted relative to the length of the basal membrane (total bronchial inflammation index expressed as number of inflammatory cells/µm basal membrane). The cellular composition of the infiltrates was analyzed and expressed as percents. By multiplying the total bronchial inflammation index with the cellular composition, an inflammatory score was obtained. Five to seven peribronchial infiltrates per animal were scored.
Lung Protein Extraction
The left lung was snap-frozen in liquid nitrogen and crushed using a Mikro-Dismembrator S (Braun Biotech Int., Melsungen, Germany). The crushed lung tissue was incubated overnight at 4°C in a solution containing 2 mol/L urea, 1 mol/L NaCl, and 50 mmol/L Tris (pH 7.5) and subsequently centrifuged 15 minutes at 16,000 x g. The supernatants were frozen and stored at -80°C before performing zymography.
Zymography
Gelatin zymography was performed as previously described.11 Gelatinase activity was detected as white lysis bands against a blue background and quantitative evaluation of the gelatinolytic activity was performed by scanning the gel using a Bio-Rad GS 700 imaging densitometer (Bio-Rad, Hercules, CA). Dilutions of culture medium conditioned by HT1080 cells were used as an internal standard. Gelatinolytic activity of MMP-2 and MMP-9 was determined by scanning the lysis band in the 72-kd and the 105-kd area, respectively.
Interleukin (IL)-13 Measurements
IL-13 was measured in the BAL using a commercially available enzyme-linked immunosorbent assay (mouse IL-13 immunoassay Quantikine M, R&D Systems, Minneapolis, MN). According to the manufacturer’s data, the lower limit of detection was less than 1.5 pg/ml.
Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for MMP and TIMP Transcripts
Total RNA was obtained from the lungs crushed in liquid nitrogen by cesium chloride ultracentrifugation.17 28S rRNA and MMP and TIMP mRNA were measured in 10-ng aliquots of total RNA by RT-PCR. An external control RNA template (synthetic RNA) was introduced in each sample to monitor the RT-PCR reaction and to allow the quantitation of each endogenous mRNA.18 RT-PCR was performed using the GeneAmp Thermostable rTth reverse transcriptase RNA PCR kit (Perkin Elmer, Branchburg, NJ) and two pairs of primers (Invitrogen, Carlsbad, CA). The primers used for RT-PCR reaction were 5'-AGATCTTCTTCTTCAAGGACCGGTT-3' (sense) and 5'-GGCTGGTCAGTGGCTTGGG-GTA-3' (anti-sense) for MMP-2, 5'-GATCTCTTCATTTTGGCCATCTCTTC-3' (sense) and 5'-CTCCAGTATTTGTC- CTCTACAAAGAA-3' (anti-sense) for MMP-3, 5'-CCAAG-TGG-GAACGCACTAACTTGA-3' (sense) and 5'-TGGAGAA-TTGTCACCGTGATCTCTT-3' (anti-sense) for MMP-8, 5'-CC-CACATTTGACGTCCAGAGAAGAA-3' (sense) and 5'-GTTTTTGATGCTATTGCTGAGATCCA-3' (anti-sense) for MMP-9, 5'-ACATTTCGCCT-CTCTGCTGATGAC-3' (sense) and 5'-CAGAAACCTTCAGCCAGAAGAACC-3' (anti-sense) for MMP-12, 5'-GGATACCCAATGCCCATTGGCCA-3' (sense) and 5'-CCA-TTGGGCATCCAGAAGAGAGC-3' (anti-sense) for MMP-14, 5'-GGCATCCTCTTGTTGC-TATCACTG-3' (sense) and 5'-GTCATCTTGATCTCATAACGCTGG-3' (anti-sense) for TIMP-1, 5'-CTCGCTGGACGTTGGAGGAAAGAA-3' (sense) and 5'-AGCCCATCTGGT-ACCTGTGGTTCA-3' (anti-sense) for TIMP-2, 5'-AGGGCTTCATGCCCCACTTCTTCA-3' (sense) and 5'-AGTAGAGGGCATTCACCAGCACCA-3' (anti-sense) for PAI-1, 5'-TATGC-AGCCCCACTACTATGGCTC-3' (sense) and 5'-GAAGTGTGAGACTCTCGTGTAGAC-3' (anti-sense) for uPA, 5'-GTTCACCCACTAATAGGGAACGTGA-3' (sense) and 5'-GAT-TCTGACTTAGAGGCGTTCAGT-3' (anti-sense) for 28S.
Reverse transcription was performed at 70°C for 15 minutes followed by 2 minutes of incubation at 95°C for denaturation of RNA-DNA heteroduplexes. Amplification started at 94°C for 15 seconds, 68°C for 20 seconds, and 72°C for 10 seconds and terminated by 2 minutes at 72°C. RT-PCR products were resolved on 10% acrylamide gels and analyzed using a Fluor-S MultImager (Bio-Rad) after staining with Gelstar (FMC Bioproducts, Rockland, ME) dye. Quantitative RT-PCR results are expressed as a ratio between the intensities of the bands corresponding to the endogenous RNA and to a synthetic RNA added in the RT-PCR reaction in known amounts. This ratio calculated for a MMP is further divided by the same ratio calculated for 28S rRNA in the same sample. With this method, the RT-PCR reaction is monitored and two systems of standard are included (28S rRNA and a synthetic RNA). All RT-PCR results shown are a mean of duplicates.
Statistical Analysis
Data were analyzed with the statistical package SPSS 6.1.2 (SPSS Inc., Chicago, IL). All results are expressed as means ± SEM. Kruskal-Wallis H-tests were used for screening significant differences between the groups. When P was < 0.05, Mann-Whitney U-tests were applied to compare the individual groups. The significance levels were adapted with Bonferroni’s conservative correction. The dose-response curves obtained from the pulmonary function tests were analyzed with the GLM univariate procedure, providing regression analysis and analysis of variance for one dependent variable by two factors and/or variables (the groups and the carbachol concentration).
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Results |
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Repeated daily exposure to aerosolized OVA induced a significant increase in total cell numbers in the BALF of both WT mice or MMP-9-/- mice when compared to their PBS-exposed counterparts (Table 1)
. Interestingly, MMP-9-/- had significantly less lymphocytes expressed either as a percentage or absolute value in their BALF than the WT mice after allergen exposure [3.07 versus 14.29% (P < 0.05) and 0.8 versus 6.9 x 104/ml (P < 0.05), respectively] (Table 1)
. In contrast, WT and MMP-9-/- mice showed a comparable increase in BALF macrophages and eosinophils (WT: 19.6 versus MMP-9-/-: 14.3%; P > 0.05). In all experimental groups, the neutrophil counts were not affected by allergen exposure.
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The airways from the WT (Figure 1a)
and MMP-9-/- (Figure 1c)
mice exposed to PBS aerosol showed normal lung histology in both groups. Sensitization and subsequent exposure to OVA resulted in a significant peribronchial and perivascular eosinophilic inflammation both in the WT (Figure 1b)
and MMP-9-/- (Figure 1d)
animals when compared to the PBS-exposed counterparts. The alveolar septa were free of inflammation. The peribronchial inflammation after OVA exposure was significantly lower in MMP-9-/- than in WT mice (P < 0.005) (Figure 2a)
. The difference in peribronchial inflammation was mainly because of a decrease in the mononuclear fraction of the inflammatory cell infiltrate (Figure 2b)
. Analysis of the airway wall thickness (normalized to the perimeter of the basal membrane) revealed no difference between the groups (data not shown).
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By zymography performed on whole lung extracts, proactivated and activated MMP-9 were detectable in each sample from the WT group and as expected undetectable in the lungs of MMP-9-/- mice. Quantification of zymograms by densitometric scanning revealed that MMP-9-related gelatinolytic activity was significantly higher in extracts from allergen-exposed mice when compared to those from PBS-exposed WT mice (P < 0.05) (Figure 3)
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AHR to increasing doses of carbachol was expressed as percent increase in pulmonary resistance. Exposure of WT mice to OVA induced an increased AHR to carbachol (P < 0.01) (Figure 4)
. By contrast, the pulmonary resistance of MMP-9-/- mice was not significantly different after exposure to OVA or PBS (Figure 4)
. It is worth noting that the airway responsiveness to intravenous carbachol injection was significantly higher in MMP-9-/- animals exposed to PBS than in their WT counterparts.
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28S rRNA was detected in each sample and quantitative analysis showed no differences between the experimental groups. Quantitative RT-PCR expressed as a ratio to 28S rRNA showed no significant differences between MMP-9-/- and WT or between OVA-exposed and unexposed mice for MMP-2, MMP-3, MMP-8, TIMP-2, and PAI-1 mRNA levels (data not shown). Interestingly, MMP-9 mRNA levels measured in WT mice were unchanged after allergen exposure (data not shown). MMP-12, MMP-14, and uPA mRNA levels were significantly increased after allergen exposure in the WT group (P < 0.05 for MMP-12 and MMP-14 and P < 0.005 for uPA), but not in MMP-9-/- mice. By contrast, TIMP-1 mRNA was similarly increased after allergen exposure in WT and MMP-9-/- groups (P < 0.005 and P < 0.05, respectively, compared to PBS-exposed mice) (Figure 5)
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IL-13 was measured in each BAL sample and shown to be increased after allergen exposure only in the WT group (P < 0.005) (Figure 6)
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Discussion |
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In the present study, although total airway inflammation scores were significantly lower in MMP-9-/- mice as compared to the WT mice after allergen challenge, BALF neutrophil and eosinophil counts were not different between MMP-9-/- and WT mice. Accordingly, it was demonstrated that MMP-9-/- mice display normal acute neutrophilic inflammation19 and that MMP inhibition does not inhibit granulocyte extravasation from the blood vessels.20 Using intranasally administered TIMP-2, Kumagai and colleagues14 found that eosinophil and macrophage migration toward the BAL was also impaired. This difference with our results could be explained by a nonspecific effect of high amounts of TIMP-2 regarding MMP inhibition and by collateral effects on cell growth.21 The fact that MMP-9-/- mice display lower AHR without any difference in the eosinophilic inflammation strongly supports previous observations showing that eosinophils are not essential for the development of AHR after allergen challenge.16,22-26 The significant decrease in lymphocyte counts in the BALF along with a failure to develop AHR in MMP-9-/- mice further supports the previously suggested key role of lymphocytes in the AHR. Lymphocyte migration through basement membranes is likely to involve MMP-9 production.27,28 Many reports in human asthmatics suggest a role for lymphocytes in the pathophysiology of AHR and cellular inflammation in the bronchi.29,30 In animal models, the same link between AHR and lymphocyte migration was observed in rats after allergen exposure31,32 and it was demonstrated that CD4+ T cells can transfer the AHR from one animal to another and from humans to SCID mice.26,33,34 In support of this notion, our experiments reveal an impaired production of IL-13 in the airways of sensitized/exposed MMP-9-/- animals. IL-13 is a prototypical product of allergen-primed (T helper 2) effector T lymphocytes. Several studies have documented the central role of IL-13 in allergen-induced AHR.35,36 IL-13 seems to be necessary and sufficient to induce the asthmatic phenotype in a manner that is independent from IL-4, eosinophils, and IgE. Recent data also shows that IL-13 introduced in the airways of naïve mice can directly induce AHR.37 Conversely, IL-13 probably plays a key role in the regulation of MMP expression because its overexpression in the airways of mice increases the expression of many MMPs including MMP-9.38,39 Interestingly, saline-exposed MMP-9-/- mice displayed higher levels of bronchial reactivity to the carbachol injection than did their WT counterparts. Similar observations were previously reported for IL-4-/- and IgE-/- animals.40,41 A hypothesis to explain this unexpected finding is that MMP-9 gene deletion would collaterally impair some negative feedback loops that regulates AHR and as described recently interfere with the activation and degradation of various mediators including some cytokines.42 We acknowledge that this latter observation is not easy to translate into a clinical significance for human asthma and warrants some caution regarding the use of selective MMP inhibitors, especially in a noninflammatory context.
We have investigated MMP-9 production at the mRNA and protein levels in the whole lungs of WT mice exposed or not exposed to allergen. MMP-9 protein was found to be increased after allergen challenge. Our observation is in accordance with the increase of MMP-9 protein previously reported in bronchial secretions from human asthmatics11,12 or after allergen challenge.13 Because MMP-9 mRNA is not concomitantly overexpressed, the MMP-9 protein modulation in whole lung extracts is likely to be related to an increased release of MMP-9 stored in the specific granules of granulocytes or to a production of the protein by other cell types during the 7 days of allergen challenges. It should be noted that MMP-9 was detected both in proactived and activated forms by zymography. The reason why the activated MMP-9 was present in the lung tissue of WT mice challenged either with allergens or PBS is unclear. Our hypothesis is, as suggested earlier, that most of the MMP-9 detected originated from granulocytes, which are potent activators of MMP-9.43,44 However, because many physical and chemical factors can account for the activation of MMP-9, we cannot rule out the possibility that the protein extraction by itself has induced a protease activation.
In accordance with previous data obtained from MMP-9-/- animals,15 the absence of MMP-9-related gelatinolytic activity in lung extracts of MMP-9-deficient mice was not compensated by an enhanced MMP-2-linked activity (data not shown). Furthermore, in the present study, the expression of the other tested MMPs was not enhanced at the mRNA level in MMP-9-/- mice when compared to WT. Further work is needed to explore the dense network of reciprocal interactions between MMPs and other classes of proteases to point out putative compensatory mechanisms.
We describe for the first time that the mRNA expression of MMP-12, MMP-14, and uPA are significantly increased after allergen challenge in sensitized WT animals. The lack of overexpression of these proteases in MMP-9-/- mice after allergen exposure together with an impaired inflammation may suggest that inflammatory cells may account for the protease production. MMP-14 and uPA could be responsible for the activation of other MMPs, such as MMP-2 and MMP-9, as demonstrated elsewhere45,46 and the absence of statistically significant differences in the levels of those proteins between the WT-OVA and knockout-PBS could potentially play a role in the increase of bronchial responsiveness to carbachol found in MMP-9-/- animals exposed to PBS. Because MMP-12 has been demonstrated to be essential for the development of emphysema in an animal model,47 this protease could also take part in the events leading to the asthmatic phenotype.
Because TIMP-1 mRNA was overexpressed after allergen challenge in both WT and MMP-9-/- mice, we hypothesize that TIMP-1 could be mainly expressed by lung resident cells rather than by blood-issued inflammatory cells. Many cells have been reported to produce TIMP-1 including neutrophils, macrophages, and epithelial cells.48-50 However, the protocol used in the present study did not allow the identification of cells responsible for the increased expression of TIMP-1 mRNA.
In summary, we have demonstrated that MMP-9 plays a key role in the cascade of events leading to pulmonary inflammation in an animal model of asthma. Our findings also confirm previous works suggesting a central role for lymphocytes in the pathogenesis of allergen-induced increases in airway responsiveness.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Fonds National de la Recherche Scientifique (Brussels, Belgium) (Fonds de la Recherche Scientifique Médicale), the FWO-Vlaanderen, the Concerted Research Initiative of the University of Ghent (project no GOA 98-6), the Fondation Leon Fredericq (University of Liege, Belgium), and fonds spéciaux de la recherche from the University of Liege, Belgium.
D. C. is a research fellow of the Fonds National de la Recherche Scientifique; K. T. and K. V. are research fellows of the Fund for Scientific Research–Flanders (Fonds voor Wetenschappelijk Onderzoek-Vlaanderen); and C. M. is a research associate and A. N. is a senior research associate of the Fonds National de la Recherche Scientifique.
D. D. C. and K. G. T. both contributed equally to this work.
Accepted for publication April 29, 2002.
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