RPE CD14 immunohistochemical, genetic, and functional expression

Abstract

CD14 is the primary receptor for lipopolysaccharide (LPS)that plays important roles in host defense and subserves other host-related biological functions. We previously identified CD14 on cultured human retinal pigment epithelial (HRPE) cells using immunocytochemical techniques. In this study, we investigated immunoreactive HRPE CD14 expression by immunohistochemically staining HRPE cells and HRPE cells in sections of human eyes with anti-CD14 monoclonal antibodies (mAb). Constitutive HRPE gene and protein expression were confirmed by semiquantitative PCR and western blotting. ELISA for cell-associated and secreted (s) HRPE CD14 revealed that specific digestion by phosphoinositol-specific phospholipase C (PI-PLC) significantly reduced ( P<0·01) cell-associated HRPE CD14 which was not modulated by LPS or γ-IFN. ELISA of the conditioned media (CM) of HRPE cells treated with PI-PLC contained significantly more ( P<0·001) sCD14, but sCD14 was not modulated by LPS or γ-IFN. FACS analysis confirmed HRPE cell surface CD14. To show functional CD14, fluorescently-labelled LPS and CD14 were demonstrated to show significant co-localization on live, cultured HRPE cells in close proximity (<7 Å) as demonstrated by resonance energy transfer of the fluorescent ligands ( P<0·0001). Significant inhibition ( P<0·001) of LPS-induced IL-8 secretion, as measured by ELISA, occurred in the presence of function blocking anti-CD14 mAb. Significant inhibition of LPS-induced HRPE IL-8 secretion by PKC, PTK, PI3 kinase, and p38 kinase inhibitors indicated cell mediators responsible for LPS-induced HRPE chemokine secretion. This study demonstrates that HRPE cells express functional CD14 in vitro and in situ along at the outer blood-retina barrier.