TLR4 and MD-2 are necessary for conferring cellular responsiveness to LPS. Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS re-stimulation, known as `endotoxin tolerance'. While induction of LPS tolerance has been reported to correlate with down-regulation of cell surface expression of TLR4/MD-2, other mechanisms of LPS tolerance have been revealed that target intracellular intermediates downstream of the TLR4/MD-2 complex. In this study, we sought to examine whether endotoxin tolerance could be induced under conditions where expression of TLR4 and MD-2 proteins is not affected by LPS. Human HEK 293T cells are completely unresponsive to LPS, but acquire high LPS sensitivity following transient transfection with CD14, TLR4, and MD-2 (293T/CD14/TLR4/MD-2 cells), as judged by NF-κB activation, ERK 1/2 phosphorylation, and TNFα gene expression. Prior exposure of 293T/CD14/TLR4/MD-2 cells to LPS resulted in a significant decrease of LPS-mediated responses, yet failed to affect expression levels of TLR4 and MD-2. Thus, altered expression and/or function of intracellular mediators downstream of the TLR4/MD-2 complex play an important role in mediating LPS tolerance.
References
1.
Medzhitov R., Janeway Jr CA. Innate immune recognition: mechanisms and pathways. Immunol Rev2000; 173: 89—97.
2.
Poltorak A., He X., Smirnova I. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science1998; 282: 2085—2088.
3.
Qureshi ST, Lariviere L., Leveque G. et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med1999; 189: 615—625.
4.
Kurt-Jones EA , Popova L., Kwinn L. et al. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol2000; 1: 398—401.
5.
Kawasaki K., Akashi S., Shimazu R., Yoshida T., Miyake K., Nishijima M.Mouse toll-like receptor 4/MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol. J Biol Chem2000; 275: 2251—2254.
6.
Okamura Y., Watari M., Jerud ES et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem2001; 276: 10229—1033.
7.
Ohashi K., Burkart V., Flohe S., Kolb H.Heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol2000; 164: 558—561.
8.
Shimazu R., Akashi S., Ogata H. et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med1999; 189: 1777—1782.
9.
Burns K., Clatworthy J., Martin L. et al. Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat Cell Biol2000; 2: 346—351.
10.
Zhang G., Ghosh S.Negative regulation of Toll-like receptor-mediated signaling by Tollip. J Biol Chem2002; 277: 7059—7065.
11.
Zhang FX, Kirschning CJ, Mancinelli R. et al. Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J Biol Chem1999; 274: 7611—7614.
12.
Medzhitov R. , Preston-Hurlburt P., Kopp E. et al. MyD88 is an adapter protein in the hToll/IL-1 receptor family signaling pathways. Mol Cell1998; 2: 253—258.
13.
Wesche H., Henzel WJ, Shillinglaw W., Li S., Cao Z.MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity1997; 7: 837—847.
14.
Muzio M., Ni J., Feng P., Dixit VMIRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science1997; 278: 1612—1615.
15.
Kopp E., Medzhitov R., Carothers J. et al. ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway. Genes Dev1999 ; 13: 2059—2071.
16.
Wadleigh DJ , Reddy ST, Kopp E., Ghosh S., Herschman HRTranscriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages. J Biol Chem2000; 275: 6259—6266.
17.
McDermott EP , O'Neill LARas participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1. J Biol Chem2002; 277: 7808—7815.
18.
Horng T., Barton GM, Medzhitov R.TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol2001; 2: 835—841.
19.
Fitzgerald KA, Palsson-McDermott EM, Bowie AG et al. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature2001; 413: 78—83.
20.
Kawai T., Adachi O., Ogawa T., Takeda K., Akira S.Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity1999; 11: 115—122.
21.
Kaisho T., Takeuchi O., Kawai T., Hoshino K., Akira S.Endotoxin-induced maturation of MyD88-deficient dendritic cells. J Immunol2001; 166: 5688—5694.
22.
Cook JAMolecular basis of endotoxin tolerance. Ann NY Acad Sci1998; 851: 426—428.
23.
Ertel W., Kremer JP, Kenney J. et al. Down-regulation of pro-inflammatory cytokine release in whole blood from septic patients. Blood1995; 85: 1341—1347.
24.
Nomura F., Akashi S., Sakao Y. et al. Endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface Toll-like receptor 4 expression. J Immunol2000; 164: 3476—3479.
25.
Li L., Cousart S., Hu J., McCall CECharacterization of interleukin-1 receptor-associated kinase in normal and endotoxin-tolerant cells. J Biol Chem2000 ; 275: 2340—2345.
26.
Mizel SB, Snipes JAGram-negative flagellin-induced self-tolerance is associated with a block in IRAK release from TLR5. J Biol Chem2002 ; 277: 22414—22420.
27.
Medvedev AE , Kopydlowski KM, Vogel SNInhibition of lipopolysaccharide-induced signal transduction in endotoxintolerized mouse macrophages: dysregulation of cytokine, chemokine, and Toll-like receptor 2 and 4 gene expression. J Immunol2000; 164: 5564—5574.
28.
Medvedev AE , Henneke P., Schromm A. et al. 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. J Immunol2001; 167: 2257—2267.
29.
Medvedev AE , Lentschat A., Wahl LM, Golenbock DT, Vogel SNDysregulation of LPS-induced TLR4-MyD88 complex formation and IRAK-1 activation in endotoxin-tolerant cells. J Immunol2002; 169: 5209—5216.
30.
McIntire FC , Sievert HW, Barlow GH, Finley RA, Lee AYChemical, physical, and biological properties of a lipopolysaccharide from Escherichia coli K235. Biochemistry1967; 6: 2363—2372.
Yang H., Young DW, Gusovsky F., Chow JCCellular events mediated by lipopolysaccharide-stimulated Toll-like receptor 4. MD-2 is required for activation of mitogen-activated protein kinases and Elk-1. JBiol Chem2000; 275: 20861—20866.
33.
Lehner MD, Morath S., Michelsen KS, Schumann RR, Hartung T.Induction of cross-tolerance by lipopolysaccharide and highly purified lipoteichoic acid via different Toll-like receptors independent of paracrine mediators . J Immunol2001; 166: 5161—5167.
34.
Schade FU, Flash R., Flohe S. et al. Endotoxin tolerance. In: Brade H, Opal SM, Vogel SN, Morrison DC. (eds) Endotoxin in Health and Disease. New York: Marcel Dekker, 1999; 50.
