Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular degeneration

Abstract

Purpose: There is evidence for complement dysfunction in age-related macular degeneration (AMD). Complement activation leads to formation of the membrane attack complex (MAC), known to assemble on retinal pigment epithelial (RPE) cells. Therefore, the effect of sub-lytic MAC on RPE cells was examined with regard to pro-inflammatory or pro-angiogenic mediators relevant in AMD.

Methods: For sub-lytic MAC induction, RPE cells were incubated with an antiserum to complement regulatory protein CD59, followed by normal human serum (NHS) to induce 5% cell death, measured by a viability assay. MAC formation was evaluated by immunofluorescence and FACS analysis. Interleukin (IL)-6, -8, monocytic chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were quantified by enzyme-linked immunosorbent assay (ELISA). Intracellular MCP-1 was analysed by immunofluorescence, vitronectin by western blotting, and gelatinolytic matrix metalloproteinases (MMPs) by zymography.

Results: Incubation of RPE cells with the CD59 antiserum followed by 5% NHS induced sub-lytic amounts of MAC, verified by FACS and immunofluorescence. This treatment stimulated the cells to release IL-6, -8, MCP-1, and VEGF. MCP-1 staining, production of vitronectin, and gelatinolytic MMPs were also elevated in response to sub-lytic MAC.

Conclusions: MAC assembly on RPE cells increases the IL-6, -8, and MCP-1 production. Therefore, sub-lytic MAC might have a significant role in generating a pro-inflammatory microenvironment, contributing to the development of AMD. Enhanced vitronectin might be a protective mechanism against MAC deposition. In addition, the increased expression of gelatinolytic MMPs and pro-angiogenic VEGF may be associated with neovascular processes and late AMD.

Figure 1

Verification of sub-lytic MAC assembly. (a) MTT viability assay measuring the amount of cell death after treating ARPE-19 cells with anti-CD59, and ascending concentrations of NHS for 1 h at 37 °C. For negative controls, cells were treated with anti-CD59/HI–NHS, and for positive controls with 1% Triton X-100. Data are plotted as means±s.d. (n=5). (b) The mean fluorescence intensity of C5b-9 using flow cytometry presented as a histogram in control (anti-CD59/HI–NHS; open histogram), and anti-CD59/NHS-treated cells (grey histogram) (n=4 for each treatment). (c) Representative images (n=4) from immunofluorescence staining for C5b-9 after MAC assembly on ARPE-19 cells. 1=HI–NHS alone, 2=NHS alone, 3=anti-CD59/HI–NHS, 4=anti-CD59/NHS. The scale bar indicates 10 μm.

Figure 2

Cytokine production by RPE. Representative (a) IL-6, (b) IL-8, and (c) MCP-1 production after various treatments as measured in the cell culture supernatant by ELISA. Duplicate data are shown as mean±s.d. (n=3). (d) Representative immunofluorescent staining of MCP-1 following treatment with HI–NHS (1), NHS (2), anti-CD59/HI–NHS (3), and anti-CD59/NHS (4) out of 4 separate experiments. The scale bar indicates 10 μm. ^*P<0.05, ^**P<0.01.

Figure 3

(a) Representative western blot for vitronectin using cell lysates of ARPE-19 cells after various treatments. (b) Semi-quantitative analysis of vitronectin western blots (n=4) normalised to α-tubulin. ^*P<0.05.

Figure 4

ELISA of representative VEGF production by sub-confluent ARPE-19 cells after treatment with anti-CD59/NHS, anti-CD59/HI–NHS, NHS alone, or HI–NHS alone. Duplicates are shown as mean±s.d., whereas the experiment was repeated two more times with comparable results. ^*P<0.05.

Figure 5

(a) Representative zymography for gelatinolytic MMP following different treatments (n=5). Data of semi-quantitative analysis of (b) MMP-2 and (c) MMP-9 from zymographs after different treatments were presented as mean±s.d. ^*P<0.05, ^**P<0.01.