Differential expression of Toll-like receptor (TLR)-2 and TLR-4 on monocytes in human sepsis

Abstract

Toll-like receptors (TLRs) are a recently described family of immune receptors involved in the recognition of pathogen-associated molecular patterns (PAMPs). The central role of TLR-2 and TLR-4 in microbial responses suggests they may be implicated in the pathogenesis of human sepsis. We hypothesized that the incidence and outcome of sepsis would be influenced by the expression of TLR-2 and TLR-4 on monocytes. We have examined the expression of TLR-2 and TLR-4 mRNA and protein and their response to pro- and anti-inflammatory agents on monocytes from subjects in the intensive therapy unit (ITU) with and without Gram-negative, Gram-positive or polymicrobial sepsis. We compared these data to ITU and healthy control subjects. TLR-2 mRNA was significantly up-regulated on monocytes from subjects with both Gram-positive and Gram-negative sepsis. Similarly, we detected increased levels of TLR-2 protein on the surface of monocytes from sepsis subjects relative to ITU controls. TLR-4 mRNA was increased in Gram-positive subjects; however, there was no corresponding increase in TLR-4 protein. Although TLR-4 mRNA expression in healthy control monocytes could be modulated in vitro by culture with lipopolysaccharide or interleukin-10, this was not observed in monocytes obtained from sepsis and ITU control subjects, suggesting that septic and ITU control milieus may alter the immunoregulation of TLR-4 mRNA expression on monocytes. TLR-2 mRNA was not modulated in culture by any stimulus in any group. We suggest that expression and regulatory response of monocyte TLR-2, and to a lesser extent TLR-4 may be abnormal in human sepsis.

Fig. 1

Representative RT-PCR gel. Each lane represents GAPDH, TLR-2 and TLR-4 expression in the same RNA sample. Lane 1 = ladder; lanes 2–4 Gram-positive sepsis; lanes 5–8 Gram-negative sepsis.

Fig. 2

Representative flow cytometry histograms depicting the monocyte expression of TLR-2 and TLR-4 (shaded) overlaid on the isotype control (white).

Fig. 3

TLR-2 mRNA expression in monocytes from sepsis and control subjects. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively. Kruskall–Wallis test P < 0·005. *P < 0·05 Gram-negative sepsis versus ITU controls and healthy controls; §P < 0·05 Gram-positive sepsis versus ITU controls and healthy controls, Dunn's mct.

Fig. 4

TLR-4 mRNA expression in monocytes from sepsis and control subjects. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively. One-way anovaP < 0·005. *P < 0·05 Gram-positive sepsis versus polymicrobial sepsis and healthy controls. §P < 0·01 Gram-positive sepsis versus ITU controls, Tukey's mct.

Fig. 5

Expression of TLR-2 and TLR-4 mRNA in survivors compared to non-survivors of sepsis. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively.

Fig. 6

Effect of LPS, polymyxin B (PB), IL-10 and IFN-γ on TLR-2 mRNA expression in monocytes cultured in vitro for 2 h. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively.

Fig. 7

Effect of LPS, polymyxin B (PB), IL-10 and IFN-γ on TLR-4 mRNA monocytes cultured in vitro for 2 h. The median values are depicted by the horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively. Healthy control monocytes one-way anovaP < 0·0001. *P < 0·001 healthy control monocytes unstimulated versus LPS treatment; §P < 0·001 unstimulated versus IL-10 treatment, Tukey's mct.

Fig. 8

TLR-2 protein expression on monocytes from sepsis and control subjects detected by flow cytometry. The median values are depicted by horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles respectively. One way anova P = 0.05. *P < 0.05 Gram-positive (G+ve) sepsis versus ITU controls. §P < 0.05 Gram-negative (G–ve) sepsis versus ITU controls (Tukey's mct).

Fig. 9

TLR-4 protein expression on monocytes from sepsis and control subjects detected by flow cytometry. The median values are depicted by horizontal line and the lower and upper edges of the boxes depict the 25 and 75 percentiles, respectively, detected by flow cytometry.