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.

FIG. 1.

Direct GBS infection of NHBE cells induces IL-8 but not iNOS mRNA. Total RNA was purified from cell lysates collected at 2, 8, and 24 h after initiation of a 2-h infection pulse with COH1-13 (100:1). RT-PCR products were resolved by electrophoresis on a 1.8% agarose-ethidium bromide gel. (Gel A) G3PDH RT-PCR products (G3PDH PCR product = 983 bp). (Gel B) IL-8 RT-PCR products (IL-8 PCR product = 289 bp). (Gel C) iNOS RT-PCR products (iNOS PCR product = 259 bp). Lanes 1, 3, and 5, uninfected cells at 2, 8, and 24 h, respectively; lanes 2, 4, and 6, GBS-infected cells at 2, 8, and 24 h, respectively; lane 7, control RNA for respective genes; lane 8, DNA molecular weight markers.

FIG. 2.

Cytomix induces iNOS mRNA in A549 cells. A549 cells were treated for 2 or 24 h with 10 ng each of IL-1, TNF-α, and IFN-γ/ml. Total RNA was extracted from cell lysates, and multiplex RT-PCR was conducted with two primer sets in a single PCR (iNOS primers plus 18S rRNA primers and competimers as an invariant endogenous control). PCR products were resolved by electrophoresis on a 1.8% agarose-ethidium bromide gel. Lane 1, DNA markers; lane 2, untreated (2 h) A549; lane 3 and 5, Cytomix-treated (2 h) A549; lane 4, same as lane 3 but without reverse transcriptase; lane 6, untreated (24 h) A549; lanes 7 and 9, Cytomix-treated (24 h) A549; lane 8, same as lane 7 but without reverse transcriptase; lane 10, 18S rRNA control (495 bp); lane 11, iNOS RNA control (349 bp).

FIG. 3.

iNOS mRNA induction in A549 cells treated with conditioned medium from GBS-treated human mononuclear cells. We centrifuged 24-h-conditioned medium from GBS-treated (strain COH1, 1:1 or 10:1 ratio) human mononuclear cells to remove GBS and added it to monolayers of A549 cells for 24 h. Total RNA was extracted from cell lysates, and multiplex RT-PCR was conducted with two primer sets in a single PCR (iNOS primers plus 18S rRNA primers and competimers as an invariant endogenous control). PCR products were resolved by electrophoresis on a 1.8% agarose-ethidium bromide gel. Lane 1, DNA markers; lane 2, A549 cells treated with conditioned medium from control mononuclear cells (uninfected); lanes 3 and 5, A549 cells treated with conditioned medium from GBS (1:1)-infected mononuclear cells; lanes 4 and 6, same as lanes 3 and 5 but without reverse transcriptase; lane 7, A549 cells treated with conditioned medium from GBS (10:1)-infected mononuclear cells; lane 8, same as lane 7 but without reverse transcriptase; lane 9, 18S rRNA control (495 bp); lane 10, iNOS RNA control (349 bp).

FIG. 4.

iNOS mRNA induction in A549 cells cocultured with GBS-treated human mononuclear cells. A total of 5 × 10⁶ mononuclear cells treated with GBS strain COH1 (1:1 ratio) were placed in 24-mm Costar Transwell culture inserts. Inserts were cocultured with monolayers of A549 cells on Transwell multiwell plates for 24 h. Total RNA was extracted from A549 cell lysates, and RT-PCR was conducted. PCR products were resolved by electrophoresis on a 1.8% agarose-ethidium bromide gel. (Gel A) RT-PCR products with Clontech iNOS primers. Lane 1, DNA markers; lane 2, A549 cocultured with uninfected mononuclear cells; lane 3, A549 cocultured with GBS in insert (without mononuclear cells); lane 4, A549 only (no coculture); lanes 5 and 6, A549 cocultured with GBS treated (1:1) mononuclear cells; lane 7, A549 cocultured with GBS-treated (10:1) mononuclear cells; lane 8, no RNA added; lane 9, iNOS RNA control (259 bp). (Gel B) The same samples as in gel A were reanalyzed with multiplex RT-PCR. Lanes 1 to 5 were as described above. Lane 6, same as lane 5 but without reverse transcriptase; lanes 7 and 8, duplicates of lanes 5 and 6; lane 9, A549 cocultured with GBS-treated (10:1) mononuclear cells; lane 10, same as lane 9 but without reverse transcriptase; lane 11, 18S rRNA control (495 bp); lane 12, iNOS RNA control (349 bp).

FIG. 5.

Morphological change in A549 cells treated with conditioned medium from GBS-treated human mononuclear cells. We centrifuged 24-h-conditioned medium from GBS-treated (strain COH1, 1:1) human mononuclear cells to remove GBS and then added it to monolayers of A549 cells for 24 h. (A) A549 cells treated with conditioned medium from control untreated mononuclear cells. (B) A549 cells treated with conditioned medium from GBS-treated mononuclear cells. Both panels are at the same magnification.