Influence of inflammasome pathway activation in macrophages on the matrix metalloproteinase expression of human hepatic stellate cells.

Inflammasomes are protein complexes that produce IL-1ß in response to damage or pathogens. As such, inflammasomes are involved in several types of hepatic fibrosis. However, the mechanisms by which these complexes drive the liver's fibrogenic status remain unclear. We co-cultured differentiated macrophages (the THP-1 cell line or human monocyte-derived macrophages (MDMs)) with human hepatic fibroblasts (either the LX-2 cell line or primary human hepatic stellate cells (HSCs)). The inflammasome pathway was activated with lipopolysaccharide (LPS) and monosodium urate (MSU) crystals, and the HSCs' responses were analyzed. Our results show that co-culture of HSCs with THP-1 cells upregulated transcription of the genes coding for metalloproteinase (MMP)-3 and MMP-9. After inflammasome pathway activation, the HSCs' phenotype was the same in the presence of THP-1 cells or MDMs (i.e. upregulation of MMP-3, MMP-9, and the pro-inflammatory cytokine IL-1ß). We found that two cytokines were involved in these changes: IL-1ß regulated MMP-3 and IL-1ß mRNA expression, whereas TNF-α regulated MMP-9 mRNA expression. Experiments with primary cells revealed that a general inflammatory environment is responsible for the downregulation of pro-fibrotic markers. Our present results suggest that inflammasome pathway activation in macrophages leads to a pro-inflammatory environment for HSCs leading to MMP/TIMP imbalance and enhanced fibrolytic properties.

Purinergic receptors: new targets for the treatment of gout and fibrosis.

Adenosine triphosphate is involved in many metabolic reactions, but it has also a role as a cellular danger signal transmitted through purinergic receptors (PRs). Indeed, adenosine 5'-triphosphate (ATP) can bind to PRs which are found in the membrane of many cell types, although the relative proportions of the receptor subtypes differ. PRs are classified according to genetic and pharmacological criteria and especially their affinities for agonists and their transduction mechanism (i.e. as metabotropic P2YRs or ionotropic P2XRs). Extracellular ATP release by activated or necrotic cells may activate various PRs and especially P2X7R, the best-characterized PR, on immune cells. P2X7R is known to regulate the activation of the Nod-like receptor (NLR)-family protein, NLRP3 inflammasome, which permit the release of IL-1ß, a potent pro-inflammatory cytokine. The P2X7R/NLRP3 pathway is involved in many inflammatory diseases, such as gout, and in fibrosis diseases associated with inflammatory process, liver or lung fibrosis. Some authors imaging also a real promising therapeutic potential of P2X7R blockage. Thus, several pharmaceutical companies have developed P2X7R antagonists as novel anti-inflammatory drug candidates. Clinical trials of the efficacy of these antagonists are now underway. A better understanding of the P2X7R/NLRP3 signalling pathways permits the identification of targets and the development of a new class of drugs able to inhibit the fibrogenesis process and collagen deposition.

Involvement of matrix metalloproteinases (MMPs) and inflammasome pathway in molecular mechanisms of fibrosis.

Fibrosis is a basic connective tissue lesion defined by the increase in the fibrillar extracellular matrix (ECM) components in tissue or organ. Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turn-over of ECM and so they are suggested to be important in tissue remodelling observed during fibrogenic process associated with chronic inflammation. Tissue remodelling is the result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of ECM components markedly controlled by the MMPs/TIMP imbalance. We previously showed an association of the differences in collagen deposition in the lungs of bleomycin-treated mice with a reduced molar pro-MMP-9/TIMP-1 ratio. Using the carbon tetrachloride (CCl4) preclinical model of liver fibrosis in mice, we observed a significant increase in collagen deposition with increased expression and release of tissue inhibitors of metalloproteinase (TIMP)-1 both at 24 h and 3 weeks later. This suggests an early altered regulation of matrix turnover involved in the development of fibrosis. We also demonstrated an activation of NLRP3-inflammasome pathway associated with the IL-1R/MyD88 signalling in the development of experimental fibrosis both in lung and liver. This was also associated with an increased expression of purinergic receptors mainly P2X7 Finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of tissue remodelling and fibrosis.

Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1ß or TNF-α Release from Human Hepatic Stellate Cells.

Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and ß, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-ß1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and ß, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-ß1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-ß1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1ß, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.

Roflumilast n-oxide associated with PGE2 prevents the neutrophil elastase-induced production of chemokines by epithelial cells.

Neutrophil chemotaxis is involved in the lung inflammatory process in conditions such as chronic obstructive pulmonary disease (COPD). Neutrophil elastase (NE), one of the main proteases produced by neutrophils, has an important role in the inflammatory process via the release of chemokines from airway epithelial cells. It was recently shown that roflumilast N-oxide has therapeutic potential in COPD. The aim of the present study was to investigate roflumilast N-oxide's effect on NE-induced chemokine production and signaling pathways in A549 epithelial cells. A549 cells were incubated with NE for 30min, washed with PBS and then cultured for 2h (for measurement of mRNA expression) and 24h (for chemokine release) or for 5 to 30min (for protein phosphorylation assays). Prior to the addition of NE, cells were also pre-incubated with prostaglandin E2 (PGE2), alone and in combination with roflumilast N-oxide. Addition of NE was associated with elevated chemokine production by A549 cells and induction of the p38α pathway. In contrast when combined with PGE2, the roflumilast N-oxide had an additive effect on the inhibition of NE-induced chemokine release and p38α and other kinases activation. In conclusion, we demonstrated that NE is able to increase the release of chemokines from epithelial cells via the activation of p38α MAP-kinase and that roflumilast N-oxide when combined with PGE2 lowers NE-induced kinase activation and chemokine production.

IL-1ß production is dependent on the activation of purinergic receptors and NLRP3 pathway in human macrophages.

The Nod-like receptor family protein 3 (NLRP3)-inflammasome pathway is known to be activated by danger signals such as monosodium urate (MSU). We investigated the role of P2 purinergic receptors in the activation of NLRP3-inflammasome pathway after MSU treatment of primary human monocyte-derived macrophages (MDMs). After initial stimulation with a low concentration of LPS (0.1 µg/ml), a 6 h treatment with MSU crystals (250, 500, and 1000 µg/ml) induced the MDMs to release IL-1ß, IL-1α, and IL-6 in a dose-dependent manner. Moreover, the caspase 1 inhibitor Z-YVAD-FMK and the cathepsin B inhibitor CA-074Me reduced production of IL-1ß in a dose-dependent manner after LPS + MSU treatment. We used real-time reverse transcription-quantitative PCR to show that treatment with LPS and MSU (500 µg/ml) induced significantly greater expression of NLRP3 and IL-1ß than after treatment with LPS. We also found that MSU treatment induced P2X purinergic receptor 7 (P2X7R) mRNA and protein expression. Furthermore, addition of the P2X7 purinergic receptor antagonist A-740003 significantly impeded IL-1ß production and pro-IL-1ß cleavage after treatment with LPS + MSU. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited the release of IL-1ß and other M1 macrophage cytokines (such as IL-1α, IL-6, and TNF-α) from MDMs stimulated with LPS + MSU. Taken as a whole, our results show that P2 purinergic receptors and the NLRP3 inflammasome pathway are involved in the secretion of IL-1ß from MSU-stimulated human macrophages. This pathway may constitute a novel therapeutic target for controlling the inflammatory process in several associated pathologies.

Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages.

Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1ß, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1ß from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1ß mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1ß, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1ß from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury.

Roflumilast N-oxide prevents cytokine secretion induced by cigarette smoke combined with LPS through JAK/STAT and ERK1/2 inhibition in airway epithelial cells.

Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD). Airway epithelial cells and macrophages are the first defense cells against cigarette smoke and these cells are an important source of pro-inflammatory cytokines. These cytokines play a role in progressive airflow limitation and chronic airways inflammation. Furthermore, the chronic colonization of airways by Gram-negative bacteria, contributes to the persistent airways inflammation and progression of COPD. The current study addressed the effects of cigarette smoke along with lipolysaccharide (LPS) in airway epithelial cells as a representative in vitro model of COPD exacerbations. Furthermore, we evaluated the effects of PDE4 inhibitor, the roflumilast N-oxide (RNO), in this experimental model. A549 cells were stimulated with cigarette smoke extract (CSE) alone (0.4% to 10%) or in combination with a low concentration of LPS (0.1 µg/ml) for 2 h or 24 h for measurement of chemokine protein and mRNAs and 5-120 min for protein phosphorylation. Cells were also pre-incubated with MAP kinases inhibitors and Prostaglandin E2 alone or combined with RNO, before the addition of CSE+LPS. Production of cytokines was determined by ELISA and protein phosphorylation by western blotting and phospho-kinase array. CSE did not induce production of IL-8/CXCL8 and Gro-α/CXCL1 from A549 cells, but increase production of CCL2/MCP-1. However the combination of LPS 0.1 µg/ml with CSE 2% or 4% induced an important production of these chemokines, that appears to be dependent of ERK1/2 and JAK/STAT pathways but did not require JNK and p38 pathways. Moreover, RNO associated with PGE2 reduced CSE+LPS-induced cytokine release, which can happen by occur through of ERK1/2 and JAK/STAT pathways. We report here an in vitro model that can reflect what happen in airway epithelial cells in COPD exacerbation. We also showed a new pathway where CSE+LPS can induce cytokine release from A549 cells, which is reduced by RNO.

Role of matrix metalloproteinases in the inflammatory process of respiratory diseases.

Matrix metalloproteinases (MMPs) are a group of proteases known to regulate the turnover of extracellular matrix and thus are suggested to be important in the process of several diseases associated with tissue remodeling. Furthermore, the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue, may be of interest as a basis for future treatment. Degradation of extracellular matrix is currently associated with structural and recruited cell activation and release of inflammatory mediators and MMPs. Indeed, a marked increase in their expression is observed associated with a variety of inflammatory diseases, including respiratory pathologies. In these conditions, we have to consider MMPs as therapeutic targets which can be inhibited by non-selective and/or selective inhibitors as anti-inflammatory compounds. The present review aims to discuss the potential interest of the inhibition of MMP in inflammatory diseases with a focus on respiratory diseases.

Macrophage metalloelastase (MMP-12) as a target for inflammatory respiratory diseases.

BACKGROUND: MMPs are known to regulate the turnover of extracellular matrix and have been suggested to be important in lung disease associated with tissue remodeling. Macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling in inflammatory respiratory diseases such as chronic obstructive pulmonary diseases (COPD), including emphysema. Recent studies using MMP-12 inhibitors have demonstrated a reduction in both the inflammatory process and airspace enlargement in lung tissue. OBJECTIVE/METHODS: This review discusses the potential involvement of MMP-12 in the pathophysiological process and proposes MMP-12 as a target for inflammatory disorders of the respiratory system. RESULTS/CONCLUSIONS: MMP-12 plays a predominant role in the inflammatory process induced by cigarette smoke, and therefore is potentially an important therapeutic target for the treatment of COPD.

MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells.

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.

Cigarette smoke-induced pulmonary inflammation is TLR4/MyD88 and IL-1R1/MyD88 signaling dependent.

Acute cigarette smoke exposure of the airways (two cigarettes twice daily for three days) induces acute inflammation in mice. In this study, we show that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signaling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4-, MyD88-, and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 activity in the bronchoalveolar space. Further, cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro, which was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activates the innate immune system via TLR4/MyD88, resulting in airway inflammation. CSC-activated macrophages released mature IL-1beta only in presence of ATP, whereas CSC alone promoted the TLR4/MyD88 signaling dependent production of IL-1alpha and pro-IL-1beta implicating cooperation between TLRs and the inflammasome. In conclusion, acute cigarette exposure results in LPS-independent TLR4 activation, leading to IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease with emphysema.

Effects of depletion of neutrophils or macrophages on the inflammatory response induced by metalloelastase (MMP-12) in mice airways.

Macrophage elastase (recombinant human matrix metalloproteinase-12, rhMMP-12), was instilled in mouse airways, inducing an early inflammatory response characterized by neutrophil recruitment and cytokine release in the bronchoalveolar lavage (BAL) fluids, followed by a delayed macrophage recruitment. We investigated the role played by alveolar macrophages and neutrophils in the delayed macrophage influx induced by rhMMP-12 (8 x 10(-3) U/mouse) in A/J mice. Mice depleted of circulating neutrophils, using a cytotoxic antibody, did not present an increase in neutrophil numbers in bronchoalveolar lavage fluids, 4 h and 24 h after rhMMP-12 instillation but the macrophage recruitment was not modified as compared to control mice at 7 days. Similar results were obtained using mice when the gene for neutrophil elastase was knocked out. Intranasal instillation of clodronate liposomes, 72 h prior to rhMMP-12 instillation, induced macrophage depletion which did not modify the macrophage recruitment at 7 days. Moreover, the stimulation of mouse macrophages by rhMMP-12 did not elicit the release of cytokines in culture supernatants. These results indicate that resident alveolar macrophages and recruited neutrophils do not play a role in the delayed macrophage recruitment induced by rhMMP-12.

Influence of early neutrophil depletion on MMPs/TIMP-1 balance in bleomycin-induced lung fibrosis.

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in interstitium resulting in respiratory failure associated with inflammation showing mainly neutrophil (PMN) recruitment. The turn over of extracellular matrix is partially regulated by proteases such as metalloproteinases (MMPs) and their inhibitors (TIMPs). We investigated the impact of PMN depletion on the MMP/TIMP-1 imbalance and the development of fibrosis in mice induced by bleomycin (0.3 mg/mouse). Administration of 200 microL of rabbit anti-mouse PMN antibody i.p. blunted the neutrophil influx detected in BAL and in whole blood one day after bleomycin administration. At day(14), hydroxyproline content was increased both in anti-PMN treated and control mice, without any difference between groups. At day one, bleomycin elicited a raise in pro-MMP-9 level in BAL that was significantly attenuated in anti-PMN depleted mice, whereas TIMP-1 and MMP-2 release were similar in both groups at day(1) and day(14). Higher RNA levels were observed in PMN-treated mice at day(1) for MMP-9 and MMP-2 and at day(14) for MMP-2 only. At day(14), bleomycin elicited a raise of TIMP-1 protein and RNA levels regardless of anti-PMN treatment, whereas MMP-9 returned to basal level. Bleomycin enhanced MMP-8 level in BAL at day(14) only for the control group. The amount of MMP-8 was more important in BAL from anti-PMN treated mice than in control mice at day(1) and day(14). PMN-depletion and the associated modifications in pro-MMP-9/TIMP-1 imbalance in lung during the early inflammatory phase do not alter susceptibility to bleomycin-induced pulmonary fibrosis.

Metalloelastase (MMP-12) induced inflammatory response in mice airways: effects of dexamethasone, rolipram and marimastat.

Direct instillation of a recombinant human form of MMP-12 (rhMMP-12) in mice airways elicited an early inflammatory response characterized by neutrophil influx, cytokine release and gelatinase activation followed by a delayed response, mainly characterized by macrophage recruitment. As this experimental model of lung inflammation partially mimics some features of chronic obstructive pulmonary disease (COPD), we have investigated the effects of treatment by anti-inflammatory compounds, dexamethasone and rolipram and a non-specific matrix metalloproteinase (MMP) inhibitor, marimastat. The compounds were administrated orally, 1 h before rhMMP-12 instillation (8 x 10(-3) U/mouse). Total and differential cell counts were evaluated in the bronchoalveolar lavage fluids. Cytokines and MMP-9 were quantified in bronchoalveolar lavage fluids and in lung homogenate supernatants. Marimastat (100 mg/kg), dexamethasone (10 mg/kg) and rolipram (0.1 and 0.3 mg/kg) were able to decrease significantly neutrophil recruitment at 4 and 24 h after rhMMP-12 instillation, but only marimastat (30 and 100 mg/kg) was effective at decreasing the macrophage recruitment occurring at day 7. Marimastat (100 mg/kg), dexamethasone (10 mg/kg) and rolipram (0.3 mg/kg) reduced significantly IL-6, KC/CXCL1, MIP-1alpha/CCL3 and MMP-9 levels in bronchoalveolar lavage fluid. Similar results were obtained in lung homogenates except with rolipram. Dexamethasone and rolipram were able to inhibit the early inflammatory response but were ineffective to limit the macrophage influx. In contrast, marimastat was able to reduce early and late response. These data indicate that MMP-12 instillation in mice could highlight some of the inflammatory response seen in COPD and could be used for the pharmacological evaluation of new anti-inflammatory mechanisms of action.

Macrophage metalloelastase (MMP-12) deficiency does not alter bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the interstitium resulting in respiratory failure. The role of remodeling mediators such as metalloproteinases (MMPs) and their inhibitors (TIMPs) in the fibrogenic process remains misunderstood. In particular, macrophage metalloelastase, also identified as MMP-12, is known to be involved in remodeling processes under pathological conditions. However, MMP-12 involvement in pulmonary fibrosis is unknown. Here we investigated fibrotic response to bleomycin in MMP-12 deficient mice. MATERIALS AND METHODS: C57BL/6 mice, Balb/c mice and MMP-12 -/- mice with a C57BL/6 background received 0.3 mg bleomycin by intranasal administration. 14 days after, mice were anesthetized and underwent either bronchoalveolear lavage (BAL) or lung removal. Collagen deposition in lung tissue was determined by Sircoltrade mark collagen assay, MMP activity in BAL fluid was analyzed by zymography, and other mediators were quantified in BAL fluid by ELISA. Real time PCR was performed to assess gene expression in lung removed one or 14 days after bleomycin administration. Student t test or Mann & Whitney tests were used when appropriate for statistical analysis. RESULTS: The development of pulmonary fibrosis in "fibrosis prone" (C57BL/6) mice was associated with prominent MMP-12 expression in lung, whereas MMP-12 expression was weak in lung tissue of "fibrosis resistant" (Balb/c) mice. MMP-12 mRNA was not detected in MMP-12 -/- mice, in conformity with their genotype. Bleomycin elicited macrophage accumulation in BAL of MMP-12 -/- and wild type (WT) mice, and MMP-12 deficiency had no significant effect on BAL cells composition. Collagen content of lung was increased similarly in MMP-12 -/- and WT mice 14 days after bleomycin administration. Bleomycin elicit a raise of TGF-beta protein, MMP-2 and TIMP-1 protein and mRNA in BAL fluids and lung respectively, and no significant difference was observed between MMP-12 -/- and WT mice considering those parameters. CONCLUSION: The present study shows that MMP-12 deficiency has no significant effect on bleomycin-induced fibrosis.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases.

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstructive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.

Macrophage elastase (MMP-12): a pro-inflammatory mediator?

As many metalloproteinases (MMPs), macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstructive pulmonary disease (COPD), have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12) in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstrutive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.

Macrophage elastase (MMP-12): a pro-inflammatory mediator?

As many metalloproteinases (MMPs), macrophage elastase (MMP-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes. Studies using animal models of acute and chronic pulmonary inflammatory diseases, such as pulmonary fibrosis and chronic obstrutive pulmonary disease (COPD), have given evidences that MMP-12 is an important mediator of the pathogenesis of these diseases. However, as very few data regarding the direct involvement of MMP-12 in inflammatory process in the airways were available, we have instilled a recombinant form of human MMP-12 (rhMMP-12) in mouse airways. Hence, we have demonstrated that this instillation induced a severe inflammatory cell recruitment characterized by an early accumulation of neutrophils correlated with an increase in proinflammatory cytokines and in gelatinases and then by a relatively stable recruitment of macrophages in the lungs over a period of ten days. Another recent study suggests that resident alveolar macrophages and recruited neutrophils are not involved in the delayed macrophage recruitment. However, epithelial cells could be one of the main targets of rhMMP-12 in our model. We have also reported that a corticoid, dexamethasone, phosphodiesterase 4 inhibitor, rolipram and a non-selective MMP inhibitor, marimastat could reverse some of these inflammatory events. These data indicate that our rhMMP-12 model could mimic some of the inflammatory features observed in COPD patients and could be used for the pharmacological evaluation of new anti-inflammatory treatment. In this review, data demonstrating the involvement of MMP-12 in the pathogenesis of pulmonary fibrosis and COPD as well as our data showing a pro-inflammatory role for MMP-12 in mouse airways will be summarized.