Ophthalmic research · 2015
Complement Stimulates Retinal Pigment Epithelial Cells to Undergo Pro-Inflammatory Changes.
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Lueck Katharina, Busch Martin, Moss Stephen E, Greenwood John, Kasper Maren, Lommatzsch Albrecht, Pauleikhoff Daniel, Wasmuth Susanne
Аннотация
BACKGROUND/AIMS: We examined the effect of human complement sera (HCS) on retinal pigment epithelial (RPE) cells with respect to pro-inflammatory mediators relevant in early age-related macular degeneration (AMD).
METHODS: RPE cells were treated with complement-containing HCS or with heat-inactivated (HI) HCS or C7-deficient HCS as controls. Cells were analysed for C5b-9 using immunocytochemistry and flow cytometry. Interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were quantified by ELISA and RT-PCR. Tumour necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), were analysed by Western blotting. The intracellular distribution of nuclear factor (NF)-x03BA;B was investigated by immunofluorescence.
RESULTS: A concentration-dependent increased staining for C5b-9 but no influence on cell viability was observed after HCS treatment. ELISA and RT-PCR analysis revealed elevated secretion and expression of IL-6, IL-8, and MCP-1. Western blot analysis showed a concentration-dependent increase in ICAM-1, VCAM-1, and TNF-α in response to HCS, and immunofluorescence staining revealed nuclear translocation of NF-x03BA;B.
CONCLUSION: This study suggests that complement stimulates NF-x03BA;B activation in RPE cells that might further create a pro-inflammatory environment. All these factors together may support early AMD development.
METHODS: RPE cells were treated with complement-containing HCS or with heat-inactivated (HI) HCS or C7-deficient HCS as controls. Cells were analysed for C5b-9 using immunocytochemistry and flow cytometry. Interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were quantified by ELISA and RT-PCR. Tumour necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), were analysed by Western blotting. The intracellular distribution of nuclear factor (NF)-x03BA;B was investigated by immunofluorescence.
RESULTS: A concentration-dependent increased staining for C5b-9 but no influence on cell viability was observed after HCS treatment. ELISA and RT-PCR analysis revealed elevated secretion and expression of IL-6, IL-8, and MCP-1. Western blot analysis showed a concentration-dependent increase in ICAM-1, VCAM-1, and TNF-α in response to HCS, and immunofluorescence staining revealed nuclear translocation of NF-x03BA;B.
CONCLUSION: This study suggests that complement stimulates NF-x03BA;B activation in RPE cells that might further create a pro-inflammatory environment. All these factors together may support early AMD development.
Предметные рубрики MeSH
Blotting, WesternCells, CulturedComplement Membrane Attack ComplexComplement System ProteinsCytokinesEnzyme-Linked Immunosorbent AssayFlow CytometryHumansImmunohistochemistryInflammation MediatorsIntercellular Adhesion Molecule-1Macular DegenerationNF-kappa BReal-Time Polymerase Chain ReactionRetinal Pigment EpitheliumVascular Cell Adhesion Molecule-1