Abstract
Purpose: :
Expression of immune molecules by neuronal cells may control central nervous system (CNS) functions. Our laboratory recently identified the immune molecules, PD-1 and PD-ligands in retinal ganglion cells (RGC). In the developing retina, interaction of PD-1 and PD-L1 is believed to play a role in neuronal cell death. However, the cellular mechanisms by which this interaction contributes to cell death are not understood. The RGC-5 cell line could serve as an in vitro model in order to identify possible downstream effects of PD-1 ligation. The purpose of the study was to test whether PD-1 or its ligands are constitutively expressed or whether expression could be induced, following differentiation of RCG5 cells towards a neuronal phenotype.
Methods: :
RGC5 cells were cultured in DMEM medium with 10% serum. Differentiation of RG-C5 cells was performed with staurosporine over a 7 day time course. PD-1 and PD-ligand mRNA and protein expression were tested using Western blot, RT-PCR and immunofluorescence.
Results: :
Exposure to staurosporine induced morphological changes in RGC-5 cells including, neurite outgrowth and induction of Thy1.1 expression, as previously described in literature. Although undifferentiated RGC-5 cells expressed PD-1 mRNA, no corresponding protein was observed by Western or immunofluorescence (IF). Following differentiation using staurosporine, PD-1 protein expression was maximally expressed at day 3. Using IF, PD-1 expression is observed after one day of exposure to staurosporine and is confined to the cell body. PD-ligand mRNA was also expressed in both differentiated and undifferentiated RGC-5 cells. Using IF techniques, PD-ligand expression is detectable in staurosporine treated RGC-5 cells, starting at day 1 and is expressed in both cell body and neurites.
Conclusions: :
Protein expression of PD-1 and PD-ligands can be induced upon differentiation with staurosporine, as evidenced by Western blot and IF. Inducted expression of these molecules will allow future studies to identify mechanisms of signal transduction following PD-1 ligation and evaluate the role that these molecules may play in neuronal cell death.
Keywords: ganglion cells • apoptosis/cell death • retinal culture