Abstract
Purpose: :
Human organotypic retinal cultures (HORCs) were used to investigate the effect of P2X7 receptor activation in the regulation of IL-1β in the human retina. IL-1β may have a neurodegenerative role in diseases such as glaucoma.
Methods: :
The HORCs were dissected from donor globes within 24 hours postmortem and were maintained in serum-free DMEM/HamF12. To investigate IL-1β expression, the HORCs were exposed to the P2X7 receptor agonist, BzATP, in serum-free medium. The expression of IL-1β mRNA was measured by real time quantitative (Taqman) RT-PCR. IL-1β secreted into the medium was measured by ELISA. Investigation of other cytokines induced by BzATP was done with a human cytokine 27-Plex Panel (Bio-Plex) array. Statistical analysis was by ANOVA.
Results: :
Exposure of the HORCs (n=4) to 100µM BzATP resulted in a time-dependent increase in IL-1β mRNA expression with a 2-fold increase at 12hrs and a 4-fold increase at 36hrs (p<0.001). The P2X7 receptor antagonist, Brilliant Blue G (BBG) (1µM), significantly inhibited the BzATP-stimulated increase in IL-1β mRNA at 36hrs (p=0.002). Secretion of mature IL-1β was measured in the medium. The mean IL-1β protein release in control retinal samples was 0.5pg/ml at 12 and 36hrs. 100µM BzATP significantly increased IL-1β protein secretion from a mean of 8.0pg/ml at 12hrs (p<0.001) to 20.6pg/ml at 36hrs, an 8-fold increase over control cultures (p=0.001). The BzATP-stimulated IL-1β protein secretion was significantly inhibited by 1µM BBG to 2.1pg/ml (p<0.001) and 3.0pg/ml (p=0.002) at 12 and 36hrs, respectively. BzATP did not cause significant up- or down-regulation of 26 other cytokines.
Conclusions: :
The HORCs retain regional morphology allowing them to be useful for the investigation of human retinal diseases. P2X7 stimulation of HORCs causes an increase in expression of IL-1β mRNA and an increase in the secretion of IL-1β protein. IL-1β secretion resulting from P2X7 stimulation may have a role in human ocular neurodegenerative diseases such as glaucoma.
Keywords: retinal culture • receptors: pharmacology/physiology • cytokines/chemokines