Purchase this article with an account.
N. Niyadurupola, P. Sidaway, L. Dawes, D. C. Broadway, J. Sanderson; P2X7 Receptor Activation in Human Organotypic Retinal Cultures (HORCs) Regulates IL-1β Expression. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4324.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
This PDF is available to Subscribers Only