Production of nitric oxide by airways neutrophils in the initial phase of murine asthma.

In experimental models of asthma, nitric oxide (NO) is produced and contributes to the physiopathology of the disease. Neutrophil is the first cell to infiltrate the lung in response to antigen stimulation, it has the capacity to produce NO but a clear demonstration that neutrophils contribute to NO production in asthma is lacking. This was the aim of the present study. At weekly intervals C57Bl/6 mice were sensitized twice with ovalbumin-alumen and challenged twice with ovalbumin aerosol. The peak of neutrophil infiltration in the bronchoalveolar lavage fluid (BALF) was 12 h after challenge, when neutrophils constituted 70% of the cell population and eosinophils only 1.5%. BALF cell preparations were stained with a NO-sensitive fluorescent dye (DAF-2) and with a nucleus marker (DAPI). Most DAF-2 stained cells could be identified as polymorphonuclear leukocytes, by the co-localization of both DAF-2 and DAPI staining. Cells from animals treated with l-NAME, were not stained for DAF-2 confirming the specificity of DAF-2 staining for NO. Moreover, the peak expression of inducible nitric oxide synthase (NOS2), in BALF cells and lung homogenates, was coincident with the peak of BALF neutrophil influx. NOS2 protein expression (arbitrary units) was detected 6 h after challenge (17.8+/-9.1 in BALF cells; 47.5+/-7.7 in lung homogenates), peak expression was at 12 h (54.5+/-8.7 and 133.7+/-10), decreasing thereafter, being no longer detected after 24 h. Thus, the neutrophils infiltrating the lung in the initial phase of murine asthma are producing NO via NOS2.