Abstract
Abstract: :
Purpose:After injury, the quiescent keratocytes of corneal stroma become activated and transform to fibroblasts and myofibroblasts. Previous work in our laboratory has shown that platelet–activating factor (PAF) is a strong inducer of apoptosis in corneal keratocytes. More recently we found that PAF also induced apoptosis in corneal myofibroblasts, although these were more resistant than keratocytes. Here we investigated how PAF, combined with TNF–α, induces corneal myofibroblast apoptosis. Methods:Pig corneal myofibroblasts were obtained from subcultured fibroblasts plated at a low density (5cells/mm2). Mouse anti–α–SMA antibody was used to identify the cell phenotype of myofibroblasts. Immunofluorescence was performed to localize PAF and TNF–α receptors in those cells. To induce myofibroblast apoptosis, 7–day–cultured corneal myofibroblasts were treated with cPAF (300 nM), TNF–α (20 ng/ml), or TNF–α plus cPAF with or without LAU0901(150 nM), a novel PAF antagonist, for 24, 48, or 72 hours. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. DAPI was used for nuclear counterstaining. Images were recorded by fluorescence microscope, Nikon Eclipse TE200 equipped with a Nikon digital camera DXM1200 using the MetaVue imaging software. Results:Immunofluorescence with a rabbit anti–PAF–receptor (N–terminus) polyclonal antibody showed that PAF receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF–α receptor 2 was localized in the cytoplasm, while TNF–receptor 1 was found in both cytoplasm and plasma membrane. Treatment with TNF–α for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells, respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and 86%, respectively, of the cells, suggesting a synergistic action between PAF and TNF–α. Blocking the PAF receptor with LAU0901 inhibited the synergistic effect induced by PAF. Conclusions:Although it has been reported that corneal keratocytes and fibroblasts both express TNF receptors, to our knowledge, this is the first study that shows the expression of TNF receptors in corneal myofibroblasts. The synergistic effect on myofibroblast apoptosis by PAF and TNF–α suggests that during prolonged inflammation, PAF acting in conjunction with other cytokines could delay stromal wound healing.
Keywords: cornea: stroma and keratocytes • wound healing • apoptosis/cell death