Synergistic effect of platelet-activating factor and tumor necrosis factor-alpha on corneal myofibroblast apoptosis.
OBJECTIVE
Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparency. In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)-alpha in corneal myofibroblast apoptosis was explored.


METHODS
Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm2). Mouse anti-alpha-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-alpha receptors in those cells. The reactivity of the antibodies was characterized by Western blot analysis. To induce myofibroblast apoptosis, PCMs were treated for 24 to 72 hours with methylcarbamyl-PAF (cPAF, 300 nM), a nonhydrolyzable PAF analogue, TNF-alpha (20 ng/mL), and TNF-alpha+cPAF, with or without LAU-0901 (150 nM), a novel PAF antagonist. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. 6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Images were recorded by fluorescence microscope.


RESULTS
Immunofluorescence with a PAF-receptor (N terminus) polyclonal antibody showed that the receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF-alpha receptor II (TNF-RII) was localized in the cytoplasm, whereas TNF-receptor I (TNF-RI) was found in both cytoplasm and plasma membrane. Treatment with TNF-alpha for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Western blot analysis showed expression of single bands corresponding to the molecular weights of the receptors. 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, demonstrating a synergistic action between PAF and TNF-alpha. Blocking the PAF receptor with LAU-0901 inhibited the synergistic effect induced by PAF.


CONCLUSIONS
Corneal myofibroblasts express a PAF receptor in the nuclear membrane, and they also express TNF-RI and RII. The synergistic effect on myofibroblast apoptosis by PAF and TNF-alpha suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.
He
Jiucheng
J
Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana, USA.
Bazan
Haydee E P
HE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism / Q000473:pathology*; D003593:Cytoplasm / Q000378:metabolism; D004095:Dihydropyridines / Q000494:pharmacology; D004338:Drug Combinations; D004357:Drug Synergism; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D020287:In Situ Nick-End Labeling; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000378:metabolism; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004095:Dihydropyridines; D004338:Drug Combinations; C487837:LAU 0901; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; C039351:platelet activating factor receptor