Cytokine, Chemokine, and Adhesion Molecule Expression Mediated by MAPKs in Human Corneal Fibroblasts Exposed to Poly(I:C)

Abstract

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. 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. 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. 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.