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Background: Macrophage elastase (MMP-12) is involved in the inflammatory process of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate in mice the effect of MMP-12 inhibition on the inflammatory process induced by cigarette smoke (CS) or by lipopolysaccharide (LPS) exposure of the airways. Experimental approach: C57BL/6 mice were given, orally, either the selective MMP-12 inhibitor AS111793 (3, 10, 30 and 100 mg kg−1), the PDE-4 inhibitor roflumilast (3 mg kg−1) or vehicle, then exposed to CS (for 3 days) or to LPS (100 μg mL−1, 30 min). Subsequent to the last smoke or LPS exposure, bronchoalveolar lavages (BAL) were performed and lungs were removed and homogenized to analyze various markers of inflammation at appropriate times. Key results: Inhibition of MMP-12 by AS111793 (10 and 30 mg kg−1) was associated with a reduction of the increase in neutrophil number in BAL fluids after 4 days and of macrophages after 11 days. On day 4, AS111793 also significantly reduced all the inflammation markers that had increased after CS exposure, including soluble TNF receptors I and II, MIP-1γ, IL-6 and pro-MMP-9 activity in BAL fluids, and KC/CXCL1, fractalkine/CX3CL1, TIMP-1 and I-TAC/CXCL11 in lung parenchyma. In contrast, inhibition of MMP-12 did not reduce neutrophil influx, pro-MMP-9 activity or KC/CXCL1 release in BAL fluids of mice exposed to LPS. Conclusion: Inhibition of MMP-12 with AS111793, reduced the inflammatory process associated with exposure of mice to CS, strongly suggesting a specific involvement of MMP-12 in lung inflammation following CS exposure. British Journal of Pharmacology (2008) 154, 1206–1215; doi:10.1038/bjp.2008.180; published online 19 May 2008

1. Several observations suggest that tachykinins are involved in the pathogenesis of bronchopulmonary alterations. We have investigated the effect of antagonists for tachykinin NK1 (SR 140333), NK2 (SR 48968) or NK3 (SR 142801) receptors on inflammatory cell recruitment, tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 release and matrix metalloproteinase (MMP)-9 activity in the bronchoalveolar lavage fluid (BALF) of mice exposed to lipopolysaccharide (LPS; 100 microg/mL aerosol for 30 min). 2. Treatment of mice with a combination of SR 140333 and SR 48968 (10(-6) mol/L, aerosol) significantly reduced the increase in the number of total cells and neutrophils and MMP-9 activity in the BALF of mice 2.5 h after LPS exposure. Treatment with the NK3 antagonist SR 142801 (10(-6) mol/L, aerosol) did not inhibit the influx of neutrophils, but markedly reduced the increase in TNF-alpha and IL-6 levels at 2.5 h and MMP-9 activity at 20 h. 3. These results show that the three tachykinin receptor antagonists may interfere with the development of airway inflammation, namely neutrophilia, TNF-alpha release or MMP-9 activity in the BALF of mice exposed to LPS and suggest that not only NK1 and NK2 receptors, but also NK3 receptors are involved in the modulation of the inflammatory response and airway remodelling.

Summary Background Chronic asthma is characterized by inflammatory cell infiltration and tissue remodelling leading to subepithelial fibrosis. Metalloproteinases (MMPs) are involved in degradation of extracellular matrix in most chronic inflammatory diseases. Objective The aim of this study was to investigate the expression of MMPs in the development of inflammatory processes associated or not with the concomitant development of subepithelial fibrosis in an experimental model of asthma. Methods Sensitized BP2 mice were challenged with ovalbumin (OA) every 2 weeks during 8 months. Several mice were removed once a month and bronchoalveolar lavages (BAL) or lung biopsies were performed. Results Lung sections stained with picrosirius and hydroxyproline measurements showed a significant collagen deposition after 16 weeks of OA challenge, demonstrating the development of subepithelial fibrosis. Pulmonary inflammation was present from the first OA challenge and was consistent throughout the 8 months of the study. Moreover, an up-regulation and activation of MMP-9 and, to a less extent, MMP-2 were observed in BAL fluid from challenged mice. The level of tissue inhibitor of metalloproteinases (TIMP)-1 increased after 12 weeks of OA challenge vs. control mice. Conclusion This study reveals that a decrease in the activation of the MMP-9 due to the increase in TIMP-1, could contribute to excessive collagen deposition following repeated antigen challenge in sensitized mice.

Background Matrix metalloproteinases (MMPs) are likely to be relevant mediators of the extracellular matrix (ECM) degradation and airway remodelling. Objective We have compared the levels of MMPs, eotaxin and soluble interleukin 2 receptor (IL-2R) in the plasma of healthy subjects, atopic patients and asthmatic patients. Methods The asthmatic patients were separated into two groups, either well controlled on inhaled therapy or acute severe asthma. Patients with acute severe disease had all received systemic corticosteroids from 12 to 48 h before the blood was taken. Blood was recovered in EDTA tubes, incubated with either f MLP, PMA or vehicle for 10 min and centrifuged. MMP-9, TIMP-1, IL-2R and eotaxin levels were measured in the plasma by ELISA. Moreover, the activity of MMPs was also evaluated by zymography. Results An increased basal level of MMP-9 and IL2-R was observed in acute severe asthma. Following stimulation with f MLP and PMA there was an enhanced production of MMP-9 in the plasma of all groups of patients. However, the MMP-9 level was significantly enhanced in acute severe asthma, compared with the others. No difference was found for the TIMP-1 level between the patients. The eotaxin level in plasma was found to be significantly lower in acute severe asthmatics compared with the others groups. Zymography technique showed a significant increased activity of MMP-9 (92 kDa) but not MMP-2 (66 kDa) in the plasma of patients with acute asthma. Conclusion The increased in MMP-9 production and activity observed in the present study suggests a process of extracellular matrix degradation in acute severe asthmatic patients and proposes MMP-9 as a non-invasive systemic marker of inflammation and airway remodelling in asthma.

Bleomycin-induced pulmonary fibrosis is known to be associated with the increased activity of two gelatinases, matrix metalloproteinase (MMP)-2 and MMP-9, in bronchoalveolar lavage (BAL). This study has investigated the effect of a synthetic inhibitor of MMP, batimastat, on the development of pulmonary fibrosis induced by bleomycin administration in mice. Animals were intranasally instilled with saline or bleomycin (0.5 mg in 100 microl per mouse). Batimastat (30 mg/kg) or vehicle alone was administered by intraperitoneal injection 24 h and 1 h before saline or bleomycin instillation, and then daily at the same dosage until the end of the study. Fifteen days after bleomycin administration, BAL was performed and the lung was removed. Treatment of mice with batimastat significantly reduced bleomycin-induced lung fibrosis, as shown in the lung by histopathological examination and by a decrease in hydroxyproline levels. Batimastat also prevented the increase in BAL macrophage and lymphocyte numbers, whereas it did not show any effect on the increased expression of active transforming growth factor-beta (TGF-beta) in BAL. Batimastat treatment was effective in reducing MMP-2 and MMP-9 activity as well as the tissue inhibitor of metalloproteinase-1 (TIMP-1) level in BAL. These results suggest that administration of the MMP inhibitor batimastat is useful in preventing experimental pulmonary fibrosis induced by bleomycin and raises the possibility of a therapeutic approach to human pulmonary fibrotic disease.

The aim of this study was to investigate the effects of IL-10, a cell permeable analogue of cyclic AMP, dibutyryl-cAMP (db-cAMP), modulators of intracellular cyclic AMP such as phosphodiesterase (PDE) inhibitors and a beta 2-adrenoceptor agonist, salmeterol, on pulmonary inflammation following acute lung injury induced by endotoxin exposure in rats. Pulmonary inflammation was induced in adult Wistar rats by a 60-min exposure to endotoxin (lipopolysaccharide, LPS, 100 micrograms/mL). 4 h later bronchoalveolar lavage (BAL) was performed. The PDE inhibitors, rolipram (3 and 5 mg/kg) and theophylline (30 and 100 mg/kg) inhibited neutrophil recruitment, TNF-alpha release and cellular activation in BAL. Salmeterol (0.5 mg/mL) and IL-10 (0.1 microgram) only inhibit TNF-alpha increase in the BAL fluid and db-AMPc (2.5 micrograms/rat) was ineffective. The present data show that the selective PDE4 inhibitor, rolipram, and the non-selective PDE inhibitor, theophylline, markedly reduced the pulmonary inflammation associated with acute lung injury in the rat. These effects may be mediated in part by IL-10 rather than by cyclic AMP, as demonstrated by the potent inhibitory activity of exogenous IL-10 on the increase in TNF-alpha release in BAL fluid of rats exposed to LPS.

Background Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness and airway inflammation. Objective This study was designed to investigate the effects of the tachykinin NK1 receptor antagonist SR 140333 (Nolpitantium) and the NK2 receptor antagonist SR 48968 (Saredutant) on the activation of alveolar macrophages in the guinea-pig. Methods Guinea-pigs sensitized and challenged by ovalbumin administered by aerosol or naive guinea-pigs were exposed by aerosol to the neutral endopeptidase, phosphoramidon and, 15 min later, to substance P. Twenty-four hours later, bronchoalveolar lavages were performed and the cell composition of bronchoalveolar lavage fluids and the arachidonate release from alveolar macrophages stimulated in vitro with fMLP were evaluated. Results Antigen challenge in sensitized guinea-pigs induced an increase in the total number of cells and granulocytes in the bronchoalveolar lavage fluids that was not reduced by pre-treatment of guinea-pigs with a single dose of SR 140333 or SR 48968 (1 mg/kg). Substance P exposure in phosphoramidon-pretreated guinea-pigs did not induce an increase in the total number of cells. In contrast, antigen or substance P exposure induced a significant increase in the in vitro fMLP-induced arachidonate release from alveolar macrophages. Pre-treatment of the guinea pigs with SR 140333 or SR 48968 did not reduce the increase in arachidonate release from fMLP-stimulated alveolar macrophages from sensitized and challenged guinea-pigs. Pre-treatment of the animals by SR 140333 and SR 48968 reduced the enhanced arachidonate release induced by fMLP from substance P-exposed guinea-pigs. Conclusion The present data demonstrate the importance of NK1- and NK2-receptor stimulation in the development of substance P-induced increased reactivity of alveolar macrophages.

The involvement of platelet activating factor (PAF) in antigen-induced bronchial hyperresponsiveness was investigated by the use of the PAF antagonists BN 52021 and BN 50730, in a guinea-pig model where sensitization and challenge were performed by aerosol. Male Hartley guinea-pigs were sensitized by two aerosol exposures at 48 hr intervals to a 0.9% NaCl solution (saline) containing 2 mg/ml ovalbumin for 30 min. Fifteen to 20 days later, guinea-pigs were challenged by exposure to five successive aerosols of increasing concentrations of ovalbumin (OA) or respectively, 10 microg/ml, 100 microg/ml, 1 mg/ml, 5 mg/ml and 10 mg/ml for 15 min each, or saline alone. Three to four hr and 18-24 hr after the aerosol challenge the guinea-pigs were prepared for recording of bronchopulmonary response and aerosol administrations were then generated with an ultrasonic nebulizer. The bronchopulmonary responses induced by successive 1-min aerosol bursts of acetylcholine (ACh) was assessed. As compared with saline-challenged guinea-pigs, an enhanced bronchopulmonary response to aerosol administration of cumulative doses of ACh was observed, 3-4 hr and 18-24 hr post-ovalbumin challenge. When the sensitized guinea-pigs were pretreated 1 hr before ovalbumin exposure with BN 52021 or BN 50730 (25 mg/kg, per os), a significant inhibition of the increase in the bronchopulmonary response to ACh was observed, both at 3-4 hr and 18-24 hr. Furthermore, when guinea-pigs were treated 3-4 hr after the ovalbumin exposure with BN 52021 or BN 50730, a significant inhibition of the hyperresponsiveness to ACh was recorded at 18-24 hr. A marked accumulation of eosinophils in the peribronchial regions was observed on histological preparations of lung specimens collected 4 hr or 24 hr after ovalbumin exposure. Pretreatment of the guinea-pigs by BN 50730 or BN 52021 did not modify the eosinophil accumulation in the peribronchial area. No significant difference in the number of eosinophils collected in the bronchoalveolar lavage fluid is observed, 24 hr post-ovalbumin challenge, under the pretreatment with BN 52021 or BN 50730. Pretreatment of guinea-pigs by BN 50730 or BN 52021 significantly reduced the PAF-induced (100 microg/ml) increase in eosinophil number in the peribronchial area. By contrast, they did not inhibit the eosinophilia induced by aerosol administration of LTB4 (5 microg/ml). These results suggest that the bronchial hyperresponsiveness observed in this study is associated with eosinophil accumulation in the lung. The potent inhibition of the bronchial hyperresponsiveness by the two unrelated antagonists of PAF suggests that the lipid mediator is involved in its triggering and duration, but not in the eosinophil infiltration.

We have studied the development of airway hyperresponsiveness and the pulmonary cell infiltration in a guinea-pig model in which both initial sensitization and subsequent exposure to the antigen were performed by aerosol. Enhanced bronchopulmonary response to aerosol administration of acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) was observed 3-4 hr and 18-24 hr after antigen exposure of sensitized animals. In contrast, when ACh and 5-HT were administered intravenously 3-4 hr after the challenge, no significant alteration of the dose-response curves was observed. However, 18-24 hr after antigen challenge, a marked leftward shift of the dose-response curve was observed on intravenous injection of ACh or 5-HT. The increased bronchial reactivity to aerosolized ACh in sensitized and challenged guinea-pigs reached a maximum by days 2-4, was still significantly increased at day 5 and returned to the basal value by day 8. No further alteration of the dose-related bronchopulmonary response to aerosol or intravenous administration of ACh was recorded 24 hr after a second antigen challenge, performed 8 days after the initial one. The analysis of bronchoalveolar lavage fluids showed a significant increase in the number of polymorphonuclear neutrophils 3-4 hr after the exposure of sensitized animals to the antigen, which was also associated with a significant eosinophilia at 18-24 hr. Histological examination of lung specimens obtained from animals 3-4 hr following challenge demonstrated eosinophil infiltration in the peribronchial regions and bronchial walls, as well as within the epithelium. Furthermore, as compared to time 3-4 hr, less eosinophils in the peribronchial area and submucosa were counted 24 hr after antigen challenge. However, a role of eosinophil-derived products in the development of bronchial hyperresponsivenss in this experimental model remains to be established.