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Chronic exposure to particulate air pollution is associated with lung function impairment. To determine the molecular mechanism(s) of this phenomenon, we investigated, in an alveolar human epithelial cell line (A549), whether diesel exhaust particles (DEPs), a main component of particulate air pollution, modulates the expression and activity of the matrix metalloprotease (MMP)-1, a collagenase involved in alveolar wall degradation. Interaction of DEPs with cigarette smoke, which also produces structural and functional lung alterations, was also investigated. A noncytotoxic concentration of DEPs induced an increase in MMP-1 mRNA and protein expression and activity in A549 cells without modifying the expression of the MMP inhibitors TIMP-1 and -2. This effect was not potentiated when cells were coexposed to noncytotoxic concentrations of cigarette smoke condensate. DEP-induced MMP-1 was associated with increased ERK 1/2 phosphorylation and upregulation of expression and activity of the NADPH oxidase analog NOX4. Cell transfection with a NOX4 small interfering RNA prevented these phenomena, showing the critical role of a NOX4 ERK 1/2 pathway in DEP-induced MMP-1 expression and activity. Similar results to those observed in A549 cells were obtained in another human lung epithelial cell line, NCI-H292. Furthermore, experiments in mice intratracheally instilled with DEPs confirmed the in vitro findings, showing the induction of NOX4 and MMP-1 protein expression in alveolar epithelial cells. We conclude that alveolar alterations secondary to MMP-1 induction could explain lung function impairment associated with exposure to particulate pollution.

Heme oxygenase (HO), the heme-degrading enzyme, has shown anti-inflammatory effects in several models of pulmonary diseases. HO is induced in airways during asthma; however, its functional role is unclear. Therefore, we evaluated the role of HO on airway inflammation [evaluated by bronchoalveolar lavage (BAL) cellularity and BAL levels of eotaxin, PGE2, and proteins], mucus secretion (evaluated by analysis of MUC5AC gene expression and periodic acid-Schiff staining), oxidative stress (evaluated by quantification of 4-hydroxynonenal adducts and carbonylated protein levels in lung homogenates), and airway responsiveness to histamine in ovalbumin (OVA)-sensitized and multiple aerosol OVA or saline-challenged guinea pigs (6 challenges, once daily, OVA group and control group, respectively). Airway inflammation, mucus secretion, oxidative stress, and responsiveness were significantly increased in the OVA group compared with the control group. HO upregulation by repeated administrations of hemin (50 mg/kg ip) significantly decreased airway responsiveness in control animals and airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These effects were reversed by the concomitant administration of the HO inhibitor tin protoporphyrin-IX (50 μmol/kg ip). Repeated administrations of tin protoporphyrin-IX alone significantly increased airway responsiveness in control animals but did not modify airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These results suggest that upregulation of the HO pathway has a significant protective effect against airway inflammation, mucus hypersecretion, oxidative stress, and hyperresponsiveness in a model of allergic asthma in guinea pigs.

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3′,5′-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.