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J. Biol. Chem., Vol. 277, Issue 25, 22814-22821, June 21, 2002

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Tumor Necrosis Factor- Increases Airway Smooth Muscle Oxidants Production through a NADPH Oxidase-like System to Enhance Myosin Light Chain Phosphorylation and Contractility^*

Gabriel Thabut, Jamel El-Benna^§, Abdoulaye Samb, Stephano Corda^¶, Jerôme Megret, Guy Leseche, Eric Vicaut^¶, Michel Aubier, and Jorge Boczkowski^**

From the  INSERM U408, ^§ INSERM U479, and Institut Féderatif de Recherche 02 "Claude Bernard," Faculté de Médecine Xavier Bichat 75870 Paris Cedex 18, France ^¶ Laboratoire d'étude de la Microcirculation, Hôpital Fernand Widal, 75010 Paris, France, and  Service de Chirurgie Thoracique, Hôpital Beaujon, 92110 Oichy, France

Tumor necrosis factor plays a critical role in airway smooth muscle hyperresponsiveness observed in asthma. However, the mechanisms underlying this phenomenon are poorly understood. We investigated if tumor necrosis factor-stimulated airway smooth muscle produced reactive oxygen species, leading to muscular hyperresponsiveness. Tumor necrosis factor increased intracellular and extracellular oxidants production in guinea pig airway smooth muscle cells and tissue homogenates. This production was abolished by inhibitors of NADPH oxidase (diphenylene iodinium or apocynin) and was enhanced by NADPH, whereas inhibitors of mitochondrial respiratory chain, nitric-oxide synthase, cyclooxygenase, and xanthine oxidase had no effect. NADPH oxidase subunits p22^phox and p47^phox were detected in smooth muscle cells and tissue homogenates by Western blot, immunohistochemistry, and spectral analysis. Furthermore, oxidants production was significantly reduced by transient transfection of smooth muscle cells with p22^phox antisense oligonucleotides. Intracellular antioxidants and diphenylene iodinium abolished tumor necrosis factor-induced muscular hyperresponsiveness and increased in phosphorylation of the myosin light chain. Finally, NADPH oxidase subunits p22^phox and p47^phox were also detected in human airway smooth muscle. Collectively, these results demonstrate that tumor necrosis factor-stimulated airway smooth muscle produces oxidants through a NADPH oxidase-like system, which plays a pivotal role in muscle hyperresponsiveness and myosin light chain phosphorylation.

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