Innate immunity in the retina: Toll-like receptor (TLR) signaling in human retinal pigment epithelial cells

Abstract

Toll-like receptors (TLRs) are crucial components of innate immunity that participate in host defense against microbial pathogens. We evaluated the expression and function of TLRs in human retinal pigment epithelial (RPE) cells. Real time PCR analysis revealed gene expression for TLRs 1-7, 9, and 10 in RPE cells. TLRs 1 and 3 were the most highly expressed TLRs. Protein expression for TLRs 2, 3, and 4 was observed on RPE cells and this expression was augmented by treatment with poly I:C or interferon-gamma (IFN-gamma). TLR 3 is the receptor for dsRNA, an intermediate of virus replication. Because RPE cells express TLR 3 and are frequently the site of virus replication within the retina, we evaluated TLR 3 signaling. RPE cells treated with poly I:C produced IFN-beta but not IFN-alpha, and this was inhibited by the treatment of RPE cells with anti-TLR 3 antibody. Human recombinant IFN-beta was shown to be biologically active on RPE cells by inhibiting viral replication. Poly I:C treatment of RPE resulted in an increase in the production of IL-6, IL-8, MCP-1, and sICAM-1. The presence of TLRs on RPE cells and the resultant TLR signaling in RPE cells suggest that these molecules may play an important role in innate and adaptive immune responses within the retina.

Fig. 1

Modulation of TLR 2, TLR 3, and TLR 4 expression in human RPE cells by poly I:C. RPE cultures were grown to confluent monolayers in 100-mm dishes and incubated without (untreated) or with poly I:C (100 μg/ml) for 24 h. The procedure for total RNA preparation and RT-PCR is described in the Materials and methods section. RNA (1 μg) was used for each RT-PCR reaction. The sequences of the PCR primers and the expected sizes of PCR products are given in the Materials and methods section. All PCR reactions were subjected to 30 cycles. The results shown are from one representative experiment.

Fig. 2

Immunofluorescent staining for TLR 3 and TLR 4 in RPE cells. Staining reactivity for TLR 3 is shown in untreated cells (A) and in cells treated for 24 h with poly I:C (100 μg/ml; B). Staining reactivity for TLR 4 is shown in untreated cells (C) and in cells treated for 24 h with IFN-γ (100 U/ml; D).

Fig. 3

Secretion of IFN-β by human RPE cells (A) and antiviral activity of IFN-β (B). (A) Human RPE cell monolayers were grown to confluence. Cultures were washed with SFM twice and incubated in the presence of various concentrations of poly I:C or poly dI:dC for 24 h. The culture supernatants were collected and analyzed for IFN-α and -β by EIA. Results from three experiments conducted in duplicate are presented as the means±S.E. (B) Human RPE cultures were grown to confluence and were infected with Vesicular Stomatitis Virus (VSV) as described in the Materials and methods section. After 24 h, cultures were fixed and stained with Giemsa, and the plaques were counted. The data are presented as the means±S.E. of triplicate cultures obtained from a representative experiment.

Fig. 4

The effect of anti-TLR 3 antibody on poly I:C-induced IFN-β production in RPE cells. RPE cell cultures grown to confluence were washed with SFM and incubated with media or anti-TLR 3 antibody (10 ug/ml) for 1 h. Then, poly I:C was added to the appropriate wells to give a final concentration of 2 or 20 ug/ml. After 24 h of incubation, supernatant fluids were collected and the concentration of IFN-β was determined by EIA. Results presented were obtained from one representative experiment with quadruplicate samples.

Fig. 5

The effects of poly I:C treatment on the production of cytokines by human RPE cells. RPE cell cultures grown to confluence in 24-well plates were washed with SFM and incubated without (control) or with SFM containing various concentrations of poly I:C or poly dI:dC. After 24 h, the culture supernatants were collected and the concentrations of IL-6, IL-8, MCP-1, and sICAM-1 were determined by EIA. Results presented for IL-6 (A), IL-8 (B), MCP-1 (C), and ICAM-1 (D) were obtained from duplicate samples of two representative experiments.