TLR4 mediates human retinal pigment epithelial endotoxin binding and cytokine expression

Abstract

Purpose: It was previously demonstrated that toll-like receptor 4 (TLR4) is involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classical role of TLR4 in HRPE endotoxin (lipopolysaccharide; LPS) binding leading to HRPE proinflammatory cytokine secretion.

Methods: Cultured HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. HRPE cells labeled with fluorescent monoclonal antibodies (mAbs) 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 (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 < 0.05) in the presence of blocking mAb to TLR4 or CD14. Upregulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14.

Conclusions: 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.