April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Effect of Terminal Complement Membrane Attack Complex/C5b-9 on RPE Cell Viability
Author Affiliations & Notes
  • P. Yang
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • S. A. Medearis
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • G. J. Jaffe
    Ophthalmology, Duke University Eye Center, Durham, North Carolina
  • Footnotes
    Commercial Relationships  P. Yang, None; S.A. Medearis, None; G.J. Jaffe, None.
  • Footnotes
    Support  P30EY05722 (Core Grant)
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 482. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal pigment epithelium • age-related macular degeneration 
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