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

Najam A. Sharif, Shouxi Xu, Peggy E. Hellberg, Iok Hou Pang, Daniel A. Gamache, John M. Yanni

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12 Scopus citations


Purpose: The aims of the study were to characterize the signal transduction responses to platelet-activating factor (PAF) and to monitor the downstream effects of PAF on the production of proinflammatory cytokines in human conjunctival epithelial cells (HCECs). Methods: The generation of inositol phosphates ([3H]IPs) from [3H]phosphoinositide (PI) hydrolysis and the mobilization of intracellular calcium ([Ca2+]i) were evaluated using ion exchange chromatography and Fura-2 fluorescence techniques, respectively. The production of the cytokines (interleukin-6 [IL-6], interleukin-8 [IL-8], and granulocyte macrophage colony-stimulating factor [GM-CSF]) from PAF-stimulated HCECs was quantified using specific ELISA assays. Specific PAF antagonists were used to study the pharmacological aspects of PAF actions in HCECs. Results: PAF (100 nM) maximally stimulated PI turnover in HCECs by 2.3±0.02 fold (n=21) above basal levels and with a potency (EC50) of 5.9±1.7 nM (n=4). PAF or its stabilized analog, methyl carbamyl (mc)PAF (EC50=0.8 nM), rapidly mobilized [Ca2+]i, which peaked within 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 (100 nM) on PI turnover and [Ca2+]i were potently antagonized by the PAF antagonists, 1-o-hexadecyl-2-o-acetyl-snglycero-3-phospho (N,N,N-trimethyl) hexanolamine (IC50=0.69 μM; Ki=38 nM), methyl 2-(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=14 μM; Ki=789 nM [n=3 for each antagonist]). PAF-induced production of IL-6, IL-8, and GM-CSF from HCECs was also blocked by these PAF antagonists (IC50=4.6-8.6 μM). Conclusions: HCECs respond to PAF by generating IPs, mobilizing [Ca2+]i, and then secreting cytokines into the extracellular medium. These results suggest that HCECs may be key target cells for the PAF released from conjunctival mast cells following ocular allergic reactions. Therefore, HCECs in culture represent suitable in vitro models for the investigation of the role of PAF in human ocular allergic and inflammatory diseases and for the discovery of therapeutically useful PAF antagonists.

Original languageEnglish
Pages (from-to)1153-1161
Number of pages9
JournalMolecular Vision
StatePublished - 19 Jun 2009


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