Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of neonatal pneumonia, sepsis, and meningitis. Early-onset GBS pneumonia is characterized by marked pulmonary epithelial and endothelial cell injury. Innate proinflammatory responses to GBS infection that may contribute to the respiratory pathology include the synthesis and release of cytokines, prostaglandins, and nitric oxide (NO). The hypothesis that NO is directly induced in lung epithelial cells by invading GBS or indirectly induced by cytokines released by GBS-infected mononuclear cells was tested. A549 transformed human respiratory epithelial cells were directly cultured with GBS, cocultured with GBS-infected human mononuclear cells or purified macrophages, or exposed to conditioned culture medium from human mononuclear cells infected by GBS. The culture medium of A549 cultures was assayed for NO secretion, and the cell lysates were tested for presence of inducible nitric oxide synthase (iNOS) mRNA by reverse transcriptase PCR (RT-PCR). GBS-treated A549 cells neither secreted detectable NO nor expressed iNOS mRNA. GBS interaction with human mononuclear cells, however, stimulated release of soluble factors that readily induced iNOS mRNA expression and NO secretion by A549 cells. Inflammatory mediator-induced nitric oxide (NO) production by alveolar epithelium may exceed that of other lung cell types such as macrophages, and induction during GBS infection may play a significant role in pulmonary defense or free-radical-mediated lung injury. Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of neonatal pneumonia, sepsis, and meningitis (26). GBS is also an important pathogen in maternal infections and in infections of nonpregnant adults with risk factors such as older age and underlying disorders (25). Early-onset GBS pneumonia is characterized by presence of numerous bacteria, an inflammatory exudate, and marked pulmonary epithelial and endothelial cell injury (1, 10). Innate proinflammatory responses to GBS infection which may contribute to the respiratory pathology include the synthesis and release of cytokines (12,22), prostaglandins (16), and nitric oxide (NO) (6, 15).In the lung, alveolar macrophages, airway epithelial cells, endothelial cells, and inflammatory cells may be sources for NO (24). Though GBS, with or without cytokines, has been shown to induce NO production in cultured murine macrophages (3,6,20), human macrophages produce far less NO (5-to 100-fold less) in response to cytokine or microbial stimuli than do murine macrophages (31). An alternate source of alveolar NO are human lung epithelial cells, which possess a nitric oxide synthase (iNOS, NOS2) that is inducible by a mixture of cytokines (21) that should, based on in vitro data, be released locally by GBS-infected alveolar macrophages and mononuclear cells (12). Alternatively, direct GBS invasion of respiratory epithelial cells (23) might also induce NO production.In this report, human respiratory epithelial cells were directly cultured w...