Toll-like receptor (TLR) expression and TLR-mediated cytokine/chemokine production by human uterine epithelial cells

Abstract

The objective of this study was to examine the expression of toll-like receptors (TLRs) by the uterine epithelial cell line ECC-1 and to determine if stimulation of the expressed TLRs induces changes in cytokine and/or chemokine secretion. The expression of TLR1 to TLR9 by ECC-1 cells was demonstrated by reverse transcription polymerase chain reaction, with only TLR10 not being expressed. Stimulation of ECC-1 cells using agonists to TLR2, TLR4 and TLR5 induced the expression of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1), as well as the pro-inflammatory cytokine IL-6, and occurred in a dose-dependent manner. In response to zymosan and flagellin, pathogen-associated molecular patterns (PAMP) that are recognized by TLR2 and TLR5 respectively, ECC-1 cells secreted significantly more IL-8, MCP-1 and IL-6 than in response to other TLR agonists. In contrast, agonists to TLR3, TLR7, and TLR9 had no effect on the secretion of the 13 cytokines or chemokines analysed. These results indicate that uterine epithelial cells are important sentinels of the innate immune system. Further it indicates that all but one of the known TLRs are expressed by ECC-1 cells and that stimulation through specific TLRs mediates changes in the expression of key chemokines and pro-inflammatory cytokines that aid in the defence of the uterus against potential pathogens.

Figure 1

Toll-like receptor expression in uterine ECC-1 epithelial cells by RT-PCR. Lanes 1–10 correspond to TLR1–TLR10, respectively. Total RNA was isolated from ECC-1 cells and examined by RT-PCR for TLR mRNA expression.

Figure 2

IL-8 production by uterine ECC-1 epithelial cells treated with ligands to several TLR. Cultured media were collected following 24-hr PAMP stimulation and analysed for IL-8 protein expression by ELISA. Cells treated with ultra pure lipopolysaccharide from Salmonella minnesota were used at a final concentration of 1 μg/ml; pam₃cys-ser-(lys)₄, 1 μg/ml; Poly(I:C), 25 μg/ml; flagellin from Escherichia coli, 100 ng/ml; zymosan from Saccharomyces cerevisiae, 10 μg/ml; peptidoglycan from Staphylococcus aureus, 10 μg/ml; CpG oligonucleotide, 1 μm; loxoribine, 100 μm. Apical and basolateral IL-8 protein expression are shown. **Significantly different (P < 0·01) from control. Number of experiments n = 4).

Figure 3

Dose-dependent apical production of IL-8 by uterine ECC-1 epithelial cells grown to maximal transepithelial resistance (TER). (a) TER following 24 hr zymosan stimulation. Cultured media were collected following 24 hr (b) zymosan or (c) flagellin stimulation and the apical media were analysed for IL-8 protein expression by ELISA. Apical and basolateral IL-8 protein expression are shown. Significantly different from control: *P < 0·05; **P < 0·01; (n = 3).

Figure 4

MCP-1 production by uterine ECC-1 epithelial cells treated with ligands to several TLR. Cultured media were collected following 24 hr PAMP stimulation and analysed for the presence of MCP-1 protein expression by ELISA. Apical and basolateral protein expression are shown. Significantly different from control: *P < 0·05; **P < 0·01; (n = 4).

Figure 5

Dose-dependent production of MCP-1 by uterine ECC-1 epithelial cells. Cultured media were collected following 24 hr (a) zymosan or (b) flagellin stimulation and analysed for MCP-1 protein expression by ELISA. Apical and basolateral MCP-1 protein expression are shown. Significantly different from control: *P < 0·05; **P < 0·01; (n = 3).

Figure 6

Apical IL-6 production by uterine ECC-1 epithelial cells treated with ligands to several TLR. Cultured media were collected following 24 hr PAMP stimulation and analysed for IL-6 protein expression by ELISA. Apical IL-6 protein expression is shown. Significantly different from control: *P < 0·05; **P < 0·01; (n = 4).

Figure 7

Dose-dependent apical production of IL-6 by uterine ECC-1 epithelial cells. Cultured media were collected following 24 hr (a) zymosan or (b) flagellin stimulation and analysed for IL-6 protein expression by ELISA. Apical IL-6 protein expression is shown. Significantly different from control: **P < 0·01; (n = 3).