PURPOSE: Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, interacts with Toll-like receptor (TLR)-3 and thereby elicits immunoinflammatory responses characteristic of viral infection. The effects of poly(I:C) on the expression of proinflammatory cytokines, chemokines, and adhesion molecules, as well as the signaling pathways that underlie such effects, were investigated in cultured human corneal fibroblasts. METHODS: Expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was evaluated by immunoblot and immunofluorescence analyses. Release of the proinflammatory cytokine IL-6 and of the chemokines interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, eotaxin, and RANTES was measured with assay kits. Subcellular localization of the p65 subunit of the transcription factor nuclear factor (NF-kappaB) was examined by immunofluorescence analysis. Expression of TLR3, phosphorylation (activation) of mitogen-activated protein kinases (MAPKs), and phosphorylation and degradation of the NF-kappaB-inhibitory protein IkappaB-alpha was assessed by immunoblot analysis. RESULTS: Poly(I:C) induced the up-regulation of TLR3, the release of IL-6, IL-8, G-CSF, MIP-1beta, eotaxin, and RANTES, and the expression of ICAM-1 and VCAM-1 in corneal fibroblasts. It also activated the MAPKs ERK, p38, and JNK and induced the phosphorylation and degradation of IkappaB-alpha and the nuclear translocation of p65 in these cells. Poly(I:C)-induced expression of IL-6, IL-8, G-CSF, MIP-1beta, exotaxin, RANTES, and ICAM-1 was inhibited differentially by the MAPK inhibitors PD98059 and SB203580 and by JNK inhibitor II. CONCLUSIONS: Poly(I:C) induces the up-regulation of TLR3, the MAPK-dependent expression of proinflammatory cytokines, chemokines, and adhesion molecules and the activation of NF-kappaB in human corneal fibroblasts. Corneal fibroblasts may thus play an important role in the modulation of local immune and inflammatory responses to viral infection in the corneal stroma.
PURPOSE:Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, interacts with Toll-like receptor (TLR)-3 and thereby elicits immunoinflammatory responses characteristic of viral infection. The effects of poly(I:C) on the expression of proinflammatory cytokines, chemokines, and adhesion molecules, as well as the signaling pathways that underlie such effects, were investigated in cultured human corneal fibroblasts. METHODS: Expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was evaluated by immunoblot and immunofluorescence analyses. Release of the proinflammatory cytokine IL-6 and of the chemokines interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, eotaxin, and RANTES was measured with assay kits. Subcellular localization of the p65 subunit of the transcription factor nuclear factor (NF-kappaB) was examined by immunofluorescence analysis. Expression of TLR3, phosphorylation (activation) of mitogen-activated protein kinases (MAPKs), and phosphorylation and degradation of the NF-kappaB-inhibitory protein IkappaB-alpha was assessed by immunoblot analysis. RESULTS:Poly(I:C) induced the up-regulation of TLR3, the release of IL-6, IL-8, G-CSF, MIP-1beta, eotaxin, and RANTES, and the expression of ICAM-1 and VCAM-1 in corneal fibroblasts. It also activated the MAPKs ERK, p38, and JNK and induced the phosphorylation and degradation of IkappaB-alpha and the nuclear translocation of p65 in these cells. Poly(I:C)-induced expression of IL-6, IL-8, G-CSF, MIP-1beta, exotaxin, RANTES, and ICAM-1 was inhibited differentially by the MAPK inhibitors PD98059 and SB203580 and by JNK inhibitor II. CONCLUSIONS:Poly(I:C) induces the up-regulation of TLR3, the MAPK-dependent expression of proinflammatory cytokines, chemokines, and adhesion molecules and the activation of NF-kappaB in human corneal fibroblasts. Corneal fibroblasts may thus play an important role in the modulation of local immune and inflammatory responses to viral infection in the corneal stroma.