Human conjunctival epithelial cell responses to platelet-activating factor (PAF): signal transduction and release of proinflammatory cytokines

[摘要] Purpose: The aims of the study were tocharacterize the signal transduction responses to platelet-activatingfactor (PAF) and to monitor the downstream effects of PAF on theproduction of proinflammatory cytokines in human conjunctivalepithelial cells (HCECs). Methods: The generation of inositolphosphates ([3H]IPs) from [3H]phosphoinositide(PI) hydrolysis and the mobilization of intracellular calcium ([Ca2+]i)were evaluated using ion exchange chromatography and Fura-2fluorescence techniques, respectively. The production of the cytokines(interleukin-6 [IL-6], interleukin-8 [IL-8], and granulocyte macrophagecolony-stimulating factor [GM-CSF]) from PAF-stimulated HCECs wasquantified using specific ELISA assays. Specific PAF antagonists wereused to study the pharmacological aspects of PAF actions in HCECs. Results: PAF (100 nM) maximallystimulated PI turnover in HCECs by 2.3±0.02 fold (n=21) above basallevels and with a potency (EC50) of 5.9±1.7 nM (n=4). PAF orits stabilized analog, methyl carbamyl (mc)PAF (EC50=0.8nM), rapidly mobilized [Ca2+]i, which peakedwithin 30–60 s and remained elevated for 3 min. PAF (10 nM–1 µM)stimulated the release of the proinflammatory cytokines, IL-6, IL-8,and GM-CSF, 1.4–3.5 fold above basal levels. The effects of PAF (100nM) on PI turnover and [Ca2+]i were potentlyantagonized by the PAF antagonists,1-o-hexadecyl-2-o-acetyl–sn-glycero-3-phospho (N,N,N-trimethyl)hexanolamine (IC50=0.69 µM; Ki=38 nM), methyl2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethyl-pyridine-3,5-dicsrboxylate(PCA-42481; IC50=0.89 µM; Ki=50 nM),rac-3-(N-octadecylcarbomoyl)-2-methoxy) propyl-(2-thiazolioethyl)phosphate (CV-3988; IC50=13 µM; Ki=771 nM), and(+/−)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl (SM-10661; IC50=14µM; Ki=789 nM [n=3 for each antagonist]). PAF-inducedproduction of IL-6, IL-8, and GM-CSF from HCECs was also blocked bythese PAF antagonists (IC50=4.6– 8.6 µM). Conclusions: HCECs respond to PAF bygenerating IPs, mobilizing [Ca2+]i, and thensecreting cytokines into the extracellular medium. These resultssuggest that HCECs may be key target cells for the PAF released fromconjunctival mast cells following ocular allergic reactions. Therefore,HCECs in culture represent suitable in vitro models for theinvestigation of the role of PAF in human ocular allergic andinflammatory diseases and for the discovery of therapeutically usefulPAF antagonists.

[学科分类] 生物化学/生物物理