Characterization of Interleukin-1 Receptor-associated Kinase in Normal and Endotoxin-tolerant Cells

Abstract

Interleukin-1 receptor-associated kinase (IRAK), a signal transducer for interleukin-1, has also been suggested to participate in the Toll-like receptor-mediated innate immune response to bacterial endotoxin lipopolysaccharide (LPS). Using the human promonocytic THP-1 cell line, we demonstrated that the endogenous IRAK is quickly activated in response to bacterial LPS stimulation, as measured by its in vitro kinase activity toward myelin basic protein. LPS also triggers the association of IRAK with MyD88, the adaptor protein linking IRAK to the Toll-like receptor/interleukin-1β receptor intracellular domain. Macrophage cells with prolonged LPS treatment become tolerant to additional dose of LPS and no longer express inflammatory cytokines. Endotoxin tolerance is a common phenomenon observed in blood from sepsis patients. We observed for the first time that the quantity of IRAK is greatly reduced in LPS-tolerant THP-1 cells, and its activity no longer responds to further LPS challenge. In addition, IRAK does not associate with MyD88 in the tolerant cells. Furthermore, application of AG126, a putative tyrosine kinase inhibitor, can substantially alleviate the LPS-induced cytokine gene expression and can also decrease IRAK level and activity. Our study indicates that IRAK is essential for LPS-mediated signaling and that cells may develop endotoxin tolerance by down-regulating IRAK. Interleukin-1 receptor-associated kinase (IRAK), a signal transducer for interleukin-1, has also been suggested to participate in the Toll-like receptor-mediated innate immune response to bacterial endotoxin lipopolysaccharide (LPS). Using the human promonocytic THP-1 cell line, we demonstrated that the endogenous IRAK is quickly activated in response to bacterial LPS stimulation, as measured by its in vitro kinase activity toward myelin basic protein. LPS also triggers the association of IRAK with MyD88, the adaptor protein linking IRAK to the Toll-like receptor/interleukin-1β receptor intracellular domain. Macrophage cells with prolonged LPS treatment become tolerant to additional dose of LPS and no longer express inflammatory cytokines. Endotoxin tolerance is a common phenomenon observed in blood from sepsis patients. We observed for the first time that the quantity of IRAK is greatly reduced in LPS-tolerant THP-1 cells, and its activity no longer responds to further LPS challenge. In addition, IRAK does not associate with MyD88 in the tolerant cells. Furthermore, application of AG126, a putative tyrosine kinase inhibitor, can substantially alleviate the LPS-induced cytokine gene expression and can also decrease IRAK level and activity. Our study indicates that IRAK is essential for LPS-mediated signaling and that cells may develop endotoxin tolerance by down-regulating IRAK. lipopolysaccharide interleukin tumor necrosis factor Toll-like receptor interleukin-1 receptor-associated kinase TNF receptor-activated factor 6 4-morpholinepropanesulfonic acid polyacrylamide gel electrophoresis myelin basic protein Upon bacterial lipopolysaccharide (LPS)1 stimulation, normal human macrophages produce various inflammatory mediators such as interleukin-1β (IL-1) and tumor necrosis factor (TNF) α (1Bone R.C. Grodzin C.J. Balk R.A. Chest. 1997; 112: 235-243Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar). Although appropriate amounts of these mediators may be essential for augmenting microcidal activities of macrophages, excessive production caused by acute bacterial infection during sepsis results in severe systematic inflammatory complications (2Bone R.C. J. Am. Med. Assoc. 1996; 276: 565-566Crossref PubMed Google Scholar). Perhaps developed as a self-protective adaptive response, human neutrophils and monocytes isolated from sepsis patients become refractory to further LPS challenge and no longer produce inflammatory cytokines (3McCall C.E. Grosso-Wilmoth L.M. LaRue K. Guzman R.N. Cousart S.L. J. Clin. Invest. 1993; 91: 853-861Crossref PubMed Scopus (172) Google Scholar). This phenomenon, referred to as endotoxin tolerance, has also been demonstrated in animal models of endotoxemia (4Beeson P.B. J. Exp. Med. 1947; 86: 29-31Crossref PubMed Scopus (205) Google Scholar) and can be induced in a variety of primary monocyte/macrophage cells and cell lines. Upon LPS treatment, the human promonocytic THP-1 cells mimic all the responses exhibited by human whole blood and blood neutrophils from sepsis patients (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). Both human leukocytes obtained from sepsis patients, and the THP-1 cells treated with LPS show an adaptation of innate immune response as reflected by the suppression of steady-state mRNAs of proinflammatory genes (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). The molecular mechanism underlying endotoxin tolerance is not known. Recently, a family of Toll-like transmembrane receptor (TLR) proteins were identified in humans and mice that respond to LPS and other microbial products, thereby modulating the innate immune response (6Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar). The TLR proteins possess leucine-rich extracellular repeats that may recognize the LPS-lipid-binding protein-CD14 complex (7Poltorak A. Ricciardi-Castagnoli P. Citterio S. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2163-2167Crossref PubMed Scopus (398) Google Scholar). Interestingly, the TLR proteins possess intracellular domains resembling the IL-1β receptor-like intracellular domain and may utilize IL-1β signaling components including adaptor protein MyD88, interleukin-1 receptor-associated kinase (IRAK), and TNF receptor-activated factor 6 (TRAF6) (6Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, 8Dushay M.S. Eldon E.D. Am. J. Hum. Genet. 1998; 62: 10-14Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Upon IL-1β stimulation, IRAK is recruited to the IL-1 receptor through the adaptor MyD88 (9Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (781) Google Scholar). IRAK subsequently undergoes phosphorylation and relays the signal downstream by interacting with TRAF6 (9Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (781) Google Scholar, 10Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Genetic analyses indicate that IRAK is shared by the IL-1/LPS signaling pathway. Overexpression of a dominant negative IRAK in human THP-1 cells inhibits the LPS-induced NFkB activation (6Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, 11Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Furthermore, overexpression of wild type IRAK in an IRAK-deficient 293 cell line renders the cell responsive to LPS stimulation as measured by NFkB promoter reporter assay (12Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). However, there has been no study regarding the endogenous IRAK response upon LPS stimulation. The inability of cells to express inflammatory genes in response to LPS in endotoxin-tolerant cells reflects a state of altered innate immunity. Disruption of the molecular signaling events linking from LPS stimulation to inflammatory gene activation could contribute to the altered innate immune response. C3H/HeJ mice, which have defects in the gene encoding the TLR4 protein, are unresponsive to LPS stimulation (13Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6577) Google Scholar, 14Qureshi S.T. Gros P. Malo D. Trends Genet. 1999; 15: 291-294Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 15Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar). MyD88 knock-out mice are also unable to induce LPS-dependent gene expression (16Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1747) Google Scholar). In addition, both the C3H/HeJ and MyD88 knock-out mice exhibit high resistance to LPS-induced shock. However, it is not known what molecular alterations are responsible for the development of endotoxin tolerance in wild type human macrophage. Examination of the molecular alteration(s) in endotoxin-tolerant cells may shed light on the mechanism of the innate adaptation developed in human septic blood cells. In this study, we examined the endogenous IRAK protein level and kinase activity in fresh and endotoxin-tolerant THP-1 cells. We observed that endogenous IRAK in fresh THP-1 cells is activated upon LPS stimulation. In contrast, IRAK level is consistently low in endotoxin-tolerant THP-1 cells and is no longer responsive to further LPS treatment. Furthermore, IRAK interacts with MyD88 upon LPS treatment, whereas such interaction is abolished in tolerant cells. Application of AG126, a putative kinase inhibitor, causes IRAK level decrease and inactivation and also decreases the cellular IL-1β expression upon LPS treatment. Our results suggest that defective IRAK may be responsible for the development of endotoxin tolerance during sepsis. LPS (Escherichia coli 0111:B4) was obtained from Sigma. AG-126 was purchased from Biomol. IRAK antibody was from Upstate Biotechnology, Inc. MyD88 and TRAF6 antibodies were obtained from Santa Cruz Biotechnology, Inc. We employed the undifferentiated human promonocytic THP-1 cells for our study. The advantages of that are 2-fold. First, our lab has previously completed extensive studies characterizing the tolerant phenotype of THP-1 cells (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar), which is similar to the tolerant phenotype seen in leukocytes from humans with sepsis. Second, undifferentiated THP-1 cells do not express IL-1 type I receptor and therefore do not respond to IL-1 stimulation (17Spriggs M.K. Lioubin P.J. Slack J. Dower S.K. Jonas U. Cosman D. Sims J.E. Bauer J. J. Biol. Chem. 1990; 265: 22499-22505Abstract Full Text PDF PubMed Google Scholar). The IRAK response we observed is solely due to LPS stimulation rather than IL-1 effect. We have found that the model of LPS tolerance in undifferentiated THP-1 cells is similar to that of vitamin D3 differentiated THP-1 cells (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). 2L. Li, S. Cousart, J. Hu, and C. E. McCall, unpublished observations. The LPS responses of both undifferentiated and differentiated THP-1 cells are CD14-dependent. Endotoxin tolerance in undifferentiated THP-1 cells can be induced by treating the cells with LPS ranging from 10 ng/ml to 1 μg/ml (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). THP-1 cells are rendered tolerant within 6 h of LPS treatment and remain tolerant up to 96 h after the initial dose of LPS (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). In our hands, intracellular events modulating LPS tolerance are optimally studied of LPS and the cells with a dose of LPS h after the initial dose of In this undifferentiated THP-1 cells were in with μg/ml and THP-1 cells were obtained by treating the cells with ng/ml endotoxin for h (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). as as endotoxin-tolerant THP-1 cells were with in fresh a of and treated with ng/ml endotoxin for various as in the was isolated the THP-1 were was on a gel in and 1 and was a was as (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). normal and endotoxin-tolerant cells were time for 10 and on for 10 in 1 of 1 1 1 1 1 1 and 1 of and were by for in a The protein in the was protein assay to the by the with of proteins were for the of antibody Biotechnology, was to of the isolated cell and for h on a of a of protein were to by for an additional h The were in a and in was of was by by to with and with The other of were with kinase 1 1 1 of kinase was to with 1 of myelin basic protein 1 of and for was after and the were to The gel was and to The of the signal was a promonocytic THP-1 cells produce inflammatory mediators such as IL-1β in response to LPS challenge. THP-1 cells with LPS are tolerant to additional dose of in from THP-1 cells after h of LPS treatment were and for expression of and IL-1β by In response to fresh THP-1 cells express IL-1β and However, with LPS greatly decreases the and expression upon dose of LPS treatment Genetic analyses indicate that the LPS response by the IL-1β signaling components MyD88, and IRAK as and and on a gel (9Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (781) Google Scholar). 10 of IL-1β stimulation, IRAK undergoes and (9Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (781) Google Scholar, 10Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Although the response of IRAK upon IL-1β stimulation has been studied there has been no study regarding the endogenous IRAK upon LPS treatment. the signaling in THP-1 cells, we examined the endogenous IRAK response to LPS stimulation in fresh THP-1 cells. undifferentiated THP-1 cells were to LPS treatment for various of IRAK protein was subsequently and to In to its IL-1β response, the level of IRAK decreases as a of protein there is no of IRAK within the first of LPS stimulation However, the of IRAK after of LPS treatment, that endogenous IRAK undergoes phosphorylation upon LPS stimulation. of LPS stimulation, IRAK decrease and remain a low We and obtained we the IRAK kinase activity in assay in and IRAK a in its kinase activity within 1 h of LPS stimulation of normal THP-1 cells, in to the IRAK activation caused by IL-1β (9Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (781) Google Scholar, 10Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). IRAK kinase activity decreases to level after of LPS treatment, with the decrease in IRAK undifferentiated THP-1 cells do not express IL-1 type 1 receptor (17Spriggs M.K. Lioubin P.J. Slack J. Dower S.K. Jonas U. Cosman D. Sims J.E. Bauer J. J. Biol. Chem. 1990; 265: 22499-22505Abstract Full Text PDF PubMed Google Scholar) and do not respond to IL-1 stimulation (17Spriggs M.K. Lioubin P.J. Slack J. Dower S.K. Jonas U. Cosman D. Sims J.E. Bauer J. J. Biol. Chem. 1990; 265: 22499-22505Abstract Full Text PDF PubMed Google M. H. H. I. H. 1999; PubMed Scopus Google Scholar), we that the observed IRAK response is caused by LPS rather than by the IL-1 THP-1 cells treated with IL-1β that IRAK protein and kinase activities are not and Furthermore, IL-1β THP-1 cells can respond to LPS stimulation, as measured by IL-1β and IRAK kinase activation and with our that LPS through endogenous IRAK in normal human cells. Our results also indicate that LPS a IRAK response with IL-1β causes IRAK and T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, H. Henzel W.J. Li S. Cao Z. Immunity. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas LPS causes a activation of IRAK. We examined the IRAK protein and its kinase activity in the endotoxin-tolerant THP-1 cells. IRAK was from tolerant THP-1 cells and by IRAK protein level is low in the tolerant cells. In addition, the of IRAK upon dose of LPS treatment We also that the IRAK as a on the and this upon dose of LPS treatment. The of the is Furthermore, IRAK protein level in tolerant cells with the level in normal cells, no IRAK kinase activation is observed in the tolerant THP-1 cells upon dose of LPS treatment We and obtained Our results indicate that there is a of IRAK response in tolerant cells. was that THP-1 cells develop endotoxin tolerance as as h after LPS treatment (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar), with our that IRAK level and activity decrease in normal THP-1 cells after of LPS stimulation. The tolerant THP-1 cells remain tolerant h after the of LPS (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). Examination of IRAK h after of LPS by the cells with the endotoxin-tolerant cells a low level of IRAK protein and low kinase activity not our results suggest that the inability of tolerant cells to express inflammatory cytokine is in by the of IRAK. MyD88 is the adaptor that both TLR and IL-1 receptor intracellular domain with IRAK (6Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar). IRAK relays the signal to TRAF6 and subsequently downstream and K. C. H. P. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We examined the interaction of endogenous IRAK with MyD88 and TRAF6 in normal as as tolerant THP-1 cells. Upon LPS stimulation, MyD88 was and through and we observed that there is a level of MyD88 protein in both the normal and tolerant cells Upon the with we found that IRAK an with MyD88 after of LPS stimulation interaction 1 h and we could not association of IRAK with MyD88 in the tolerant THP-1 cells the TRAF6 protein was antibody and by and MyD88, we observed that TRAF6 is also a protein with a level in both normal and tolerant cells Upon the with we can the of IRAK This is in with the that IRAK undergoes phosphorylation and association with TRAF6 protein. Interestingly, the of IRAK that with TRAF6 in normal and tolerant THP-1 cells. However, we the that IRAK may during LPS stimulation in normal and tolerant cells. has been that of AG126, a putative tyrosine kinase inhibitor, can decrease the IL-1β and production in mice macrophages upon LPS stimulation A. A. M. A. A. Science. 1994; PubMed Scopus Google Scholar). AG-126 can also the mice from LPS-induced septic A. A. M. A. A. Science. 1994; PubMed Scopus Google Scholar). signaling is for cellular LPS response, we examined AG-126 can with this signaling with the we that the THP-1 cells with for 1 h can decrease the IL-1β production upon LPS stimulation 6 We examined the of on IRAK protein level and kinase activity. Interestingly, treatment of THP-1 cells with for 1 h consistently causes a decrease in the IRAK protein level In addition, there is no of IRAK kinase activity upon LPS treatment 6 The IRAK level as as activity decrease and of LPS-induced cytokine gene expression further indicates that of signaling the of IRAK to endotoxin tolerance model of the of IRAK in signaling in normal and tolerant IRAK is a putative it is that AG-126 inhibits IRAK may other and that in IRAK level decrease and of IRAK phosphorylation and of other of IRAK the of IRAK during signaling in normal and tolerant cells. Genetic analyses indicate that LPS signaling and IL-1 signaling MyD88, and TRAF6 (6Medzhitov R. Preston-Hurlburt P. Kopp E. Stadlen A. Chen C. Ghosh S. Janeway Jr., C.A. Mol. Cell. 1998; 2: 253-258Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar). Using the undifferentiated human promonocytic THP-1 cells, which do not respond to IL-1 stimulation, we that LPS stimulation to the activation of endogenous IRAK kinase and its interaction with Our study that LPS a IRAK response with studies IL-1 responsive cell as that IL-1β causes IRAK and T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, H. Henzel W.J. Li S. Cao Z. Immunity. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we observed that LPS causes a IRAK activation and no decrease in IRAK level within the first of LPS stimulation T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Furthermore, it was that the association of MyD88 and IRAK within after IL-1β stimulation and quickly within of IL-1β treatment H. Henzel W.J. Li S. Cao Z. Immunity. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, we found that LPS-induced interaction 1 h after LPS stimulation. THP-1 cells express low of and are therefore responsive to The IRAK response we observed be caused by its low also from events on IRAK by there is no study regarding the of IRAK. of the phosphorylation of IRAK upon LPS and IL-1β stimulation regarding the of IRAK activity and its in these signaling of the signaling components can to endotoxin C3H/HeJ mice, which a in TLR4 are unresponsive to LPS stimulation (13Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6577) Google Scholar). mice with MyD88 gene also to respond to LPS challenge (16Kawai T. Adachi O. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1747) Google Scholar). However, it is not known wild type human monocytes and promonocytic cell endotoxin tolerance upon with Our study indicates that tolerant THP-1 cells normal of MyD88 as as TRAF6 IRAK protein level is with that in normal THP-1 cells. Furthermore, additional LPS challenge no longer IRAK kinase activity in tolerant cells. We also that IRAK no longer with MyD88 in the tolerant cells upon further LPS stimulation. results indicate that the signaling components MyD88, and alterations in IRAK with THP-1 cells endotoxin further the of IRAK in cells endotoxin First, THP-1 cells develop endotoxin tolerance as as h after LPS treatment (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar), with our that IRAK level and activity are in normal THP-1 cells after h of LPS stimulation. Second, the tolerant THP-1 cells remain tolerant h after the of LPS (5LaRue K.E. McCall C.E. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (101) Google Scholar). Examination of IRAK that the endotoxin-tolerant cells low level of IRAK protein and low kinase activity h after of application of a putative tyrosine kinase inhibitor, can LPS Examination of IRAK that causes decrease in IRAK signal and also IRAK kinase activation upon LPS treatment. we suggest that the inability of tolerant cells to express inflammatory cytokines in response to LPS may be by the of IRAK The IRAK kinase activation in signaling is not has been that during signaling IRAK kinase activity may not be essential J. 1999; PubMed Scopus Google Scholar). a IRAK in cells, it was that IRAK could B. K. K. J. 1999; PubMed Scopus Google Scholar). has been therefore suggested that IRAK as an essential adaptor protein during The kinase activity of IRAK may its and of IL-1 signaling T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The decrease in IRAK level after h of LPS stimulation observed in this study may also be caused by a similar effect. IRAK may not be the that is and responsible for endotoxin are other and Although are overexpression of of these can the IL-1 and LPS response in a 293 cell line IRAK (12Wesche H. Gao X. Li X. Kirschning C.J. Stark G.R. Cao Z. J. Biol. Chem. 1999; 274: 19403-19410Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). In with the study, mice with IRAK gene was to response to with a decrease M. T. J. Cao Z. J. Immunol. 1999; Google Scholar). studies suggest that may in LPS-mediated However, it is also that endogenous can respond to there are no antibodies for and there has been no study regarding these endogenous the antibodies are it be to the of these in and LPS-mediated be also to study cells have to to endotoxin Disruption of components of IRAK may also be and for development of LPS Recently, it was that the level of TLR4 receptor was reduced in LPS-tolerant macrophages S. Y. S. T. M. K. Takeda K. Akira S. J. Immunol. 2000; PubMed Scopus Google Scholar). However, the mechanism of TLR4 is not known. there is no in TLR4 level normal and tolerant cells S. Y. S. T. M. K. Takeda K. Akira S. J. Immunol. 2000; PubMed Scopus Google Scholar), receptor and for the the TLR4 receptor in macrophages from C3H/HeJ mice was also upon LPS challenge S. Y. S. T. M. K. Takeda K. Akira S. J. Immunol. 2000; PubMed Scopus Google Scholar). This that other LPS events are for TLR4 and development of endotoxin In addition, The TLR4 level decreases upon LPS challenge and was reduced after h of LPS S. Y. S. T. M. K. Takeda K. Akira S. J. Immunol. 2000; PubMed Scopus Google Scholar). The that endotoxin tolerance can be induced with a of h of LPS that LPS tolerance does not solely of suppression of the not be to in the that a of including LPS and intracellular components contribute to the development of endotoxin In LPS stimulation of human promonocytic THP-1 cells to the activation of endogenous IRAK kinase and its interaction with The decrease of IRAK level and its to further LPS stimulation with the THP-1 cells endotoxin the that endogenous IRAK is in the signaling and that of IRAK is mechanism by which cells may endotoxin