Abstract
Purpose: :
A2E, the major fluorophore of lipofuscin, accumulates in retinal pigment epithelial cells with increasing age. This accumulation is particularly marked in Stargardt disease, but has also been implicated in the pathogenesis of age related macular degeneration. Causative factors in age related macular degeneration include oxidative stress and inflammation. A2E has been shown to induce oxidative stress in retinal pigment epithelial cells. The purpose of this study was to assess whether it could also induce inflammation, through the release of interleukin-1ß.
Methods: :
A2E was synthesized and purified using established methods. Purity of greater than 95% was confirmed using high performance liquid chromatography. Molecular weight was confirmed using MALDI mass spectrometry. ARPE-19 cells were incubated with A2E for 24 hours and IL-1ß production was assessed in cell culture supernatant using enzyme linked immunosorbant assay. Immunohistochemical techniques were used to detect the activation of cellular pathways in the ARPE-19 cells. Specific inhibitors were used to confirm signalling pathways involved.
Results: :
Incubation of 10 µM and 25 µM of A2E with ARPE-19 cells produced 13 pg/ml and 90 pg/ml of IL-1ß in the cell culture supernatant respectively. Incubation with 10 µM of A2E, in the presence of a caspase-1 specific inhibitor, produced inhibition of IL-1ß production in a dose dependent manner. Cells stimulated with A2E also showed increased immunohistochemical staining for the NLRP3 inflammasome and ASC protein, compared with unstimulated cells.
Conclusions: :
A2E induces IL-1ß production in ARPE-19 cells through caspase-1 activation. This appears to be dependent on activation of the NLRP3 inflammasome. Accumulation of A2E in the retinal pigment epithelium is associated with Stargardt disease and age related macular degeneration. A2E may therefore have a pathogenic role in these diseases through the release of the proinflammatory cytokine IL-1ß.
Keywords: retinal pigment epithelium • inflammation • age-related macular degeneration