Recent studies have implicated local inflammation and activation of complement amongst the processes involved in the pathogenesis of age-related macular degeneration (AMD). Several lines of investigation also indicate that bis-retinoid pigments, such as A2E, that accumulate as lipofuscin in retinal pigment epithelial (RPE) cells, contribute to the disease process. In an investigation of a potential trigger for complement activation in AMD, we explored the notion that the complex mixture of products resulting from photooxidation of A2E might include a range of fragments that could be recognized by the complement system as “foreign” and that could serve to activate the complement system, leading to low-grade inflammation. To this end, we established an in vitro assay by using human serum as a source of complement, and we measured products of C3 activation by enzyme immunoassay. Accordingly, we found that the C3 split products inactivated C3b (iC3b) and C3a were elevated in serum, overlying ARPE-19 cells that had accumulated A2E and were irradiated to induce A2E photooxidation. Precoating of microtiter plates with two species of oxidized A2E, peroxy-A2E, and furano-A2E, followed by incubation with serum, also activated complement. We suggest that products of the photooxidation of bis-retinoid lipofuscin pigments in RPE cells could serve as a trigger for the complement system, a trigger than would predispose the macula to disease and that, over time, could contribute to chronic inflammation. These findings link four factors that have been posited as being associated with AMD: inflammation, oxidative damage, drusen, and RPE lipofuscin.

The bis-retinoid pigments that accumulate in retinal pigment epithelial cells as lipofuscin are associated with inherited and age-related retinal disease. In addition to A2E and related cis isomers, we previously showed that condensation of two molecules of all-trans -retinal leads to the formation of a protonated Schiff base conjugate, all-trans -retinal dimer-phosphatidylethanolamine. Here we report the characterization of the related pigments, all-trans -retinal dimer-ethanolamine and unconjugated all-trans -retinal dimer, in human and mouse retinal pigment epithelium. In eyecups of Abcr ^−/− mice, a model of recessive Stargardt macular degeneration, all-trans -retinal dimer-phosphatidylethanolamine was increased relative to wild type and was more abundant than A2E. Total pigment of the all-trans -retinal dimer series (sum of all-trans -retinal dimer-phosphatidylethanolamine, all-trans -retinal dimer-ethanolamine, and all-trans -retinal dimer) increased with age in Abcr ^−/− mice and was modulated by amino acid variants in Rpe65. In in vitro assays, enzyme-mediated hydrolysis of all-trans -retinal dimer-phosphatidylethanolamine generated all-trans -retinal dimer-ethanolamine, and protonation/deprotonation of the Schiff base nitrogen of all-trans -retinal dimer-ethanolamine was pH-dependent. Unconjugated all-trans -retinal dimer was a more efficient generator of singlet oxygen than A2E, and the all-trans -retinal dimer series was more reactive with singlet oxygen than was A2E. By analyzing chromatographic properties and UV-visible spectra together with mass spectrometry, mono- and bis-oxygenated all-trans -retinal dimer photoproducts were detected in Abcr ^−/− mice. The latter findings are significant to an understanding of the adverse effects of retinal pigment epithelial cell lipofuscin.