Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Abstract

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.

Fig. 1

Effect of Staphylococcus aureus enterotoxin B (SEB) on CD14 and CD16 expression on human monocytes. Whole blood was stimulated with 100 ng/ml of SEB for (a) zero, (b) two and (c) 20 hours. Cells were stained with CD14 PerCP and CD16 (APC) for flow cytometry. Dot plots are gated on monocytes based on their light-scatter properties. Bold figures represent percentages of CD14⁺ CD16^– cells and CD14^dimCD16⁺ cells. Data shown are representative of five individual experiments.

Fig. 2

Change in the percentage of the CD14^dimCD16⁺ monocyte subpopulation with culture and stimulation. Whole blood cultures were stimulated with 100 ng/ml lipopolysaccharide (LPS), peptidoglycan (PGN) or Staphylococcus aureus enterotoxin B (SEB) for 0, 2 and 20 h. The CD14^dimCD16⁺ monocyte subpopulation was measured by flow cytometry. Experiments represent the mean and standard error for five individual experiments. Statistical significance was compared with the unstimulated cells (*P < 0·05).

Fig. 3

Stimulation of whole blood cultures with lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) induces cytokine. Whole blood cultures were stimulated with 100 ng/ml of LPS, peptidoglycan (PGN) or SEB for 0, 2 and 20 h. Tumour necrosis factor (TNF)-α and interleukin (IL)-6 enzyme-linked immunosorbent assays (ELISAs) were performed on culture supernatants. The results represent the mean and standard error for five individual experiments. Statistical significance was compared with the unstimulated cells (*P < 0·05).

Fig. 4

Correlation between tumour necrosis factor (TNF)-α and the percentage of CD14^dimCD16⁺ monocytes. Whole blood cultures were stimulated for 20 h with or without 100 ng/ml of lipopolysaccharide (LPS), peptidoglycan (PGN) or Staphylococcus aureus enterotoxin B (SEB). Each dot represents data from an individual experiment. The percentage of CD14^dimCD16⁺ monocytes were determined by flow cytometry and TNF-α was determined by enzyme-linked immunosorbent assay (ELISA) on the supernatants from the same cells. Correlation coefficient r = 0·72.

Fig. 5

Toll-like receptor (TLR)2 and TLR4 on monocyte subpopulations in whole blood culture. Whole blood cultures were stimulated for 0, 2 and 20 h with 100 ng/ml lipopolysaccharide (LPS), peptidoglycan (PGN) or Staphylococcus aureus enterotoxin B (SEB). Using four-colour flow cytometry, TLR2 and TLR4 expression was measured on the total CD14⁺ population and the two subpopulations. Relative fluorescence was calculated by subtracting the mean fluorescence of the unstimulated cells from the mean fluorescence of the stimulated cells. Data represent the mean and standard error of five separate experiments. Statistical significance was compared with the unstimulated cells (*, P < 0·05)

Fig. 6

Stimulation of whole blood cultures with 20 ng/ml of recombinant tumour necrosis factor (TNF)-α and 100 ng/ml of Staphylococcus aureus enterotoxin B (SEB) for 20 h. The results represent the mean and standard error for five individual experiments. The statistical significance is in comparison with unstimulated cells (*P < 0·05).

Fig. 7

Differential expression of Toll-like receptors (TLRs) on monocyte subpopulations of sepsis patients. Expression of (a) TLR2 and (b) TLR4 was measured on individual patient monocyte subpopulations by four-colour flow cytometry. Relative fluorescence was calculated by subtracting the mean fluorescence of the isotype matched control from the mean fluorescence of the specific TLR stain. Lines represent the mean of 10 sepsis patients (*P < 0·05).