Immunohistochemical, Genetic and Functional Expression of CD14 on Human Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: To study the immunohistochemical, genetic, and functional expression of CD14 on human retinal pigment epithelial cells. Methods: Cultured HRPE cells and HRPE cells in sections of human eyes were immunohistochemically stained with CD14 monoclonal antibodies (mAb). ELISA for cell-associated and secreted sCD14 was performed on cells and conditioned media (CM) from HRPE cultures after treatment with PI-PLC, LPS, or γ-IFN. FACS analysis was used to confirm HRPE cell surface CD14. Semiquantitative PCR was performed to show HRPE CD14 gene expression. Co-labeling with fluorescent LPS and anti-CD14 mAb was used to simultaneously detect these ligands on HRPE cells. Blocking CD14 mAb was used to show functional blockage of LPS- induced IL-8 secretion as measured by ELISA. PKC, PTK, PI3 kinase, and MAPK inhibitors were used to indicate cell mediators responsible for LPS-induced HRPE IL-8 secretion. Results: Immunoreactive CD14 was present on cultured HRPE cells and on HRPE in normal human eyes in which CD14 was predominantly along the basolateral cell membrane. Specific digestion by PI-PLC reduced cell-associated HRPE CD14 which was not modulated by LPS or γ-IFN. FACS confirmed cell surface HRPE CD14. CM of HRPE cells treated with PI-PLC contained more sCD14, but sCD14 was not modulated by LPS or γ-IFN. PCR confirmed CD14 mRNA in HRPE cells. Fluorescently-labeled LPS and CD14 co-localized on live, cultured HRPE cells and are in close proximity as demonstrated by resonance energy transfer. LPS-induced IL-8 was inhibited by anti-CD14 mAb and inhibitors of PKC, PTK, PI3 kinase and p38 kinase. Conclusions:This study demonstrates that HRPE cells express functional CD14 in vitro and in situ along the basolateral cell membrane at the outer blood-retina barrier.

Keywords: retinal pigment epithelium • cytokines/chemokines • immunomodulation/immunoregulation