Purchase this article with an account.
H.R. Petty, V.M. Elner, S.G. Elner, B.A. Hughes, A.L. Kindzelskii; Toll-Like Receptor 4 (TLR4) on RPE Cells Participates in Transmembrane Signaling in Response to Photoreceptor Outer Segments . Invest. Ophthalmol. Vis. Sci. 2003;44(13):370.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the role of TLR4 in HRPE transmembrane signaling in response to photoreceptor outer segment (POS) binding. Methods: Human and bovine (control) POS were isolated from retinal homogenates by centrifugation and labeled with FITC. TLR4 and CD36 mAbs were conjugated with FITC or TRITC. Live primary to second passaged HRPE cells, seeded onto glass coverslips, were exposed to fluorescently labeled POS, TLR4 mAb, and/or CD36 mAb. Cells were observed by fluorescence microscopy and resonance energy transfer (RET) was determined between human POS and TLR4 or CD36. NADPH autofluorescence and calcium ion oscillations due to human POS binding were detected using a cooled photomultiplier tube (PMT) attached to the fluorescence microscope. Western blot analysis and RT-PCR were used to detect HRPE TLR4 protein and gene expression, respectively. Results: During POS-to-HRPE binding, homologous human POS/HRPE combinations stimulated transmembrane metabolic and calcium signaling within RPE cells, but the heterologous combination, bovine POS/HRPE, did not. CD36 collected at the POS-HRPE interface in response to bovine or human POS binding, but HRPE TLR4 only collected at the human POS/HRPE and not bovine POS/HRPE interfaces. Kinetic experiments of human POS/HRPE combinations revealed the following sequence: CD36 arrives at the human POS/HRPE interface first, followed by TLR4 accumulation within 2 minutes, which is then immediately followed by changes in NADPH metabolic and calcium signaling. Western blot and RT-PCR analysis of HRPE cells confirmed TLR4 expression. Conclusions: Our results suggest that TLR4 at the HRPE cell surface participates in the transmembrane signaling events associated with human POS recognition.
This PDF is available to Subscribers Only