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The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO 2 ) and titanium dioxide (nano-TiO 2 ) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO 2 or nano-TiO 2 caused a significant increase in P2Y1, P2Y2, A2 A and/or A2 B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1 β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2 A or A2 B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1 β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.

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

Chronic obstructive pulmonary disease (COPD) is characterized by an inflammatory process and airway remodeling involving matrix metalloproteinases (MMPs). Thus, we analyzed the expression and release of MMP-12 (macrophage metalloelastase) in bronchoalveolar lavage (BAL) and lung tissue from COPD patients and control subjects. Immunocytochemistry and immunochemistry were performed to analyze the expression of MMP-12 in BAL cells and bronchial biopsies. Western blotting was used for the evaluation of MMP-12 in BAL fluids. The number of MMP-12 expressing macrophages together with the staining intensity was higher in BAL samples from COPD patients than in controls subjects. Similar results were noted in bronchial biopsies with higher MMP-12 expression in COPD subjects than in controls. Enhanced MMP-12 level was also observed in BAL fluids from patient with COPD in comparison to control subjects. This study demonstrated that COPD patients produce greater quantities of MMP-12 than controls, which may be a critical step in the pathogenesis of COPD and emphysema.

Pulmonary fibrosis has an aggressive course and is usually fatal an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components in the lung interstitium. Matrix metalloproteinases (MMPs) are a major group of proteinases known to regulate the ECM remodeling and so they are hypothesized to be important in the process of lung fibrosis. These led to the concept that modulation of airway remodeling including excessive proteolytic damage of the tissue may be of interest for future treatment. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could argue in favor of antiprotease treatments. Moreover, these observations emphasize that effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of expensive lung destruction and fibrosis.

Dysregulation of matrix metalloproteinases (MMPs) has been implicated in lung injury associated with inflammatory disorders and several lung diseases such as pulmonary fibrosis. We studied a murine model of lipopolysaccharide (LPS)-induced chronic inflammation in order to analyse the relationship between MMP activity in bronchoalveolar lavage fluid and collagen deposition in lung tissue. BP2 mice were exposed to repeated aerosols of LPS of E. coli for 8 months. The inflammatory reaction induced by LPS increased throughout the time of exposure and was associated after 10 weeks with collagen deposition in the alveolar walls. Meantime, we observed in BAL fluid from LPS-exposed mice an early induction of MMP-9 correlated with neutrophil recruitment. MMP-2 increased during the early inflammatory phase, and also during the development of the fibrotic phase. Repeated exposure of mice to an aerosol of LPS can lead to pulmonary interstitial fibrosis and MMPs seem to be associated with this process.

Background β 2 -adrenoceptor agonists have been shown to reduce the lipopolysaccharide (LPS)-induced cytokine release by human monocyte-derived macrophages (MDMs). We compare the expression of β 2 -adrenoceptors and the inhibitory effect of formoterol and salmeterol on the LPS-induced release of tumor necrosis factor (TNF)- α , interleukin (IL)-1β, IL-6 and a range of chemokines (CCL2, 3, 4, and IL-8) by human lung macrophages (LMs) and MDMs. Methods LMs were isolated from patients undergoing resection and MDMs were obtained from blood monocytes in the presence of GM-CSF. LMs and MDMs were incubated in the absence or presence of formoterol or salmeterol prior to stimulation with LPS. The effects of formoterol were also assessed in the presence of the phosphodiesterase inhibitor roflumilast. Results LPS-induced cytokine production was higher in LMs than in MDMs. Salmeterol and formoterol exerted an inhibitory effect on the LPS-induced production of TNF- α , IL-6, CCL2, CCL3, and CCL4 in MDMs. In contrast, the β 2 -adrenoceptor agonists were devoid of any effect on LMs - even in the presence of roflumilast. The expression of β 2 -adrenergic receptors was detected on Western blots in MDMs but not in LMs. Conclusions Concentrations of β 2 -adrenoceptor agonists that cause relaxation of the human bronchus can inhibit cytokine production by LPS-stimulated MDMs but not by LMs.

The aim of this study was to determine whether neurokinin B (NKB) or specific agonists of tachykinin NK3 receptors, [MePhe7]NKB and senktide, were able to induce airway hyperresponsiveness in guinea‐pigs. The effects of these compounds were compared to those of substance P (SP), neurokinin A (NKA) and the preferential tachykinin NK1 ([Sar9, Met(02)11]SP) or NK2 ([βAla8]NKA (4‐10)) receptor agonists. In guinea‐pigs pretreated with phosphoramidon (10−4 M aerosol for 10 min) and salbutamol (8.7×10−3 M for 10 min), all tachykinins administrated by aerosol (3×10−7 to 10−4 M) induced airway hyperresponsiveness 24 h later, displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine (i.v.). The rank order of potency was: [βAla8]NKA (4‐10)>NKA=NKB=senktide=[MePhe7]NKB=[Sar9,Met(02)11]SP>SP. Airway hyperresponsiveness induced by [MePhe7]NKB was prevented by the tachykinin NK3 (SR 142801) and NK2 (SR 48968) receptor antagonists. Bronchoconstriction induced by tachykinins administered by aerosol was also determined. SP, NKA, NKB and the tachykinin NK1 and NK2 receptor agonist induced bronchoconstriction. The rank order of potency was: NKA=[βAla8]NKA (4‐10)>NKB=SP=[Sar9,Met(02)11]SP. Under similar conditions, and for concentrations which induce airway hyperresponsiveness, senktide and [MePhe7]NKB failed to induce bronchoconstriction. It is concluded that tachykinin NK3‐receptor stimulation can induce airway hyperresponsiveness and that this effect is not related to the ability of tachykinins to induce bronchoconstriction. British Journal of Pharmacology (2000) 130, 49–56; doi:10.1038/sj.bjp.0703278

This study was undertaken to investigate the possible contribution of the blockade of eotaxin generation to the anti‐eosinophilotactic effect of phosphodiesterase (PDE) type 4 inhibitors. In some experiments, the putative synergistic interaction between PDE type 4 inhibitors and the β2‐agonist salbutamol was also assessed. Sensitized guinea‐pigs aerosolized with antigen (5% ovalbumin, OVA) responded with a significant increase in eotaxin and eosinophil levels in the bronchoalveolar lavage fluid (BALF) at 6 h. Eosinophil recruitment was inhibited by both PDE type 4 inhibitors rolipram (5 mg kg−1, i.p.) and RP 73401 (5 mg kg−1, i.p.) treatments. In contrast, only rolipram inhibited eotaxin production. Sensitized rats intrapleurally challenged (i.pl.) with antigen (OVA, 12 μg cavity−1) showed a marked eosinophil infiltration at 24 h, preceded by eotaxin generation at 6 h. Intravenous administration of a rabbit anti‐mouse eotaxin antibody (0.5 mg kg−1) significantly reduced allergen‐evoked eosinophilia in this model. Local pretreatment with rolipram (40 μg cavity−1) or RP 73401 (40 μg cavity−1) 1 h before challenge reduced eosinophil accumulation evaluated in the rat pleural effluent, but only the former was active against eotaxin generation. The inhibitors of PDE type 3 (SK&F 94836) and type 5 (zaprinast) failed to alter allergen‐evoked eosinophil recruitment in rats. Local injection of β2‐agonist salbutamol (20 μg cavity−1) inhibited both eosinophil accumulation and eotaxin production following pleurisy. The former was better inhibited when salbutamol and rolipram were administered in combination. Treatment with rolipram and RP 73401 dose‐dependently inhibited eosinophil adhesion and migration in vitro. These effects were clearly potentiated by salbutamol at concentrations that had no effect alone. Our findings indicate that although rolipram and RP 73401 are equally effective in inhibiting allergen‐induced eosinophil infiltration only the former prevents eotaxin formation, indicating that PDE 4 inhibitors impair eosinophil accumulation by mechanisms independent of eotaxin production blockade. British Journal of Pharmacology (2001) 134, 283–294; doi:10.1038/sj.bjp.0704233

1. The aim of this study was to determine whether neurokinin B (NKB) or specific agonists of tachykinin NK(3) receptors, [MePhe(7)]NKB and senktide, were able to induce airway hyperresponsiveness in guinea-pigs. The effects of these compounds were compared to those of substance P (SP), neurokinin A (NKA) and the preferential tachykinin NK(1) ([Sar(9), Met(0(2))(11)]SP) or NK(2) ([betaAla(8)]NKA (4-10)) receptor agonists. 2. In guinea-pigs pretreated with phosphoramidon (10(-4) M aerosol for 10 min) and salbutamol (8.7x10(-3) M for 10 min), all tachykinins administrated by aerosol (3x10(-7) to 10(-4) M) induced airway hyperresponsiveness 24 h later, displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine (i.v.). The rank order of potency was: [betaAla(8)]NKA (4-10)>NKA=NKB=senktide=[MePhe(7)]NKB=[Sar(9),Met(0(2))(11)]SP>SP. 3. Airway hyperresponsiveness induced by [MePhe(7)]NKB was prevented by the tachykinin NK(3) (SR 142801) and NK(2) (SR 48968) receptor antagonists. 4. Bronchoconstriction induced by tachykinins administered by aerosol was also determined. SP, NKA, NKB and the tachykinin NK(1) and NK(2) receptor agonist induced bronchoconstriction. The rank order of potency was: NKA=[betaAla(8)]NKA (4-10)>NKB=SP=[Sar(9), Met(0(2))(11)]SP. Under similar conditions, and for concentrations which induce airway hyperresponsiveness, senktide and [MePhe(7)]NKB failed to induce bronchoconstriction. 5. It is concluded that tachykinin NK(3)-receptor stimulation can induce airway hyperresponsiveness and that this effect is not related to the ability of tachykinins to induce bronchoconstriction.