Intratracheal Instillation of Silica Up-regulates Inducible Nitric Oxide Synthase Gene Expression and Increases Nitric Oxide Production in Alveolar Macrophages and Neutrophils

Alveolar macrophages (AM) exposed to cytokines or bacterial lipopolysaccharide (LPS) produce the free radical nitric oxide (NO·) by an inducible nitric oxide synthase (iNOS). They also release reactive oxygen free radicals following exposure to silica dust. The purpose of the present study was to determine whether NO· is produced by rat AM and/or recruited leukocytes following the intratracheal (IT) instillation of silica. Male Sprague-Dawley rats (175 to 225 g) were IT instilled with either silica dust (10 mg/100 g body wt) or LPS (0.25 mg/100 g body wt). After 24 h, bronchoalveolar lavage cells (BALC) and lavaged lung tissue were assayed for iNOS mRNA. Cell counts of BALC and iNOS-dependent (Nw-nitro-Larginine methyl ester [L-NAME]-inhibitable) chemiluminescence generated by AM were also determined. Northern blot analysis demonstrated that the steady-state levels of BALC iNOS mRNA were significantly increased by 3-fold following IT silica and by 7-fold following IT LPS. Partially enriched fractions of either AM or leukocytes from silica-treated rats both exhibited significantly elevated iNOS mRNA in Northern analysis. iNOS-dependent chemiluminescence was significantly increased in AM by 36-fold following IT silica and by 89-fold following IT LPS. Differential counts of BALC showed that AM numbers did not change in any of the treatments; however, red blood cells increased by 30-fold following IT silica and by 23-fold following IT LPS. Total leukocytes (polymorphonuclear leukocytes plus lymphocytes) increased by 58-fold following IT silica and by 274-fold following IT LPS. The results demonstrate that AM and neutrophils iNOS steady-state mRNA levels, AM iNOS-dependent chemiluminescence, and lavagable red blood cells and leukocytes all increased following IT silica. These findings suggest that NO· production in rat AM and neutrophils is increased in response to silica. NO· has the ability to then combine with superoxide and form a highly reactive toxic species, peroxynitrite, which may be responsible in part for silica-induced lung damage.