TLR4 mediates human retinal pigment epithelial endotoxin binding and cytokine expression.

PURPOSE: We previously demonstrated toll-like receptor 4 (TLR4) to be involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classic role of TLR4 in HRPE endotoxin (lipopolysaccharide [LPS]) binding leading to HRPE proinflammatory cytokine secretion. METHODS: Cultured human HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). HRPE cells labeled with fluorescent monoclonal antibodies (mAb) to TLR4 and its associated adhesion molecule, CD14, were analyzed by real-time microscopy and resonance energy transfer (RET), determining associations of fluorescent LPS, TLR4, and CD14. LPS-induced HRPE secretion of interleukin-8 (IL-8) was measured with and without specific blocking mAb to TLR4 and CD14. HRPE TLR4 expression was measured after LPS exposure in the presence and absence of blocking antibodies to TLR4 and CD14. RESULTS: All three HRPE cell lines demonstrated constitutive TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. Examination of HRPE cells labeled with fluorescent mAb to TLR4, CD14, and LPS demonstrated RET among the three molecules, indicating that LPS-CD14 binding preceded LPS-TLR4 binding and the close association of CD14 and TLR4. LPS-induced IL-8 was significantly reduced (P < .05) in the presence of blocking mAb to TLR4 or CD14. Up-regulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14. CONCLUSION: HRPE TLR4 is a multifunctional molecule that participates in photoreceptor outer segment membrane recognition, oxidant production, LPS recognition, and cytokine production. These roles indicate potential involvement in retinal degenerative and inflammatory processes.

Activated monocytes induce human retinal pigment epithelial cell apoptosis through caspase-3 activation.

Dysfunction and loss of human retinal pigment epithelial (HRPE) cells is a significant component of many ocular diseases, in which mononuclear phagocyte infiltration at the HRPE-related interface is also observed. In this study, we investigated whether HRPE cell apoptosis may be induced by overlay of IFN-gamma-activated monocytes. Human monocytes primed with IFN-gamma overlaid directly onto HRPE cells elicited significant increases in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive HRPE cells (p < 0.0001) and decreases of proliferating cell nuclear antigen-positive (p < 0.0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by the caspase-3 inhibitor, Z-DEVD-fmk. However, co-incubations in which activated monocytes were prevented from direct contact with HRPE cells or in which the monocytes were separated from the HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Function-blocking anti-CD18 and anti-intercellular adhesion molecule-1 (ICAM-1) antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells by 48% (p = 0.0051) and 38% (p = 0.046), respectively. Anti-CD18 and anti-ICAM-1 antibodies significantly inhibited caspase-3 activity by 56% (p < 0.0001) and 45% (p < 0.0001), respectively. However, antibodies to vascular cell adhesion molecule-1, TNF-alpha, IL-1beta, or TNF-related apoptosis-inducing ligand did not inhibit apoptosis or caspase-3 activation. Direct overlay of monocytes also induced reactive oxygen metabolites (ROM) within HRPE cells. The intracellular HRPE cell ROM production was inhibited by the anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase, presumably due to its failure to penetrate into HRPE cells. Accordingly, neither superoxide dismutase nor N(G)-monomethyl-L-arginine had significant effects on HRPE cell apoptosis or caspase-3 activation. Our results suggest that activated monocytes may induce ROM in HRPE cells through cell-to-cell contact, in part via CD18 and ICAM-1, and promote HRPE cell apoptosis. These mechanisms may compromise HRPE cell function and survival in a variety of retinal diseases.

RPE CD14 immunohistochemical, genetic, and functional expression.

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 gamma-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 gamma-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 (<7A) 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.

Human RPE cell apoptosis induced by activated monocytes is mediated by caspase-3 activation.

PURPOSE: To determine the effects of activated monocytes on the induction of human retinal pigment epithelial (HRPE) cell reactive oxygen metabolite (ROM) production and apoptosis. METHODS: HRPE cells were co-cultured with interferon-gamma (IFN-gamma)-stimulated human monocytes. HRPE apoptosis was detected by propidium iodide, proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labeling (TUNEL) staining, caspase-3 activation, and Western blot analysis. HRPE cell ROMs were imaged using the fluorescent marker dihydrotetramethylrosamine (H2TMRos). RESULTS: IFN-gamma-activated monocytes in direct contact with HRPE cells elicited significant increases in TUNEL-positive (P < .0001) and decreases in PCNA-positive (P < .0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by inhibitor Z-DEVD-fmk. Co-incubations, in which monocytes were either prevented from direct contact with HRPE cells or separated from HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Anti-CD18 and anti-ICAM-1 antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells, by 48% (P = .0051) and 38% (P = .046), respectively, and caspase-3 activity by 56% (P < .0001) and 45% (P < .0001), respectively. Overlay of monocytes induced HRPE cell ROM that was inhibited by anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase (SOD) or nitric oxide (NO) inhibitors. Accordingly, neither SOD nor NO inhibitors had significant effects on HRPE cell apoptosis or caspase-3 activation. CONCLUSIONS: We demonstrated that IFN-gamma-activated monocytes may induce ROM in HRPE cells through cell-to-cell contact, in part via CD18 and ICAM-1, and promote HRPE cell apoptosis via caspase-3 activation. These mechanisms may compromise HRPE cell function and survival in retinal diseases in which mononuclear phagocyte infiltration at the HRPE interface is observed.