Matrix Metalloproteinase-9 Deficiency Impairs Cellular Infiltration and Bronchial Hyperresponsiveness during Allergen-Induced Airway Inflammation
2002; Elsevier BV; Volume: 161; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64205-8
ISSN1525-2191
AutoresDidier Cataldo, Kurt G. Tournoy, Karim Vermaelen, Carine Munaut, Jean‐Michel Foidart, Renaud Louis, Agnès Noël, Romain Pauwels,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoWe investigated the specific role of matrix metalloproteinase (MMP)-9 in allergic asthma using a murine model of allergen-induced airway inflammation and airway hyperresponsiveness in MMP-9−/− mice and their corresponding wild-type (WT) littermates. After a single intraperitoneal sensitization to ovalbumin, the mice were exposed daily either to ovalbumin (1%) or phosphate-buffered saline aerosols from days 14 to 21. Significantly less peribronchial mononuclear cell infiltration of the airways and less lymphocytes in the bronchoalveolar lavage fluid were detected in challenged MMP-9−/− as compared to WT mice. In contrast, comparable numbers of bronchoalveolar lavage fluid eosinophils were observed in both genotypes. After allergen exposure, the WT mice developed a significant airway hyperresponsiveness to carbachol whereas the MMP-9−/− mice failed to do so. Allergen exposure induced an increase of MMP-9-related gelatinolytic activity in WT lung extracts. Quantitative reverse transcriptase-polymerase chain reaction showed increased mRNA levels of MMP-12, MMP-14, and urokinase-type plasminogen activator after allergen exposure in the lung extracts of WT mice but not in MMP-9-deficient mice. In contrast, the expression of tissue inhibitor of metalloproteinases-1 was enhanced after allergen exposure in both groups. We conclude that MMP-9 plays a key role in the development of airway inflammation after allergen exposure. We investigated the specific role of matrix metalloproteinase (MMP)-9 in allergic asthma using a murine model of allergen-induced airway inflammation and airway hyperresponsiveness in MMP-9−/− mice and their corresponding wild-type (WT) littermates. After a single intraperitoneal sensitization to ovalbumin, the mice were exposed daily either to ovalbumin (1%) or phosphate-buffered saline aerosols from days 14 to 21. Significantly less peribronchial mononuclear cell infiltration of the airways and less lymphocytes in the bronchoalveolar lavage fluid were detected in challenged MMP-9−/− as compared to WT mice. In contrast, comparable numbers of bronchoalveolar lavage fluid eosinophils were observed in both genotypes. After allergen exposure, the WT mice developed a significant airway hyperresponsiveness to carbachol whereas the MMP-9−/− mice failed to do so. Allergen exposure induced an increase of MMP-9-related gelatinolytic activity in WT lung extracts. Quantitative reverse transcriptase-polymerase chain reaction showed increased mRNA levels of MMP-12, MMP-14, and urokinase-type plasminogen activator after allergen exposure in the lung extracts of WT mice but not in MMP-9-deficient mice. In contrast, the expression of tissue inhibitor of metalloproteinases-1 was enhanced after allergen exposure in both groups. We conclude that MMP-9 plays a key role in the development of airway inflammation after allergen exposure. Allergen-induced airway inflammation, which is orchestrated by activated T cells and T-cell-derived cytokines, is thought to be the cornerstone in the pathogenesis of allergen-induced airway hyperresponsiveness (AHR) in asthma.1Wardlaw AJ Dunnette S Gleich GJ Collins JV Kay AB Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Relationship to bronchial hyperreactivity.Am Rev Respir Dis. 1988; 137: 62-69Crossref PubMed Scopus (793) Google Scholar, 2Louis R Lau LC Bron AO Roldaan AC Radermecker M Djukanovic R The relationship between airways inflammation and asthma severity.Am J Respir Crit Care Med. 2000; 161: 9-16Crossref PubMed Scopus (487) Google Scholar Murine models of asthma mimic some features of human asthma such as the development of airway inflammation and AHR after allergen exposure and might therefore be useful to investigate the role of individual cell types and mediators.3Kips JC Tournoy KG Pauwels RA Gene knockout models of asthma.Am J Respir Crit Care Med. 2000; 162: S66-S70Crossref PubMed Scopus (18) Google Scholar 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.4Nagase H Activation mechanisms of matrix metalloproteinases.Biol Chem. 1997; 378: 151-160PubMed Google Scholar 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.5Kjeldsen L Sengelov H Lollike K Nielsen MH Borregaard N Isolation and characterization of gelatinase granules from human neutrophils.Blood. 1994; 83: 1640-1649Crossref PubMed Google Scholar, 6Legrand C Gilles C Zahm JM Polette M Buisson AC Kaplan H Birembaut P Tournier JM Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.J Cell Biol. 1999; 146: 517-529Crossref PubMed Scopus (211) Google Scholar, 7Mautino G Oliver N Chanez P Bousquet J Capony F Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics.Am J Respir Cell Mol Biol. 1997; 17: 583-591Crossref PubMed Scopus (204) Google Scholar, 8Ohno I Ohtani H Nitta Y Suzuki J Hoshi H Honma M Isoyama S Tanno Y Tamura G Yamauchi K Nagura H Shirato K Eosinophils as a source of matrix metalloproteinase-9 in asthmatic airway inflammation.Am J Respir Cell Mol Biol. 1997; 16: 212-219Crossref PubMed Scopus (212) Google Scholar 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.9Delclaux C Delacourt C d'Ortho MP Boyer V Lafuma C Harf A Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane.Am J Respir Cell Mol Biol. 1996; 14: 288-295Crossref PubMed Scopus (366) Google Scholar, 10Ivanoff A Ivanoff J Hultenby K Sundqvist KG Infiltrative capacity of T leukemia cell lines: a distinct functional property coupled to expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1).Clin Exp Metastasis. 1999; 17: 695-711Crossref PubMed Scopus (37) Google Scholar 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-9.7Mautino G Oliver N Chanez P Bousquet J Capony F Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics.Am J Respir Cell Mol Biol. 1997; 17: 583-591Crossref PubMed Scopus (204) Google Scholar MMP-9 concentration is elevated in induced sputum from patients with asthma.11Cataldo D Munaut C Noël A Frankenne F Bartsch P Foidart J Louis R MMP-2 and MMP-9 derived gelatinolytic activity in the sputum from patients with asthma and COPD.Int Arch Allergy Immunol. 2000; 123: 259-267Crossref PubMed Scopus (194) Google Scholar, 12Vignola AM Riccobono L Mirabella A Profita M Chanez P Bellia V Mautino G D'accardi P Bousquet J Bonsignore G Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis.Am J Respir Crit Care Med. 1998; 158: 1945-1950Crossref PubMed Scopus (340) Google Scholar Allergen challenge results in an increase of MMP-9 in the bronchoalveolar lavage fluid (BALF) in asthmatics13Becky Kelly EA Busse WW Jarjour NN Increased matrix metalloproteinase-9 in the airway after allergen challenge.Am J Respir Crit Care Med. 2000; 162: 1157-1161Crossref PubMed Scopus (170) Google Scholar and the inhibition of MMPs by intranasally administered TIMP-2 decreased the airway responsiveness in a mouse model of asthma.14Kumagai K Ohno I Okada S Ohkawara Y Suzuki K Shinya T Nagase H Iwata K Shirato K Inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma.J Immunol. 1999; 162: 4212-4219PubMed Google Scholar 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. MMP-9−/− mice and matched wild-type (WT) control (MMP-9+/+) littermates were generated as described previously15Vu TH Shipley JM Bergers G Berger JE Helms JA Hanahan D Shapiro SD Senior RM Werb Z MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1474) Google Scholar 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. 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 × 20 × 15 cm). The aerosol was produced by an ultrasonic nebulizer (Ultraschallvernebler Sirius Nova; Heyer Medizintechnologie, Bad Ems, Germany) as described elsewhere.16Tournoy KG Kips JC Schou C Pauwels RA Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness.Clin Exp Allergy. 2000; 30: 79-85Crossref PubMed Scopus (158) Google Scholar 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 to carbachol was measured 24 hours after the final allergen exposure as previously described.16Tournoy KG Kips JC Schou C Pauwels RA Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness.Clin Exp Allergy. 2000; 30: 79-85Crossref PubMed Scopus (158) Google Scholar 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. 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. 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. As previously described,16Tournoy KG Kips JC Schou C Pauwels RA Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness.Clin Exp Allergy. 2000; 30: 79-85Crossref PubMed Scopus (158) Google Scholar 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.16Tournoy KG Kips JC Schou C Pauwels RA Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness.Clin Exp Allergy. 2000; 30: 79-85Crossref PubMed Scopus (158) Google Scholar 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. 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 × g. The supernatants were frozen and stored at −80°C before performing zymography. Gelatin zymography was performed as previously described.11Cataldo D Munaut C Noël A Frankenne F Bartsch P Foidart J Louis R MMP-2 and MMP-9 derived gelatinolytic activity in the sputum from patients with asthma and COPD.Int Arch Allergy Immunol. 2000; 123: 259-267Crossref PubMed Scopus (194) Google Scholar 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. 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. Total RNA was obtained from the lungs crushed in liquid nitrogen by cesium chloride ultracentrifugation.17Chirgwin JM Przybyla AE MacDonald RJ Rutter WJ Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16610) Google Scholar 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.18Lambert CA Colige AC Munaut C Lapiere CM Nusgens BV Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.Matrix Biol. 2001; 20: 397-408Crossref PubMed Scopus (89) Google Scholar 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. 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). 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 × 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.Table 1BALF Cell CountsWT/PBSWT/OVAKO/PBSKO/OVATotal cells (× 104/ml)12.5 ± 3.637.5 ± 12.5*, P < 0.05 versus PBS-exposed counterparts.11.6 ± 3.226.0 ± 4.3*, P < 0.05 versus PBS-exposed counterparts.Macrophage (× 104/ml)12.4 ± 3.623.8 ± 8.3*, P < 0.05 versus PBS-exposed counterparts.11.5 ± 3.221.6 ± 3.2*, P < 0.05 versus PBS-exposed counterparts.Lymphocyte (× 104/ml)0.0 ± 0.06.9 ± 3.1*, P < 0.05 versus PBS-exposed counterparts.†, P < 0.05 versus OVA-exposed KO mice.0.0 ± 0.00.8 ± 0.2*, P < 0.05 versus PBS-exposed counterparts.Neutrophil (× 104/ml)0.0 ± 0.00.3 ± 0.20.1 ± 0.10.0 ± 0.0Eosinophil (× 104/ml)0.0 ± 0.06.4 ± 1.6*, P < 0.05 versus PBS-exposed counterparts.0.0 ± 0.04.3 ± 1.9*, P < 0.05 versus PBS-exposed counterparts.Total cell numbers and cellular composition of BALF performed 24 hour after the last allergen exposure in actively sensitized WT and MMP-9 deficient (KO) mice exposed to OVA (n = 7 and n = 6, respectively), aerosol, or PBS (n = 7 and n = 6, respectively). Kruskal-Wallis:* , P < 0.05 versus PBS-exposed counterparts.† , P < 0.05 versus OVA-exposed KO mice. Open table in a new tab Total cell numbers and cellular composition of BALF performed 24 hour after the last allergen exposure in actively sensitized WT and MMP-9 deficient (KO) mice exposed to OVA (n = 7 and n = 6, respectively), aerosol, or PBS (n = 7 and n = 6, respectively). Kruskal-Wallis: 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).Figure 2Quantification of histological inflammation. Mean peribronchial inflammation scores (a) and cellular composition of the infiltrates (b) were determined in the four groups as described in the Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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). 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. 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). 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). Although clinical observations suggest a putative role for MMP-9 in the cascade of events leading to clinical asthma, the exact role of MMP-9 in the pathogenesis of asthma remains to be determined. In the current experiments, MMP-9−/− mice showed a decreased lymphocytic inflammation and peribronchial mononuclear cell infiltration as compared to WT animals sensitized and exposed to allergen and failed to develop allergen-induced AHR. 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 inflammation19Betsuyaku T Shipley JM Liu Z Senior RM Neutrophil emigration in the lungs, peritoneum, and skin does not require gelatinase B.Am J Respir Cell Mol Biol. 1999; 20: 1303-1309Crossref PubMed Scopus (126) Google Scholar and that MMP inhibition does not inhibit granulocyte extravasation from the blood vessels.20Mackarel AJ Cottell DC Russell KJ FitzGerald MX O'Connor CM Migration of neutrophils across human pulmonary endothelial cells is not blocked by matrix metalloproteinase or serine protease inhibitors.Am J Respir Cell Mol Biol. 1999; 20: 1209-1219Crossref PubMed Scopus (77) Google Scholar Using intranasally administered TIMP-2, Kumagai and colleagues14Kumagai K Ohno I Okada S Ohkawara Y Suzuki K Shinya T Nagase H Iwata K Shirato K Inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma.J Immunol. 1999; 162: 4212-4219PubMed Google Scholar 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.21Edwards DR Beaudry PP Laing TD Kowal V Leco KJ Leco PA Lim MS The roles of tissue inhibitors of metalloproteinases in tissue remodelling and cell growth.Int J Obes Relat Metab Disord. 1996; 20: S9-S15PubMed Google Scholar 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.16Tournoy KG Kips JC Schou C Pauwels RA Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness.Clin Exp Allergy. 2000; 30: 79-85Crossref PubMed Scopus (158) Google Scholar, 22Matsubara S Fushimi K Ogawa K Kikkawa H Nakata A Kameda R Kikuchi M Naito K Ikezawa K Inhibition of pulmonary eosinophilia does not necessarily prevent the airway hyperresponsiveness induced by Sephadex beads.Int Arch Allergy Immunol. 1998; 116: 67-75Crossref PubMed Scopus (15) Google Scholar, 23Lefort J Bachelet CM Leduc D Vargaftig BB Effect of antigen provocation of IL-5 transgenic mice on eosinophil mobilization and bronchial hyperresponsiveness.J Allergy Clin Immunol. 1996; 97: 788-799Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 24Coyle AJ Kohler G Tsuyuki S Brombacher F Kopf M Eosinophils are not required to induce airway hyperresponsiveness after nematode infection.Eur J Immunol. 1998; 28: 2640-2647Crossref PubMed Scopus (55) Google Scholar, 25Hessel EM Van Oosterhout AJ Van AI Van Esch B Hofman G Van Loveren H Savelkoul HF Nijkamp FP Development of airway hyperresponsiveness is dependent on interferon-gamma and independent of eosinophil infiltration.Am J Respir Cell Mol Biol. 1997; 16: 325-334Crossref PubMed Scopus (187) Google Scholar, 26Tournoy KG Kips JC Pauwels RA The allergen-induced airway hyperresponsiveness in a human-mouse chimera model of asthma is T cell and IL-4 and IL-5 dependent.J Immunol. 2001; 166: 6982-6991PubMed Google Scholar 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. 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