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P. Yang, S. A. Medearis, G. J. Jaffe; Effect of Terminal Complement Membrane Attack Complex/C5b-9 on RPE Cell Viability. Invest. Ophthalmol. Vis. Sci. 2010;51(13):482.
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Complement activation has been implicated increasingly in the pathogenesis of age-related macular degeneration (AMD). Complement membrane attack complex (MAC)/C5b-9, the end product of complement cascade, is present in drusen and in experimental CNV mouse models. Interestingly, sublytic MAC/C5b-9 induces cell proliferation by survival pathways such as PI3K/Akt or MAPK pathways in some non-ocular nucleated cells. Herein, we investigate the effect of MAC/C5b-9 deposition on RPE viability and signal transduction pathways.
MAC/C5b-9 was assembled on cultured human RPE cells with either purified complement components (C5b6-C9) or emmprin antibody and 10% normal human serum (NHS, a complement source). MAC/C5b-9 complement components minus C7 (a MAC/C5b-9 complement component) and heat-inactived NHS (HiNHS) were used as negative controls. MAC/C5b-9 formation was determined by immunofluorescent stain using C5b-9 antibody. The lytic activity of MAC/C5b-9 was examined in sheep erythrocytes. The effect of MAC/C5b-9 deposition on RPE cell viability was assessed by tetrazolium salt (WST-1) assay and a lactate dehydrogenase (LDH) release assay. MAC/C5b-9 deposition-induced phosphorylation of Erk, Akt, P38, and JNK was examined by Western blot. MAC/C5b-9 deposition-induced NF-ΚB translocation was evaluated by immunofluorescent double stain.
There was specific immunofluorescent C5b-9 membrane staining in the emmprin-NHS and C5b6-C9 groups, but not in the emmprin-HiNHS or minus C7 groups 15 minutes, 30 minutes, and 1 hour after complement incubation. MAC/C5b-9 deposition-mediated cell lysis was confirmed in sheep erythrocytes 30 minute following complement treatment. However, there was no significant LDH release or decreased cell viability in the emmprin-NHS and C5b6-C9 groups 1 hour and 24 hours, respectively, after complement addition. Phosphorylation of Erk, Akt, P38, and JNK, measured 15 minutes and 30 minutes following complement treatment was not detected in the C5b6-C9 group. NF-ΚB translocation was not detected in the C5b6-C9 or emmprin-NHS groups 1 hour and 2 hours after complement treatment.
RPE cells are resistant to MAC/C5b-9 deposition-mediated cell lysis. This resistance may help to protect RPE cells from complement-mediated injury normally, and in diseases such as AMD.
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