Induction of Tolerance to Lipopolysaccharide and Mycobacterial Components in Chinese Hamster Ovary/CD14 Cells Is Not Affected by Overexpression of Toll-Like Receptors 2 or 4

Abstract

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-κB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-κB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-κB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-κB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.

Corticotropin-Releasing Factor and the Urocortins Induce the Expression of TLR4 in Macrophages via Activation of the Transcription Factors PU.1 and AP-1

“…Forced overexpression of TLR4 in macrophages results in increased proinflammatory cytokine secretion in response to LPS (23). Increased expression of TLR4 has also been reported in several experimental models of inflammation including intestinal inflammation (39) and respiratory syncytial virus-induced inflammation (40).…”

“…LPS transduces signals via TLR4 while forced overexpression of TLR4 results in increased cytokine secretion in response to LPS (23). Therefore, the aim of the present study was to examine the role of CRF and its related peptides UCN1 and UCN2 on the regulation of TLR4 expression by macrophages.…”

Differential effects of CpG‐DNA in Toll‐like receptor‐2/‐4/‐9 tolerance and cross‐tolerance

“…8,[10][11][12] In light of the finding that 10 cellular responses to different microbial stimuli are mediated by different 11 TLRs, studies were initiated to determine whether, in analogy to LPS tolerance, pretreatment with microbial non-LPS stimuli also induces hyporesponsiveness to subsequent restimulation. Indeed, it has been reported that stimulation with prototypical ligands for TLR-2 (+TLR-1 or -6), [13][14][15][16][17][18] TLR-4, TLR-5 19 and TLR-9 20,21 also induces this state of hyporesponsiveness towards subsequent stimulation with the same ligand. Moreover, stimuli signalling via TLR-2 and TLR-4, 14,15 as well as TLR-4 and TLR-9, 20 can substitute for each other, mediating cross-tolerance in vitro as well as in vivo.…”

“…However, additional adaptor proteins have been identified which mediate TLR-specific signal transduction. [24][25][26][27][28][29] Thus, in TLR-4 signalling, toll-interleukin 1 receptor domain containing adaptor protein (TIRAP)/ MyD88 adaptor like protein (Mal) 13 can partly substitute for MyD88. Furthermore, TLR-3 and TLR-4 make use of 14 toll-interleukin 1 receptor domain containing adaptor inducing interferon beta (TRIF)/toll-interleukin 1 receptor domain (TIR) containing adaptor molecule (TICAM)-1 and TRIF related adaptor molecule (TRAM) 15 /TICAM-2, thereby inducing interferon (IFN)-b, which in turn activates a set of IFN-inducible genes in a paracrine mode.…”

“…7 These receptors mediate activation of the innate immune system by microbial compounds. 8 To date, 13 TLRs have been identified, most of which have been studied in detail and have had specific ligands identified 6 . 8,9 Thus, TLR-2 mediates recognition of such microbial components as lipoproteins, lipopeptides 7,8 (e.g.…”