Premium
Functional Expression of Toll‐Like Receptors in Human Retinal and Choroidal Vascular Endothelial Cells
Author(s) -
Stewart E.,
Wei R.,
Branch M.,
Sidney L.,
Amoaku W.
Publication year - 2015
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2015.0630
Subject(s) - tlr2 , flow cytometry , tlr3 , biology , receptor , toll like receptor , innate immune system , blot , retinal , microbiology and biotechnology , immunology , pathology , immune system , medicine , gene , biochemistry
Purpose Toll‐like receptors ( TLR s) are a family of proteins that initiate the innate immune response in reaction to invading microbes. Studies confirm the expression of TLR s in a variety of ocular tissues and cells, and it has also been suggested that selected TLR s may be associated with geographic atrophy and neovascularisation in age‐related macular degeneration, diabetic retinopathy and other vascular and inflammatory diseases of the ocular posterior segment. However, TLR expression and localisation in the retinal and choroidal vasculature has not been defined. Methods In this study the gene ( mRNA ) expression of TLR s 1‐10 was investigated using RT ‐ PCR and comparative qPCR and the protein expression and localisation of selected TLR s (3, 4, 6 and 9) were examined using western blotting, flow cytometry and immunofluorescent staining. Results PCR showed gene expression of TLR 1‐6 and 9 in human choroidal endothelial cells ( hCEC ) and TLR 2‐6, 9 and 10 in human retinal endothelial cells ( hREC ). Western blotting detected TLR 3, 4 and 9 proteins in both hCEC and hREC with higher levels in hCEC , whilst TLR 6 protein was not detectable in either cell type. Flow cytometry detected all four TLR s (3, 4, 6 and 9) on the cell surface and intracellularly, TLR 6 expression was detectable but low. The expression and localisation of TLR 3, 4 and 9 were confirmed by immunofluorescent staining and TLR functionality tested by expression of IL ‐6 ( ELISA ) in response to TLR ligands. Conclusions This study has, for the first time, identified the differential expression and localisation of TLR s in intraocular endothelial cells. This profiling will help inform our understanding of different retinal and choroidal vascular diseases, as well as the development of future treatments for intraocular vascular diseases.