Complement and UV-irradiated photoreceptor outer segments increase the cytokine secretion by retinal pigment epithelial cells.
OBJECTIVE
Age-related macular degeneration (AMD) is accompanied by increased complement activation, and by lipofuscin accumulation in retinal pigment epithelial (RPE) cells due to incomplete degradation of photoreceptor outer segments (POS). The influence of POS, ultraviolet (UV)-irradiated POS and human complement sera (HCS) on cytokine secretion from RPE cells was therefore examined.


METHODS
RPE cells were incubated with POS or UV-POS every other day for 1 week. The autofluorescence (AF) was measured photometrically and by flow cytometry. Senescence-associated genes were analyzed by RT-PCR. Internalization and degradation of POS were determined using phagocytosis and degradation assays, and lysosomal function by neutral red uptake. RPE cells in polycarbonate cell culture inserts were incubated apically with POS or UV-POS and afterward basally with HCS. C7-deficient HCS was used as control. The integrity of the cell monolayer was assessed by measuring the transepithelial electrical resistance (TER) and the permeability. Interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor were quantified by ELISA.


RESULTS
POS treatment led to an increased AF and senescence marker expression, which were further elevated in response to UV-POS. UV-POS were preferentially accumulated over POS and the lysosomal function was impaired due to UV-POS. HCS intensified the cytokine production compared with controls. POS had no effect, though UV-POS combined with HCS induced a significant increase in all cytokines.


CONCLUSIONS
RPE cultivation with UV-POS might serve as a model to investigate the accumulation of lipofuscin-like structures. The enhanced cytokine secretion due to UV-POS with HCS may account for an increased susceptibility for lipofuscin-loaded cells to complement, inducing a proinflammatory environment as observed in AMD.
Ophtha-Lab, St. Franziskus Hospital, Muenster, Germany.
Katharina
K
Lueck
Maren
M
Hennig
Albrecht
A
Lommatzsch
Daniel
D
Pauleikhoff
Susanne
S
Wasmuth
D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D003165:Complement System Proteins / Q000494:pharmacology*; D016207:Cytokines / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D010587:Phagocytosis / Q000502:physiology; D055214:Retinal Photoreceptor Cell Outer Segment / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014466:Ultraviolet Rays
D015415:Biomarkers; D016207:Cytokines; D003165:Complement System Proteins