Platelet-Activating Factor-Induced Intracellular Signaling and Release of Cytokines and Prostaglandin E 2 in Immortalized Human Corneal Epithelial Cells

Abstract

Purpose and Methods:

Immortalized human corneal epithelial (CEPI-17-CL4) cells were exposed to different concentrations of platelet-activating factor (PAF) and the mobilization of intracellular calcium ([Ca²⁺]_i) was studied using fluorometrics. Additionally, the production of the cytokines [interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α)], and matrix metalloproteinase-1 (MMP-1) and prostaglandin E₂ (PGE₂) from PAF-stimulated cells was also determined using ELISA assays.

Results:

While PAF, histamine, and bradykinin stimulated the mobilization of [Ca²⁺]_i in these cells, PAF was the least efficacious. [Ca²⁺]_i mobilization induced by PAF was inhibited by 2 PAF receptor antagonists, PCA-42481 and CV-6209 (both 10 μM), and by a phospholipase C inhibitor, U73122 (60% at 4 μM). PAF increased the production of PGE₂ (with maximum effect at 30 and 100 nM) and GM-CSF (maximum effect at 1 μM) in CEPI-17-CL4 cells. However, PAF did not stimulate the generation of IL-6, IL-8, TNF-α, and MMP-1 to any significant level and in a consistent manner. PAF increased the incorporation of [³H]-thymidine into CEPI-17-CL4 cells with a maximal effect at 30 nM.

Conclusions:

These data indicate that functional PAF receptors are present on CEPI-17-CL4 cells that can activate mobilization of [Ca²⁺]_i. Other consequences of PAF receptor activation in CEPI-17-CL4 cells are the generation of PGE₂ and certain proinflammatory cytokines such as GM-CSF, and increasing cell proliferation.