Lipopolysaccharide (LPS) signals through Toll-like receptors (TLRs) in the course of sepsis, resulting in the release of inflammatory factors. In cell lines and murine models, parts of the signaling pathways involved have been elucidated with MyD88, Mal/TIRAP and IKK2 playing an important role in the induction of NF-κB. By focusing on primary human cells, we have shown that there are fundamental signaling differences between human and murine macrophages and between cells of myeloid and non-myeloid origins. In primary human cells, there are no available knockouts so we employed the use of dominant negatives to investigate the signaling cascades. We show that in primary human macrophages MyD88, Mal/TIRAP and IKK2-independent alternative pathways activate NF-κB and induce the expression of inflammatory cytokines, whereas in non-myeloid synovial fibroblasts MyD88 and/or Mal/TIRAP are essential adaptors for LPS signaling.
Cohen J.The immunopathogenesis of sepsis. Nature2002 ; 420: 885—891.
2.
Waage A.Production and clearance of tumor necrosis factor in rats exposed to endotoxin and dexamethasone. Clin Immunol Immunopathol1987; 45: 348—355.
3.
Waage A., Brandtzaeg P., Halstensen A., Kierulf P., Espevik T.The complex pattern of cytokines in serum from patients with meningococcal septic shock. Association between interleukin 6, interleukin 1, and fatal outcome. Production and clearance of tumor necrosis factor in rats exposed to endotoxin and dexamethasone. J Exp Med1989; 169: 333—338.
4.
Van Zee KJ, DeForge LE, Fischer E. et al. IL-8 in septic shock, endotoxemia, and after IL-1 administration . J Immunol1991; 146: 3478—3482.
5.
Padkin A., Goldfrad C., Brady AR, Young D., Black N., Rowan K.Epidemiology of severe sepsis occurring in the first 24 hrs in intensive care units in England, Wales, and Northern Ireland. Crit Care Med2003; 31: 2332—2338.
6.
Wright SDCD14 and innate recognition of bacteria. J Immunol1995; 155: 6—8.
7.
Juan TS, Hailman E., Kelley MJ et al. Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64. J Biol Chem1995; 270: 5219—5224.
8.
Haziot A., Ferrero E., Kontgen F. et al. Resistance to endotoxin shock and reduced dissemination of Gram-negative bacteria in CD14-deficient mice. Immunity1996; 4: 407—414.
9.
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.
10.
Perera PY, Mayadas TN, Takeuchi O. et al. CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression . J Immunol2001; 166: 574—581.
Alexopoulou L., Holt AC, Medzhitov R., Flavell RARecognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature2001; 413: 732—738.
13.
Hemmi H., Takeuchi O., Kawai T. et al. A Toll-like receptor recognizes bacterial DNA. Nature2000; 408: 740—745.
14.
Hayashi F., Smith KD, Ozinsky A. et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature2001; 410: 1099—1103.
15.
Takeda K., Kaisho T., Akira S.Toll-like receptors. Annu Rev Immunol2003 ; 21: 335—376.
16.
Heguy A., Baldari C., Bush K. et al. Internalization and nuclear localization of interleukin 1 are not sufficient for function. Cell Growth Differ1991; 2: 311—315.
17.
Bartfai T., Behrens MM, Gaidarova S., Pemberton J., Shivanyuk A., Rebek Jr J.A low molecular weight mimic of the Toll/IL-1 receptor/resistance domain inhibits IL-1 receptor-mediated responses. Proc Natl Acad Sci USA2003; 100: 7971—7976.
18.
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.
19.
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.
20.
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.
21.
Horng T., Barton GM, Medzhitov R.TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol2001; 2: 835—841.
22.
Yamamoto M., Sato S., Mori K. et al. A novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol2002; 169: 6668—6672.
23.
Oshiumi H., Matsumoto M., Funami K., Akazawa T., Seya T.TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat Immunol2003 ; 4: 161—167.
24.
Fitzgerald KA , Rowe DC, Barnes BJ et al. LPS-TLR4 signaling to IRF-3/7 and NF-{kappa}B involves the Toll adapters TRAM and TRIF. J Exp Med2003; 198: 1043—1055.
25.
Bin LH, Xu LG, Shu HBTIRP, a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling. J Biol Chem2003; 278: 24526—24532.
26.
Oshiumi H., Sasai M., Shida K., Fujita T., Matsumoto M., Seya T.TICACM-2: a bridging adapter recruiting to Toll-like receptor 4 TICAM-1 that induces interferon-beta. J Biol Chem2003
27.
Yamamoto M. , Sato S., Hemmi H. et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science2003; 301: 640—643.
28.
Mink M., Fogelgren B., Olszewski K., Maroy P., Csiszar K.A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans. Genomics2001; 74: 234—244.
29.
Toshchakov V., Jones BW, Perera PY et al. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol2002; 3: 392—398.
30.
Kawai T., Adachi O., Ogawa T., Takeda K., Akira S.Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity1999; 11: 115—122.
31.
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.
32.
Ninomiya-Tsuji J., Kishimoto K., Hiyama A., Inoue J., Cao Z., Matsumoto K.The kinase TAK1 can activate the NIK-I kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature1999; 398: 252—256.
33.
Yamamoto M. , Sato S., Hemmi H. et al. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol2003; 4.
34.
Fitzgerald KA, McWhirter SM, Faia KL et al. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol2003; 4: 491—496.
35.
Sharma S., tenOever BR, Grandvaux N., Zhou GP, Lin R., Hiscott J.Triggering the interferon antiviral response through an IKK-related pathway . Science2003; 300: 1148—1151.
36.
Butler DM, Feldmann M., Di Padova F., Brennan FMp55 and p75 tumor necrosis factor receptors are expressed and mediate common functions in synovial fibroblasts and other fibroblasts. Eur Cytokine Netw1994; 5: 441—448.
37.
Foxwell B., Browne K., Bondeson J. et al. Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent. Proc Natl Acad Sci USA1998; 95: 8211—8215.
38.
Aoki K., Barker C., Danthinne X., Imperiale MJ, Nabel GJEfficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro. Mol Med1999; 5: 224—231.
39.
Burns K., Martinon F., Esslinger C. et al. MyD88, an adapter protein involved in interleukin-1 signaling . J Biol Chem1998; 273: 12203—12209.
40.
Oitzinger W. , Hofer-Warbinek R., Schmid JA, Koshelnick Y., Binder BR, de Martin R.Adenovirus-mediated expression of a mutant IkappaB kinase 2 inhibits the response of endothelial cells to inflammatory stimuli. Blood2001; 97: 1611—1617.
41.
Smith C., Andreakos E., Crawley JB, Brennan FM, Feldmann M., Foxwell BMNF-kappaB-inducing kinase is dispensable for activation of NF-kappaB in inflammatory settings but essential for lymphotoxin beta receptor activation of NF-kappaB in primary human fibroblasts. J Immunol2001 ; 167: 5895—5903.
42.
Ciesielski CJ, Andreakos E., Foxwell BM, Feldmann M.TNF-induced macrophage chemokine secretion is more dependent on NF-κB expression than lipopolysaccharide-induced macrophage chemokine secretion. Eur J Immunol2002; 32: 2037—2045.
43.
Graham FL, Prevec L.Methods for construction of adenovirus vectors. Mol Biotechnol1995; 3: 207—220.
44.
Feldmann M. , Andreakos E., Smith C. et al. Is NF-kappaB a useful therapeutic target in rheumatoid arthritis? Ann Rheum Dis2002 ; 61 (Suppl 2): ii13—ii18.
45.
Mosmann T.Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods1983; 65: 55—63.
46.
Whiteside ST , Visvanathan KV, Goodbourn S.Identification of novel factors that bind to the PRD I region of the human beta-interferon promoter . Nucleic Acids Res1992; 20: 1531—1538.
47.
Clarke CJ, Taylor-Fishwick DA, Hales A. et al. Interleukin-4 inhibits kappa light chain expression and NF kappa B activation but not I kappa B alpha degradation in 70Z/3 murine pre-B cells . Eur J Immunol1995; 25: 2961—2966.
48.
Andreakos E. , Smith C., Monaco C., Brennan FM, Foxwell BM, Feldmann M.Ikappa B kinase 2 but not NF-kappa B-inducing kinase is essential for effective DC antigen presentation in the allogeneic mixed lymphocyte reaction. Blood2003; 101: 983—991.
49.
Hirschfeld M., Ma Y., Weis JH, Vogel SN, Weis JJRepurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J Immunol2000; 165: 618—622.
50.
Guillot L., Medjane S., Le-Barillec K. et al. Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. J Biol Chem2004; 279: 2712—2718.
51.
Andreakos E. , Smith C., Monaco C., Brennan FM, Foxwell BM, Feldmann M.IκB kinase 2 but not NF-κB-inducing kinase is essential for effective DC antigen presentation in the allogeneic mixed lymphocyte reaction. Blood2003; 101: 983—991.
52.
Andreakos E. , Smith C., Kiriakidis S. et al. Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy. Arthritis Rheum2003; 48: 1901—1912.
53.
Andreakos E. , Sacre SM, Smith C. et al. Distinct pathways of LPSinduced NF-{kappa}B activation and cytokine production in human myeloid and nonmyeloid cells defined by selective utilization of MyD88 and Mal/TIRAP . Blood2004; 103: 2229—2237.
54.
Takeda K., Kaisho T., Akira S.Toll-like receptors. Annu Rev Immunol2003 ; 21: 335—376.
55.
Horwood NJ, Mahon T., McDaid JP et al. Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production. J Exp Med2003; 197: 1603—1611.
56.
Jefferies CA , Doyle S., Brunner C. et al. Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4. J Biol Chem2003; 278: 26258—26264.
