Cytokine Responses to Group B Streptococci Induce Nitric Oxide Production in Respiratory Epithelial Cells

Abstract

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...

Modulation of Human β-Defensin-2 Transcription in Pulmonary Epithelial Cells by Lipopolysaccharide-Stimulated Mononuclear Phagocytes Via Proinflammatory Cytokine Production

“…8B). Thus, our observation may complement the earlier report that A549 cells did not respond to Gram-positive group B Streptococci and their products (44,45).…”

“…Likewise, this promoter was not activated by coincubation with 250 U/ml IFN-␥ and 10 g/ml LPS (data not shown). In the lungs, mononuclear phagocytes such as alveolar macrophages respond to bacteria and their products, and activate epithelial cells by producing various cytokines and chemokines (1,3,44,45). Therefore, we tested whether the coculture of A549 cells with the MM6 monocytic cell line affects hBD-2 promoter activity following LPS stimulation.…”

Nitric Oxide Is a Key Determinant of Group B Streptococcus–Induced Murine Macrophage Apoptosis

“…After nonopsonic phagocytosis of GBS, macrophages produce cytokines such as tumor necrosis factor (TNF)-a [7] and nitric oxide (NO) [8,9]. GBS also stimulates NO production in respiratory epithelial cells via the paracrine effects of soluble factors released by GBS-infected mononuclear cells [10]. Despite this, GBS persists inside macrophages for long periods and induce macrophage apoptosis [11].…”

Intracellular viability in human non-polarized respiratory epithelial 16 HBE 14o- cells by group B Streptococcus serotype III clinical isolates presenting 162-kb and 183-kb virulence markers

“…Monolayers were post-fixed with 1% OsO 4 , 5 mM CaCl 2 and 0.8% K 4 [Fe(CN 6 )] in cacodylate buffer for 1 h at 22˚C, and dehydrated through a series of graded ethanol solutions and embedded in Epon. Ultra-thin sections were examined under Zeiss EM 906 transmission electron microscope (Zeiss, Oberkochen, Germany) (20).…”

“…The serotype-specific capsular polysaccharides are essential for pathogenesis, and serotype III is prevalent among strains isolated from infants with invasive infections (5). Failure of polymorphonuclear leukocytes and pulmonary macrophages to effectively eliminate GBS from the alveolar space and the ability of bacteria to adhere to respiratory epithelial cells are essential for the development of pneumonia (6). The mechanism(s) used by GBS to cause invasive disease are not completely understood, but is clear that GBS are able to invade polarized epithelial cells (7,8).…”