Toll-like receptors and TLR-mediated signaling: more questions than answers

Zhixing K. Pan

Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614

PATHOGEN-ASSOCIATED MOLECULAR PATTERNS (PAMPs), bacterial components or products, are powerful activators of the immune system. These include lipopolysaccharide (LPS) from gram-negative bacteria; peptidoglycan and lipoteichoic acid (LTA) from gram-positive bacteria; formylated peptides from both gram-negative and gram-positive bacteria; lipoarabinomannan from mycobacteria; and mannans from yeast cell wall. Recognition of pathogens is primarily mediated by a set of receptors on innate immune cells referred to as pattern recognition receptors (PRRs). Recognition of PAMPs by PRRs results in the activation of different intracellular signaling molecules, which leads to the expression of various effector molecules, such as proinflammatory cytokines, nitric oxide, and eicosanoids. These molecules mediate inflammatory responses and drive T cell development, which leads to activation of adaptive immunity (21).

Recent studies revealed that mammalian Toll-like receptors (TLRs) are key molecules for recognizing microbial PAMPs to evoke the inflammatory response (14). Several lines of evidence support the view that TLR4 is the receptor for gram-negative bacterial LPS and TLR2 is the receptor for gram-positive peptidoglycan and lipoproteins (29, 30, 33). As the receptor for LPS, TLR4 is the best-characterized member of the TLR family. LPS-binding protein (LBP) binds to LPS on the surface of gram-negative bacteria. The LPS-LBP complex is then transferred to CD14 located on the surface of monocytes/macrophages (1).

Toll is a transmembrane receptor in Drosophila that is involved in the induction of an antifungal response (17). Activation of the Toll receptor results in the stimulation of several signaling molecules that are homologous to proteins involved in NF-{kappa}B in mammalian cells (4). The cloning of a family of human receptors structurally related to Drosophila Toll revealed cytoplasmic domains with sequence homology to the intracellular portion of the IL-1 receptor (26, 27). Similarly, the signaling pathway of the TLR family is highly homologous to that of the IL-1 receptor family. Both TLR and IL-1 receptor interact with an adaptor protein, myeloid differentiation factor 88 (MyD88), in their intracellular domains (2). The death domain of MyD88 then recruits downstream IL-1 receptor-associated kinase (IRAK) to the receptor complex (2). IRAK is then autophosphorylated and dissociates from the receptor complex and recruits TNF receptor-associated factor 6 (TRAF6) that in turn activates downstream kinases (23). Several such kinases and enzymes have been found to be involved in TLR-mediated signaling pathways, including activation of NF-{kappa}B and MAPKs (6, 11). Although MyD88 plays an important role in TLR signaling pathways, recent studies have shown that LPS-induced responses do not require MyD88, and NF-{kappa}B activation is delayed in MyD88-deficient cells (12). A molecule termed Toll receptor-IL-1 receptor domain-containing adpater protein (TIRAP) or MyD88 adapter-like protein (MAL) has recently been identified (9, 10). Overexpression of TIRAP/MAL activates NF-{kappa}B, and dominant negative mutants of the protein inhibit NF-{kappa}B activation mediated by TLR (9, 10). Dominant negative mutant of TIRAP/MAL blocks LPS-induced maturation of dendritic cells from both wild-type and MyD88-deficient mice (10). Together, these data demonstrate that TIRAP/MAL is responsible for MyD88-independent TLR signaling.

In addition to NF-{kappa}B activation, TLR can also activate MAPK signaling cascades. The MAPKs transduce extracellular signals into cellular responses and play important roles in cell proliferation, apoptosis, differentiation, cell migration, and gene expression (8, 32). Mammalian cells express three types of MAPKs, including ERKs, p38 MAPKs, and c-Jun NH2-terminal kinases. Recent studies have shown that MAPKs, including ERK, c-Jun NH2-terminal kinases, and p38 can be activated by LPS. MAPKs p42 and p44, referred to as ERK1/ERK2, are activated by MAPK/ERK kinase 1 (MEK1). The activation of p38 MAPK is regulated by upstream MAPKK (MKK3, MKK6, and probably MKK4) via phosphorylation of a TGY phosphorylation site (15, 24). The p38 MAPK effects are carried out by downstream substrates, including protein kinases and transcription factors (20, 24). Recently, the p38 MAPK signaling pathway has also been demonstrated to play an important role in regulating mRNA stability (13, 19, 31). However, the role of TLRs in LTA-induced cell activation and TLR-mediated signaling events is not completely understood. It remains controversial whether LTA stimulates the immune system via TLR2 (5, 28) or TLR4 (30). Takeuchi et al. (30) have reported that LTA stimulates nitric oxide and cytokine IL-6 gene expression in macrophages from TLR2-deficient mice. In contrast, Schwandner et al. (29) suggested that LTA from a highly purified butanol-extracted procedure (22) stimulates NF-{kappa}B activation via TLR2 (25), and this function of LTA is enhanced by LBP and CD14 (28).

LTA is the major component of the cell wall of gram-positive bacteria and stimulates inflammatory responses in the lung (16, 18). Therefore, understanding the mechanisms that regulate LTA-mediated cell activation is crucial for understanding the pathogenesis of lung inflammatory disease and the interactions between lung innate immunity and inflammatory responses. The report by Lee and coworkers, one of the current articles in focus (Ref. 14a, see p. L921 in this issue), provides further evidence that LTA (from the gram-positive bacteria Staphylococcus aureus)-stimulated MAPK activation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca2+, and phosphatidylinositol 3-kinase in human tracheal smooth muscle cells. The authors demonstrated expression of both TLR2 and TLR4 receptor genes in human tracheal smooth muscle cells. Treatment of these cells with TLR2 antibody significantly blocked LTA-induced p42/p44 MAPK phosphorylation, but TLR4-blocking antibody had no effect on this response. Further demonstration of the requirement of TLR2 for LTA-induced MAPK activation was obtained by overexpressing a human mutant of TLR2. The authors also transfected a human dominant mutant of TLR2 in human tracheal smooth muscle cells and found that LTA-stimulated MAPK activity was blocked in these transfected cells. These data strongly suggest that LTA-activated MAPK is mediated through TLR2.

Lee and coworkers (14a) also examined the signaling molecules in TLR2-mediated MAPK activation stimulated with LTA. They provided evidence that tyrosine kinase, PLC, PKC, Ca2+, MEK, and phosphatidylinositol 3-kinase are important molecules in LTA-induced MAPK activation in human tracheal smooth muscle cells. Interestingly, although TLR2 by itself has no tyrosine kinase activity, it possesses several phosphotyrosine residues in the cytosolic domain. Arbibe et al. (3) have shown that heat-killed S. aureus (HKSA) stimulation of HEK-293-TLR2 and THP1 cells led to transient tyrosine phosphorylation of the TLR2 cytosolic domain. Mutation of both tyrosine residues (Tyr616, 761) on TLR2 abolished NF-{kappa}B activation in HEK-293 cells stimulated with HKSA (3). In another study, it was reported that LPS-induced NF-{kappa}B activation and cytokine gene expression utilize a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4 (7). Thus it is possible that tyrosine phosphorylation of TLRs plays an important role in the activation of TLRs. The present experiments involve the use of genistein, a tyrosine kinase inhibitor. Further experiments might provide additional information of TLR activation, such as whether overexpressing a mutation of TLR2 (Tyr616, 761) in human tracheal smooth muscle cells can abolish LTA-induced MAPK activation, or whether p85 subunit of phosphatidylinositol 3-kinase associates with tyrosine-phosphorylated motifs on TLR2 in LTA-stimulated human tracheal smooth muscle cells (3). Of particular interest, this paper contains data from the use of human primary airway cells in which the LTA-stimulated MAPK activation is mediated through a TLR2 receptor in human tracheal smooth muscle cells. Future studies are expected to address and delineate the relationship between MyD88, IRAKs, TRAF6, and TLR2, and the function of MyD88, IRAKs, and TRAF6 in LTA-induced MAPK activation in human tracheal smooth muscle cells.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Z. K. Pan, Dept. of Microbiology and Immunology, Medical College of Ohio, Toledo, OH 43614 (E-mail: zkpan{at}mco.edu).


    REFERENCES
 TOP
 REFERENCES
 

  1. Aderem A and Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 406: 782-787, 2000.[CrossRef][ISI][Medline]
  2. Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol 12: 13-19, 2000.[CrossRef][ISI][Medline]
  3. Arbibe L, Mira JP, Teusch N, Kline L, Guha M, Mackman N, Godowski PJ, Ulevitch RJ, and Knaus UG. Toll-like receptor 2-mediated NF-{kappa}B activation requires a Rac1-dependent pathway. Nat Immun 1: 533-540, 2000.[CrossRef][ISI]
  4. Belvin MP and Anderson KV. A conserved signaling pathway: the Drosophila toll-dorsal pathway. Annu Rev Cell Dev Biol 12: 393-416, 1996.[CrossRef][ISI][Medline]
  5. Brightbill HD, Libraty DH, Krutzik SR, Yang RB, Belisle JT, Bleharski JR, Maitland M, Norgard MV, Plevy SE, Smale ST, Brennan PJ, Bloom BR, Godowski PJ, and Modlin RL. Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. Science 285: 732-736, 1999.[Abstract/Free Full Text]
  6. Chen LY, Zuraw BL, Liu FT, Huang S, and Pan ZK. IL-1 receptor-associated kinase and low molecular weight GTPase RhoA signal molecules are required for bacterial lipopolysaccharide-induced cytokine gene transcription. J Immunol 169: 3934-3939, 2002.[Abstract/Free Full Text]
  7. Chen LY, Zuraw BL, Zhao M, Liu FT, Huang S, and Pan ZK. Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-{kappa}B activation in human peripheral blood monocytes. Am J Physiol Lung Cell Mol Physiol 284: L607-L613, 2003.[Abstract/Free Full Text]
  8. English J, Pearson G, Wilsbacher J, Swantek J, Karandikar M, Xu S, and Cobb MH. New insights into the control of MAP kinase pathways. Exp Cell Res 253: 255-270, 1999.[CrossRef][ISI][Medline]
  9. Fitzgerald KA, Palsson-McDermott EM, Bowie AG, Jefferies CA, Mansell AS, Brady G, Brint E, Dunne A, Gray P, Harte MT, McMurray D, Smith DE, Sims JE, Bird TA, and O'Neill LA. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 413: 78-83, 2001.[CrossRef][ISI][Medline]
  10. Horng T, Barton GM, and Medzhitov R. TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immun 2: 835-841, 2001.[CrossRef][ISI]
  11. Karin M and Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu Rev Immunol 18: 621-663, 2001.[CrossRef][ISI]
  12. Kawai T, Adachi O, Ogawa T, Takeda K, and Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11: 115-122, 1999.[ISI][Medline]
  13. Kontoyiannis D, Kotlyarov A, Carballo E, Alexopoulou L, Blackshear PJ, Gaestel M, Davis R, Flavell R, and Kollias G. Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology. EMBO J 20: 3760-3770, 2001.[Abstract/Free Full Text]
  14. Kopp EB and Medzhitov R. The Toll-receptor family and control of innate immunity. Curr Opin Immunol 11: 13-18, 1999.[CrossRef][ISI][Medline]
  15. Lee C-W, Chien C-S, and Yang C-M. Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 286: L921-L930, 2004.[Abstract/Free Full Text]
  16. Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, and Adams JL. Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology 47: 185-201, 2000.[CrossRef][ISI][Medline]
  17. Leemans JC, Vervoordeldonk MJ, Florquin S, van Kessel KP, and van der Poll T. Differential role of interleukin-6 in lung inflammation induced by lipoteichoic acid and peptidoglycan from Staphylococcus aureus. Am J Respir Crit Care Med 165: 1445-1450, 2002.[Abstract/Free Full Text]
  18. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, and Hoffmann JA. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86: 973-983, 1996.[ISI][Medline]
  19. Lemjabbar H and Basbaum C. Platelet-activating factor receptor and ADAM10 mediate responses to Staphylococcus aureus in epithelial cells. Nat Med 8: 41-46, 2002.[CrossRef][ISI][Medline]
  20. Mahtani KR, Brook M, Dean JL, Sully G, Saklatvala J, and Clark AR. Mitogen-activated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor {alpha} mRNA stability. Mol Cell Biol 21: 6461-6469, 2001.[Abstract/Free Full Text]
  21. Martin-Blanco E. p38 MAPK signalling cascades: ancient roles and new functions. Bioessays 22: 637-645, 2000.[CrossRef][ISI][Medline]
  22. Medzhitov R and Janeway CA Jr. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 9: 4-9, 1997.[CrossRef][ISI][Medline]
  23. Morath S, Geyer A, and Hartung T. Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. J Exp Med 193: 393-397, 2001.[Abstract/Free Full Text]
  24. Muzio M, Natoli G, Saccani S, Levrero M, and Mantovani A. The human toll signaling pathway: divergence of nuclear factor {kappa}B and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J Exp Med 187: 2097-2101, 1998.[Abstract/Free Full Text]
  25. Ono K and Han J. The p38 signal transduction pathway: activation and function. Cell Signal 12: 1-13, 2000.[CrossRef][ISI][Medline]
  26. Opitz B, Schroder NW, Spreitzer I, Michelsen KS, Kirschning CJ, Hallatschek W, Zahringer U, Hartung T, Gobel UB, and Schumann RR. Toll-like receptor-2 mediates Treponema glycolipid and lipoteichoic acid-induced NF-{kappa}B translocation. J Biol Chem 276: 22041-22047, 2001.[Abstract/Free Full Text]
  27. Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, and Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088, 1998.[Abstract/Free Full Text]
  28. Rock FL, Hardiman G, Timans JC, Kastelein RA, and Bazan JF. A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA 95: 588-593, 1998.[Abstract/Free Full Text]
  29. Schroder NW, Morath S, Alexander C, Hamann L, Hartung T, Zahringer U, Gobel UB, Weber JR, and Schumann RR. Lipoteichoic acid of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor-2, lipopolysaccharide-binding protein, and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 278: 15587-15594, 2003.[Abstract/Free Full Text]
  30. Schwandner R, Dziarski R, Wesche H, Rothe M, and Kirschning CJ. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 274: 17406-17409, 1999.[Abstract/Free Full Text]
  31. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, and Akira S. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11: 443-451, 1999.[ISI][Medline]
  32. Vasudevan S and Peltz SW. Regulated ARE-mediated mRNA decay in Saccharomyces cerevisiae. Mol Cell 7: 1191-1200, 2001.[CrossRef][ISI][Medline]
  33. Widmann C, Gibson S, Jarpe MB, and Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 79: 143-180, 1999.[Abstract/Free Full Text]
  34. Yoshimura A, Lien E, Ingalls RR, Tuomanen E, Dziarski R, and Golenbock D. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J Immunol 163: 1-5, 1999.[Abstract/Free Full Text]




This Article
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Pan, Z. K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Pan, Z. K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.