Abstract
Purpose: :
Apoptosis plays a causative role for development and progression of certain retinal diseases such as retinitis pigmentosa (RP). Alterations in the microenvironment produced by dead or dying cells can precipitate photoreceptor death in RP. In this study, we determine whether Fas ligand (FasL) and its cognate receptor Fas are expressed and functional in cone photoreceptors.
Methods: :
Expression of FasL/Fas was examined in posterior segments of postmortem human eye samples and culture of murine cone photoreceptor cell line (661W) by immunofluorescent co-labeling experiments using antibodies specific for FasL/Fas and cone marker proteins. For functional evaluation, cultured 661W cells in chamber slides were treated with either recombinant FasL at different doses (15ng/ml to 3.3µg/ml) and time of exposure from 6 to 48 hrs, or Fas agonist Jo2 (20µg/ml) for 6 hrs. Non-treated cells or cells treated with FasL neutralizing antibody NOK-2, or isotype IgG2 for Jo2 were controls. Apoptosis was examined by TUNEL assay and quantified in cells showing typical DNA strand nicks and fragmentation. Cell morphology and growth characteristics were compared before and after treatment by light microscopy. Active executioner caspase 3 was detected by immunofluorescence in the presence or absence of co-treatment with caspase 8 inhibitor Z-IETD-FMK.
Results: :
Immunofluorescent signal for Fas is predominantly in outer segment of the photoreceptor layer in human retina including cones. In cultured 661W cells, the receptor is localized at the cell surface membrane. Expression of endogenous FasL is barely detectable. Treatment with recombinant FasL or Fas agonist Jo2 causes apoptotic changes such as clustering of cells, shrinkage of cell body and loss of cell-cell contact. The number of TUNEL positive cells after FasL treatment shows positive correlation with dose and time of exposure, and is saturable by 48 hrs of exposure to nearly 65% of total cells. In addition, executioner caspase 3 is activated, which can be blocked by co-treatment with caspase 8 inhibitor.
Conclusions: :
Specific expression of Fas in cone photoreceptors was found in human retina. Treatment of 661W cells with recombinant FasL causes apoptosis, suggesting a pro-apoptotic function for the FasL/Fas system in cone photoreceptors. The data lay out the rationale to delineate the pathogenic contribution of extrinsic apoptotic signaling mediated by FasL/Fas in various genetic forms of retinitis pigmentosa.
Keywords: photoreceptors • apoptosis/cell death • retinal degenerations: cell biology