Complement activation by RPE cells preexposed to TNFα and IFNγ
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Complement activation by RPE cells preexposed to TNFα and IFNγ. / Udsen, Maja; Tagmose, Christian; Garred, Peter; Nissen, Mogens Holst; Faber, Carsten.
In: Experimental Eye Research, Vol. 218, 108982, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Complement activation by RPE cells preexposed to TNFα and IFNγ
AU - Udsen, Maja
AU - Tagmose, Christian
AU - Garred, Peter
AU - Nissen, Mogens Holst
AU - Faber, Carsten
N1 - Publisher Copyright: © 2022 The Authors
PY - 2022
Y1 - 2022
N2 - Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.
AB - Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.
KW - Complement
KW - Interferons
KW - Membrane attack complex
KW - Retinal pigment epithelium
KW - Tumor necrosis factor
U2 - 10.1016/j.exer.2022.108982
DO - 10.1016/j.exer.2022.108982
M3 - Journal article
C2 - 35183540
AN - SCOPUS:85125468217
VL - 218
JO - Experimental Eye Research
JF - Experimental Eye Research
SN - 0014-4835
M1 - 108982
ER -
ID: 300287056