Expression and modulation of RPE cell membrane complement regulatory proteins.
OBJECTIVE
Complement, inflammation, and oxidant injury contribute to age-related macular degeneration (AMD). Membrane complement regulatory proteins (mCRPs) such as CD46, CD55, and CD59, protect host cells from complement attack. The factors that regulate RPE mCRP expression are not well understood. In this study, the authors sought to determine whether cytokines and hydroquinone (HQ) affect mCRP expression in cultured human RPE (hRPE) and cultured mouse RPE (mRPE) cells.


METHODS
Cultured hRPE and mRPE cells were stimulated with cytokines for various times or with HQ for multiple 6-hour periods. mRNA and protein expression of mCRPs in cultured hRPE cells from 10 donors, native hRPE, and mouse eyecups and native mRPE cells were evaluated by real-time RT-PCR, Western blot analysis, and flow cytometry, respectively.


RESULTS
Three mCRPs were expressed in cultured hRPE cells (CD59>CD46>CD55). CD46 and CD59 protein were detected in native hRPE cells. CD59 protein levels in cultured hRPE cells were higher than in native hRPE cells. CD46 protein polymorphisms were observed in cultured hRPE cells. Cultured hRPE cell mCRP expression was upregulated by TNF-alpha, IL-1beta, and a repetitive nonlethal dose of HQ. CD59a levels were higher in mouse eyecups than in nonocular tissues. Mouse mCRP mRNA and protein were detected in native mRPE cells. Responsiveness to cytokines in cultured mRPE cells differed from that in cultured hRPE cells.


CONCLUSIONS
Human and mouse RPE cell mCRPs are upregulated by inflammatory cytokines and repetitive nonlethal oxidant exposure in a species-specific manner. Increased cell mCRPs may help to protect RPE cells from complement- and oxidant-mediated injury in diseases such as AMD.
Yang
Ping
P
Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Tyrrell
Jillian
J
Han
Ian
I
Jaffe
Glenn J
GJ
D000818:Animals; D015153:Blotting, Western; D018958:CD55 Antigens / Q000235:genetics* / Q000378:metabolism; D018957:CD59 Antigens / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology*; D051916:Membrane Cofactor Protein / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000378:metabolism; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D011994:Recombinant Proteins / Q000494:pharmacology; D012160:Retina / Q000187:drug effects / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
C497857:CD46 protein, human; D018958:CD55 Antigens; D018957:CD59 Antigens; C482970:CR1L protein, human; C081470:Cr1l protein, mouse; D016207:Cytokines; D006873:Hydroquinones; C497859:Mcp protein, mouse; D051916:Membrane Cofactor Protein; D012333:RNA, Messenger; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D011994:Recombinant Proteins; C051590:CD59 protein, human; C031927:hydroquinone