Toll-like receptor 4 (TLR4) of retinal pigment epithelial cells participates in transmembrane signaling in response to photoreceptor outer segments

Abstract

Retinal pigment epithelial (RPE) cells mediate the recognition and clearance of effete photoreceptor outer segments (POS), a process central to the maintenance of normal vision. Given the emerging importance of Toll-like receptors (TLRs) in transmembrane signaling in response to invading pathogens as well as endogenous substances, we hypothesized that TLRs are associated with RPE cell management of POS. TLR4 clusters on human RPE cells in response to human, but not bovine, POS. However, TLR4 clustering could be inhibited by saturating concentrations of an inhibitory anti-TLR4 mAb. Furthermore, human POS binding to human RPE cells elicited transmembrane metabolic and calcium signals within RPE cells, which could be blocked by saturating doses of an inhibitory anti-TLR4 mAb. However, the heterologous combination of bovine POS and human RPE did not trigger these signals. The pattern recognition receptor CD36 collected at the POS-RPE cell interface for both homologous and heterologous samples, but human TLR4 only collected at the human POS-human RPE cell interface. Kinetic experiments of human POS binding to human RPE cells revealed that CD36 arrives at the POS-RPE interface followed by TLR4 accumulation within 2 min. Metabolic and calcium signals immediately follow. Similarly, the production of reactive oxygen metabolites (ROMs) was observed for the homologous human system, but not the heterologous bovine POS-human RPE cell system. As (a) the bovine POS/human RPE combination did not elicit TLR4 accumulation, RPE signaling, or ROM release, (b) TLR4 arrives at the POS-RPE cell interface just before signaling, (c) TLR4 blockade with an inhibitory anti-TLR4 mAb inhibited TLR4 clustering, signaling, and ROM release in the human POS-human RPE system, and (d) TLR4 demonstrates similar clustering and signaling responses to POS in confluent RPE monolayers, we suggest that TLR4 of RPE cells participates in transmembrane signaling events that contribute to the management of human POS.

Figure 1.

Expression of TLR4 by human RPE cells. (A) RT-PRC demonstrates the presence of TLR4 message. (B) Western blots demonstrate the presence of TLR4 antigen in RPE cells.

Figure 2.

TLR4 clusters at the sites of POS binding. Representative DIC (A and E), fluorescence (B, C and F, G), and RET (D and H) micrographs are shown. RPE cells were trace labeled with TRITC-conjugated anti-TLR4 (clone HTA1216) at 80 ng/ml and then incubated for 2 h at 37°C. In panels E–H, cells were trace labeled using anti-TLR4 clone HTA1216 and then incubated with anti-TLR4 clone HTA125 at 30 μg/ml for 2 h at 37°C to block TLR4. RPE cells were then incubated with FITC-conjugated human POS, which were found to randomly label the surface of RPE cells (B and F). In the absence of anti-TLR4 clone HTA125, TLR4 clustered at sites of POS binding (C), and the anti-TLR4 clone HTA1216 mAb was found in close proximity with the POS (D). However, incubation with a saturating dose of the anti-TLR4 clone HTA125 blocked clustering of the anti-TLR4 clone HTA1216 (G) and RET with POS (H) (×375) (n = 3).

Figure 3.

TLR4 accumulates at sites of human but not bovine POS on human RPE cells. Representative DIC (column 1), fluorescence (columns 2 and 3), and RET (column 4) micrographs are shown. RPE cells were labeled with TRITC-conjugated anti-TLR4 (clone HTA1216). Human RPE cells were incubated with FITC-conjugated human POS (A–D), no POS (E–H), or FITC-conjugated bovine POS (I–L). In the absence of POS, TLR4 was uniformly distributed on RPE cells (G). Human POS bound to human RPE cells (B), which was accompanied by the coclustering of TLR4 (C). When RET was examined, RET was observed between the surface of the POS and TLR4 (D). When fluorescent bovine POS were incubated with human RPE cells, no coclustering with TLR4 could be detected. Thus, in contrast to the results found in Fig. 4 for CD36, only human POS promote the coclustering of TLR4 (n = 9) (×736).

Figure 4.

Human and bovine POS promote CD36 clustering on human RPE cells. Representative DIC (column 1), fluorescence (columns 2 and 3), and RET emission (column 4) micrographs are shown. RPE cells were labeled with TRITC-conjugated anti-CD36. Samples were then incubated in the absence of POS (A–D), FITC-conjugated human POS (E–H), or FITC-conjugated bovine POS (I–L). In the absence of POS, CD36 was distributed about the perimeter of RPE cells (C). Human POS bound to human RPE cells (F), which was accompanied by the coclustering of CD36 (G). When RET was evaluated, RET was observed between the surface of the POS and CD36 (H). When fluorescent bovine POS were incubated with human RPE cells, a similar set of observations were made (I–L). Thus, both human and bovine POS promote the coclustering of CD36 in their vicinity (n = 8) (×736).

Figure 5.

Human POS promote coclustering of CD36 and TLR4 on human RPE cells. Representative DIC (column 1), fluorescence (columns 2 and 3), and RET (column 4) micrographs are shown. RPE cells were labeled with TRITC-conjugated anti-CD36 and FITC-conjugated anti-TLR4. Samples were then incubated with human or bovine POS. CD36 and TLR4 were distributed about the perimeter of RPE cells (B and C). In the presence of human POS, CD36 (F) and TLR4 (G) coclustered on human RPE cells. RET microscopy demonstrated low levels of energy transfer on untreated cells (D). However, bright areas of RET could be observed on RPE cells in the presence of human POS (H). In contrast, bovine POS did not induce coclustering of CD36 and TLR4 or RET between these labels (J–L). Thus, human POS promote the coclustering of CD36 and TLR4 (n = 6) (×736).

Figure 6.

Quantitative kinetic analysis of receptor complex assembly and signaling at sites of POS binding. Using a PMT detector, photon count rates in the vicinity of bound POS were measured. Intensity is plotted at the ordinate, whereas time is given at the abscissa. The region surrounding the POS was selected by an iris in a back focal plane of the microscope. (A) Samples were labeled with both TRITC-conjugated anti-CD36 and FITC-conjugated anti-TLR4. The kinetics of CD36 and TLR4 accumulation at the site of POS binding is shown. CD36 arrives at the site of POS binding before TLR4. (B) Correspondence between the times of anti-CD36-to-anti-TLR4 RET and the exhibition of NAD(P)H oscillations during POS binding. (C) Kinetics of RET acquisition between anti-CD36 and anti-TLR4 at a site of POS binding. (D) Cells were labeled with anti-CD36 and anti-TLR4 and with the calcium indicator indo-1. Correspondence between the times of anti-CD36-to-anti-TLR4 RET and the initiation of calcium spikes in RPE cells (n > 4).

Figure 7.

Representative NAD(P)H oscillations and oxidant release profiles of RPE cells. Human RPE cells were incubated with bovine (a–d) or human (e–p) POS. Cells were monitored for NAD(P)H concentration (a, e, i, and m), intracellular calcium levels (b, f, j, and n), oxidant release (c, g, k, and o), and oxidant release in the presence of superoxide dismutase (SOD) (d, h, l, and p). Although bovine POS did not affect NAD(P)H and calcium signals of human RPE cells (a and b), these signals were apparent when cells were incubated with human POS (e and f). The release of oxidants was detected with H₂-TMRose as previously described (Kindzelskii et al., 1998). In parallel with their effects on signaling, bovine POS did not induce oxidant release (c), whereas human POS promoted oxidant release (g). This oxidant release could be inhibited by SOD (h). The ability of POS to stimulate metabolic oscillations, calcium signaling, and ROM release was not affected by prior labeling with anti-TLR4 (clone HTA1216; 80 ng/ml) (i–l). However, labeling with anti-TLR4 clone HTA1216 followed with treatment with anti-TLR4 clone HTA125 at 30 μg/ml blocked metabolic oscillations, calcium signaling, and ROM release (m–o) (n = 5). Bar, 30 s.

Figure 8.

Representative NAD(P)H oscillations and calcium spikes of RPE cells. The intensity is plotted at the ordinate, whereas the time in seconds is given at the abscissa. Experiments were performed as described in the preceding figure. Human RPE cells were incubated with human POS followed by analyses of NAD(P)H oscillations (A and C) or calcium spikes (B and D). Single cells (A and B) and cell layers (C and D) were evaluated. In the case of cell layers, more than one cell was illuminated, thereby leading to the higher amplitudes of the oscillations. Nonetheless, similar behaviors were noted for single cells and monolayers.