Human Complement Factor H (CFH) Is Expressed in ARPE 19 Cells

Abstract

Purpose: : A common haplotype in the gene for the regulator of the alternative pathway of complement CFH has been linked to individual predisposition to age–related macular degeneration. CFH is primarily synthesised in the liver. PCR–analysis revealed CFH and its truncated form FHL–1 transcripts in human RPE cells. In this study we analyzed immunehistochemically expression of CFH and FHL–1 in the ARPE 19 cell line.

Methods: : ARPE 19 cell line was obtained from ATCC depository and cultured in Dulbeccos modified Eagles medium and Ham’s F 12 medium containing 20 % fetal calf serum (FCS). All experiments were performed between the 10^th and 18^th passage. After removing serum remnants cells were incubated in serum free medium containing increasing interleukin 6 concentrations for 24 h. The supernatants were used for western blot analysis. For immunohistochemical detection of CFH the cells were fixed in acetone. Goat antibody directed against human CFH and rabbit anti FHL–1 were incubated for 2 h at RT. The secondary antibody conjugated to Alexa 555 was incubated for 1 h and was followed by a nucleus staining with DAPI 5 µl /ml in PBS for 15 min. Medium supernatants were concentrated 10 fold in Amicon Ultra chamber (Millipore) 10.000 Da. Western blot was performed in a 10 % SDS Gel under non–reducing conditions.

Results: : Immunohistochemistry of ARPE cells was positive for CFH and FHL–1. Interleukin 6 induced CFH and FHL–1 expression in a dose dependent manner, although a basal CFH and FHL–1 production could be observed also without interleukin stimulation. Western blot analysis showed presence of CFH and FHL–1 in supernatants. Controls for human albumin and media were negative.

Conclusions: : ARPE 19 cells produce CFH and FHL–1. As CFH polymorphism is linked to age related macular degeneration, a low level complement activation at the RPE–choroid interface might be involved in the pathogenesis. Local production of CFH and other complement components within the RPE might be of pathological value as well.

Keywords: retina • age-related macular degeneration • immunohistochemistry