Gamma Interferon and Granulocyte/Monocyte Colony-stimulating Factor Prevent Endotoxin Tolerance in Human Monocytes by Promoting Interleukin-1 Receptor-associated Kinase Expression and Its Association to MyD88 and Not by Modulating TLR4 Expression
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m200705200
ISSN1083-351X
AutoresMinou Adib‐Conquy, Jean‐Marc Cavaillon,
Tópico(s)Immune cells in cancer
ResumoEndotoxin tolerance is characterized by a decreased production of proinflammatory cytokines by cultured leukocytes in response to lipopolysaccharide (LPS) following a first exposure to the same stimulus. Gamma interferon (IFNγ) and granulocyte/monocyte colony-stimulating factor (GM-CSF) are immunostimulatory cytokines that prime monocytes and prevent endotoxin tolerance. In this study, we show that the deactivating effects of LPS, as well as the priming effects of IFNγ and GM-CSF or their capacity to restore tumor necrosis factor (TNF) production by LPS-tolerized human monocytes are independent of the modulation of TLR2, TLR4, or MD-2. In monocytes pretreated with IFNγ or GM-CSF, interleukin-1 receptor-associated kinase (IRAK) expression is up-regulated. After LPS stimulation, an increased IRAK kinase activity, a higher MyD88/IRAK association, and a stronger NF-κB activation are observed. In contrast, in LPS-tolerized monocytes, IRAK expression and kinase activity, IRAK/MyD88 association, and NF-κB activation are inhibited. Furthermore, the prevention of tolerance by IFNγ and GM-CSF was independent of IRAK kinase activity. Our results suggest that these cytokines prevent endotoxin tolerance induced by low but not by high doses of LPS by inhibiting IRAK degradation and by promoting its association with MyD88 after a second LPS stimulation, which in turn leads to NF-κB activation and TNF production. Endotoxin tolerance is characterized by a decreased production of proinflammatory cytokines by cultured leukocytes in response to lipopolysaccharide (LPS) following a first exposure to the same stimulus. Gamma interferon (IFNγ) and granulocyte/monocyte colony-stimulating factor (GM-CSF) are immunostimulatory cytokines that prime monocytes and prevent endotoxin tolerance. In this study, we show that the deactivating effects of LPS, as well as the priming effects of IFNγ and GM-CSF or their capacity to restore tumor necrosis factor (TNF) production by LPS-tolerized human monocytes are independent of the modulation of TLR2, TLR4, or MD-2. In monocytes pretreated with IFNγ or GM-CSF, interleukin-1 receptor-associated kinase (IRAK) expression is up-regulated. After LPS stimulation, an increased IRAK kinase activity, a higher MyD88/IRAK association, and a stronger NF-κB activation are observed. In contrast, in LPS-tolerized monocytes, IRAK expression and kinase activity, IRAK/MyD88 association, and NF-κB activation are inhibited. Furthermore, the prevention of tolerance by IFNγ and GM-CSF was independent of IRAK kinase activity. Our results suggest that these cytokines prevent endotoxin tolerance induced by low but not by high doses of LPS by inhibiting IRAK degradation and by promoting its association with MyD88 after a second LPS stimulation, which in turn leads to NF-κB activation and TNF production. tumor necrosis factor interleukin lipopolysaccharide Toll-like receptor gamma interferon interleukin-1 receptor-associated kinase granulocyte/monocyte colony-stimulating factor peripheral blood mononuclear cells fluorescence-activated cell sorter reverse transcription monoclonal antibody glyceraldehyde-3-phosphate dehydrogenase Despite the major progress achieved thanks to antibiotherapy, the frequency and severity of nosocomial infections and the occurrence of sepsis syndrome remain major problems in Western countries (1Randel-Frausto M. Infect. Dis. Clin. North Am. 1999; 13: 299-312Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). There have been many reports on the reduced capacity of circulating leukocytes from septic patients to produce cytokines as compared with cells from healthy controls. This hyporeactivity, also termed deactivation, has been particularly well studied in isolated monocytes and in whole blood assays. Monocytes from septic patients have a diminished capacity to release TNFα,1 IL-1α and -β, IL-6, IL-10, and IL-12 (2Muñoz C. Carlet J. Fitting C. Misset B. Bleriot J.P. Cavaillon J.M. J. Clin. Invest. 1991; 88: 1747-1754Crossref PubMed Scopus (638) Google Scholar, 3Van Deuren M. Van Der Ven-Jongekrijg H. Demacker P.N.M. Baterlink A.K.N. Van Dalen R. Sauerwein R.W. Gallati H. Vannice J. van Der Meer J.W.M. J. Infect. Dis. 1994; 169: 157-161Crossref PubMed Scopus (145) Google Scholar, 4Ertel W. Keel M. Neidhardt R. Steckholzer U. Kremer J.P. Ungethuem U. Trentz O. Blood. 1997; 89: 1612-1620Crossref PubMed Google Scholar); however, this is not the case for IL-1ra (3Van Deuren M. Van Der Ven-Jongekrijg H. Demacker P.N.M. Baterlink A.K.N. Van Dalen R. Sauerwein R.W. Gallati H. Vannice J. van Der Meer J.W.M. J. Infect. Dis. 1994; 169: 157-161Crossref PubMed Scopus (145) Google Scholar). Although a defect in the activation of transcription factor NF-κB has been reported (5Adib-Conquy M. Adrie C. Moine P. Asehnoune K. Fitting C. Pinsky M.R. Dhainaut J.-F. Cavaillon J.-M. Am. J. Respir. Crit. Care Med. 2000; 162: 1877-1883Crossref PubMed Scopus (168) Google Scholar), the precise mechanism leading to this hyporeactivity remains unknown.This deactivation found in leukocytes from septic patients resembles the phenomenon called endotoxin tolerance. Endotoxin tolerance is defined by a reduced capacity of the host (in vivo) or of cultured leukocytes (in vitro) to respond to lipopolysaccharide (LPS) activation following a first exposure to this stimulus. Endotoxin tolerance is thought to be an adaptive response that tends to limit the overwhelming inflammation that occurs during bacterial infection, but it may also favor subsequent infections in survivors of septic shock. It is associated with a decreased production of proinflammatory cytokines and with changes in the cellular levels and composition of NF-κB. Indeed, its has been shown that unlike naive cells, tolerized cells have a predominance of the p50 homodimer of NF-κB (6Ziegler-Heitbrock H.W.L. Wedel A. Schraut W. Ströbel M. Wendelgass P. Sterndorf T. Bäuerle P.A. Haas J.G. Riethmüller G. J. Biol. Chem. 1994; 269: 17001-17004Abstract Full Text PDF PubMed Google Scholar), which in contrast to the p65p50 heterodimer, has a minimal transactivation capacity (7Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar, 8Ballard D.W. Dixon E.P. Peffer N.J. Bogerd H. Doerre S. Stein B. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1875-1879Crossref PubMed Scopus (217) Google Scholar). However, other studies have shown that endotoxin tolerance was associated with a depletion of both forms of NF-κB (9Blackwell T.S. Blackwell T.R. Christman J.W. J. Leukocyte Biol. 1997; 62: 885-891Crossref PubMed Scopus (43) Google Scholar).CD14, a glycosylphosphatidylinositol-linked cell surface protein binds specifically the endotoxins (10Wright S. Ramos R. Tobias P. Ulevitch R. Mathison J. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3376) Google Scholar). However, CD14 lacks an intracellular domain, and LPS signal transduction was shown to be mediated by Toll-like receptors (TLR) (11Poltorak A., He, X. Smirnova I. Liu M.Y. Van Huffel C., 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 (6380) Google Scholar, 12Yang R.-B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1098) Google Scholar). TLR family members are transmembrane proteins with an extracellular domain containing leucine-rich repeats and a cytoplasmic domain homologous to that of the IL-1R type I (13Medzhitov R. Preston-Hurlburt P. Janeway C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4378) Google Scholar). Furthermore, it has been reported that TLRs and IL-1R share several signaling molecules including adaptor protein MyD88, interleukin-1 receptor-associated kinase (IRAK), and TNF receptor-activated factor 6 (TRAF6) (14Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar). Upon activation, IRAK is recruited to the receptor through MyD88, becomes highly phosphorylated (by autophosphorylation and/or by the action of another kinase), and then relays the signal downstream by interacting with TRAF6. The kinase activity of IRAK does not seem to be essential for signal transduction, because kinase-inactive mutants of IRAK have been shown to induce NF-κB activation (15Knop J. Martin M.U. FEBS Lett. 1999; 448: 81-85Crossref PubMed Scopus (90) Google Scholar, 16Maschera B. Ray K. Burns K. Volpe F. Biochem. J. 1999; 339: 227-231Crossref PubMed Scopus (79) Google Scholar). It is now clear that TLR4 is necessary for LPS signaling, whereas TLR2 is involved in Gram-positive bacteria signaling (17Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2759) Google Scholar) except for LPS from Leptospira interrogansand Porphyromonas gingivalis, which have been shown to signal through TLR2 (18Werts C. Tapping R.I. Mathison J.C. Chuang T.-H. Kravchenko V. Saint Girons I. Haake D.A. Godowski P.J. Hayashi F. Ozinsky A. Underhill D.M. Kirschning C.J. Wagner H. Aderem A. Tobias P.S. Ulevitch R.J. Nat. Immunol. 2001; 2: 346-352Crossref PubMed Scopus (567) Google Scholar, 19Hirschfeld M. Weis J.J. Toshchakov V. Salkowski C.A. Cody M.J. Ward D.C. Qureshi N. Michalek S.M. Vogel S.N. Infect. Immun. 2001; 69: 1477-1482Crossref PubMed Scopus (555) Google Scholar). MD-2, a soluble protein associated with TLR4 at the cell surface, is needed for an efficient signaling in response to endotoxin (20Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1730) Google Scholar). The effect of endotoxin tolerance on the expression of TLR4 has been studied in macrophages of LPS-tolerized mice and has led to conflicting results. One study reported the down-regulation of TLR4 both in terms of mRNA and membrane expression (21Nomura F. Akashi S. Sakao Y. Sato S. Kawai T. Matsumoto M. Nakanishi K. Kimoto M. Miyake K. Takeda K. Akira S. J. Immunol. 2000; 164: 3476-3479Crossref PubMed Scopus (648) Google Scholar), whereas in another report, down-regulation of TLR4 was not observed and TLR2 mRNA was found to be up-regulated (22Medvedev A.E. Kopydlowski K.M. Vogel S.N. J. Immunol. 2000; 164: 5564-5574Crossref PubMed Scopus (446) Google Scholar). More recently, it has been shown that endotoxin tolerance in the monocytic cell line THP-1 was associated with a defect in the formation of the MyD88·IRAK complex (23Li L. Cousart S., Hu, J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar).Endotoxin tolerance can be prevented or reversed in vitro by immunostimulatory cytokines such as IFNγ or GM-CSF (24Haas J.G. Meyer N. Riethmüller G. Ziegler-Heitbrock H.W.L. Eur. J. Immunol. 1990; 20: 1181-1184Crossref PubMed Scopus (26) Google Scholar, 25Randow F. Docke W.D. Bundschuh D.S. Hartung T. Wendel A. Volk H.D. J. Immunol. 1997; 158: 2911-2918PubMed Google Scholar). The monocyte desensitization is prevented if these cytokines are added during the first contact with LPS and is reversed if they are added subsequent to LPS preculture and before the second LPS challenge. Furthermore, GM-CSF was shown to reactivate in vitro the impaired TNFα production by whole blood samples from patients after trauma or cardiopulmonary bypass but not in samples from patients with severe sepsis (26Flohé S. Börgermann J. Dominguez F.E. Majetschak M. Lim L. Kreuzfelder E. Obertacke U. Nast-Kolb D. Schade F.U. Shock. 1999; 12: 17-24Crossref PubMed Scopus (48) Google Scholar). IFNγ seems to be more potent than GM-CSF, as it can restore TNFα secretion in monocytes from septic patients (27Döcke W.D. Randow F. Syrbe U. Krausch D. Asadullah K. Reinke P. Volk H.D. Kox W. Nat. Med. 1997; 3: 678-681Crossref PubMed Scopus (957) Google Scholar). IFNγ and GM-CSF can also prevent LPS tolerance inducedin vivo (28Bundschuh D.S. Barsig J. Hartung T. Randow F. Docke W.D. Volk H.D. Wendel A. J. Immunol. 1997; 158: 2862-2871PubMed Google Scholar).The aim of this study was to gain insight into the mechanisms of prevention of endotoxin tolerance by INFγ and GM-CSF. For this purpose, we studied the expression of TLR4, TLR2, and MD-2 mRNA by RT-PCR, as well as that of TLR4 on the surface of monocytes after exposure to low or high doses of LPS, IFNγ, or GM-CSF. We compared the capacity of these monocytes to activate NF-κB and produce TNF in response to a secondary LPS challenge. We also studied the expression of IRAK both at mRNA and protein levels. Finally, IRAK kinase activity and its association with MyD88 following LPS stimulation were analyzed.DISCUSSIONEndotoxin tolerance in monocytes is characterized by a transient down-regulation of several functions, such as the capacity to produce proinflammatory cytokines (32Cavaillon J.M. Trends Microbiol. 1995; 3: 320-324Abstract Full Text PDF PubMed Scopus (105) Google Scholar). This state of hyporesponsiveness to LPS following an exposure to bacterial products can be reversed by IFNγ or GM-CSF. These two cytokines possess immunostimulatory properties; they not only prime resting monocytes but also restore monocytic functions after LPS tolerization (24Haas J.G. Meyer N. Riethmüller G. Ziegler-Heitbrock H.W.L. Eur. J. Immunol. 1990; 20: 1181-1184Crossref PubMed Scopus (26) Google Scholar, 25Randow F. Docke W.D. Bundschuh D.S. Hartung T. Wendel A. Volk H.D. J. Immunol. 1997; 158: 2911-2918PubMed Google Scholar). IFNγ and GM-CSF have been shown to up-regulate TNF mRNA (24Haas J.G. Meyer N. Riethmüller G. Ziegler-Heitbrock H.W.L. Eur. J. Immunol. 1990; 20: 1181-1184Crossref PubMed Scopus (26) Google Scholar, 26Flohé S. Börgermann J. Dominguez F.E. Majetschak M. Lim L. Kreuzfelder E. Obertacke U. Nast-Kolb D. Schade F.U. Shock. 1999; 12: 17-24Crossref PubMed Scopus (48) Google Scholar). Furthermore, priming of monocytes with IFNγ enhanced the activation of NF-κB and increased the half-life of its p65 subunit mRNA (33Hayes M.P. Freeman S.L. Donnelly R.P. Cytokine. 1995; 7: 427-435Crossref PubMed Scopus (90) Google Scholar, 34de Wit H. Hoogstraten D. Halie R.M. Vellenga E. Exp. Hematol. 1996; 24: 228-235PubMed Google Scholar). In agreement with these previous studies, we found that IFNγ, and also GM-CSF, enhanced NF-κB nuclear translocation in response to LPS. Nuclear translocation of NF-κB was much more intense after priming with IFNγ than with GM-CSF, although comparable levels of TNF were found in the culture supernatants for both cytokines. Perhaps other mechanisms, such as an enhanced mRNA stability, may contribute to the increased production of TNF in GM-CSF-primed monocytes. In addition, we showed that IFNγ and GM-CSF could restore NF-κB activation, which was otherwise defective, in monocytes tolerized with low doses of LPS. Several authors have shown that LPS tolerance induces modifications in the expression of NF-κB. Some reports show an increase in p50p50 homodimer (6Ziegler-Heitbrock H.W.L. Wedel A. Schraut W. Ströbel M. Wendelgass P. Sterndorf T. Bäuerle P.A. Haas J.G. Riethmüller G. J. Biol. Chem. 1994; 269: 17001-17004Abstract Full Text PDF PubMed Google Scholar), the inactive form of this transcription factor, whereas other studies show a depletion in both p65p50 and p50p50 (9Blackwell T.S. Blackwell T.R. Christman J.W. J. Leukocyte Biol. 1997; 62: 885-891Crossref PubMed Scopus (43) Google Scholar, 35Takasuka N. Matsuura K. Yamamoto S. Akagawa K.S. J. Immunol. 1995; 154: 4803-4812PubMed Google Scholar). Similar to the latter studies, we found that NF-κB nuclear expression was barely induced by LPS in endotoxin-tolerized cells.In the present study, we aimed to gain insights in the precise mechanism of action of IFNγ and GM-CSF in terms of TLR modulation and post-receptor signaling. We showed that the priming effects of IFNγ and GM-CSF were independent of the modulation of TLR4 or MD-2 mRNA. Our results show that MD-2 mRNA, like TLR2, which is not involved in E. coli LPS signaling, is not significantly modulated by IFNγ, GM-CSF, and high or low doses of LPS. We found a significant up-regulation of TLR4 mRNA by IFNγ but not with GM-CSF, whereas both cytokines primed monocytes similarly. Conversely, LPS at 100 ng/ml did not down-regulate TLR4 mRNA, but this concentration of LPS led to a profound defect in TNF production in response to a subsequent endotoxin stimulation. The literature differs on the effects of LPS on TLR4 expression, but this is most likely because of differences in timing or the cell types. Indeed, an up-regulation of TLR4 mRNA has been observed in the hours following LPS stimulation of monocytes, neutrophils, or endothelial cells (36Muzio M. Bosisio D. Polentarutti N. D'amico G. Stoppacciaro A. Mancinelli R. van't Veer C. Penton-Rol G. Ruco L.P. Allavena P. Mantovani A. J. Immunol. 2000; 164: 5998-6004Crossref PubMed Scopus (895) Google Scholar, 37Faure E. Thomas L., Xu, H. Medvedev A.E. Equils O. Arditi M. J. Immunol. 2001; 166: 2018-2024Crossref PubMed Scopus (401) Google Scholar). However, for mouse macrophages, the levels of TLR4 mRNA after 20–24 h were similar to those found in untreated cells (22Medvedev A.E. Kopydlowski K.M. Vogel S.N. J. Immunol. 2000; 164: 5564-5574Crossref PubMed Scopus (446) Google Scholar, 38Sato S. Nomura F. Kawai T. Takeuchi O. Mühlradt P.F. Takeda K. Akira S. J. Immunol. 2000; 165: 7096-7101Crossref PubMed Scopus (347) Google Scholar) or even below base line (39Matsuguchi T. Musikacharoen T. Ogawa T. Yoshikai Y. J. Immunol. 2000; 165: 5767-5772Crossref PubMed Scopus (249) Google Scholar). TLR4 mRNA was still up-regulated 24 h after LPS stimulation in one study dealing with cardiac myocytes and endothelial cells (40Frantz S. Kobzik L. Kim Y.-D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Invest. 1999; 104: 271-280Crossref PubMed Scopus (555) Google Scholar). Similarly to cardiac myocytes and endothelial cells (37Faure E. Thomas L., Xu, H. Medvedev A.E. Equils O. Arditi M. J. Immunol. 2001; 166: 2018-2024Crossref PubMed Scopus (401) Google Scholar, 40Frantz S. Kobzik L. Kim Y.-D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Invest. 1999; 104: 271-280Crossref PubMed Scopus (555) Google Scholar), we observed that IFNγ up-regulated TLR4 mRNA expression in human monocytes, but in contrast to endothelial cells, it had no effect on TLR2.We did not find an agreement between mRNA levels and surface expression of TLR4, which emphasizes that the analysis of TLR should not be limited to the mRNA expression as seen in a number of previous studies. Our data indicate that the priming effects of IFNγ and GM-CSF and the reversal of endotoxin tolerance by these cytokines were independent of TLR4 surface expression. Our observation of a dissociation between LPS-induced deactivation and TLR4 expression is consistent with a recent report showing that overexpression of TLR4 and MD-2 in a CD14-positive Chinese hamster ovary cell line could not overcome endotoxin tolerance (41Medvedev A.E. Henneke P. Schromm A. Lien E. Ingalls R. Fenton M. Golenbock D.T. Vogel S.N. J. Immunol. 2001; 167: 2257-2267Crossref PubMed Scopus (140) Google Scholar). Similarly, a mycoplasmal lipopeptide has been reported to induced cross-tolerance to a subsequent LPS stimulation without affecting the surface expression of the TLR4·MD2 complex on mouse peritoneal macrophages (38Sato S. Nomura F. Kawai T. Takeuchi O. Mühlradt P.F. Takeda K. Akira S. J. Immunol. 2000; 165: 7096-7101Crossref PubMed Scopus (347) Google Scholar). However, some studies performed on mouse peritoneal macrophages, or the THP-1 cell line, have shown that tolerization with E. coli LPS was associated with a down-regulation of TLR4 surface expression (21Nomura F. Akashi S. Sakao Y. Sato S. Kawai T. Matsumoto M. Nakanishi K. Kimoto M. Miyake K. Takeda K. Akira S. J. Immunol. 2000; 164: 3476-3479Crossref PubMed Scopus (648) Google Scholar, 38Sato S. Nomura F. Kawai T. Takeuchi O. Mühlradt P.F. Takeda K. Akira S. J. Immunol. 2000; 165: 7096-7101Crossref PubMed Scopus (347) Google Scholar, 42Martin M. Katz J. Vogel S.N. Michalek S.M. J. Immunol. 2001; 167: 5278-5285Crossref PubMed Scopus (151) Google Scholar). We did not find this to be the case for human monocytes; perhaps the effect of LPS on TLR4 expression also depends on the differentiation stage of the cells and/or the compartment they derive from.As LPS tolerance prevention by IFNγ and GM-CSF was not linked to TLR4 expression, we further analyzed the effect of these cytokines on the signaling pathway downstream the receptor. The immunostimulatory effect of IFNγ and GM-CSF may be explained by their capacity to up-regulate IRAK expression and to induce a more important and sustained MyD88·IRAK association in response to LPS. On the contrary, LPS-tolerized monocytes expressed less IRAK than untreated cells. When the cells were tolerized with a low dose of endotoxin (1 ng/ml), MyD88·IRAK association was still found; however, the complex rapidly dissociated after the second LPS stimulation. IRAK down-regulation was more pronounced for the cells tolerized with 100 ng/ml LPS; consequently we barely detected any IRAK·MyD88 complexes in these cells. These results are in accordance with a recent study on the monocytic cell line THP-1, in which a defect of association between MyD88 and IRAK was found after LPS tolerization (23Li L. Cousart S., Hu, J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). In the former study, the cells were tolerized with 100 ng/ml LPS. In the present study, using human monocytes, we show that at this concentration of endotoxin, IFNγ or GM-CSF could not reverse the defect in IRAK expression. In contrast, the reversal was possible in cells tolerized with a low concentration of LPS, where both IFNγ and GM-CSF induced an up-regulation of IRAK and its association with MyD88 in response to LPS challenge. The time course analysis of IRAK expression at protein and mRNA levels suggests that the up-regulation of this protein by IFNγ and GM-CSF at 1 ng/ml LPS is due to an inhibition of its degradation rather than to increased transcription. IFNγ has been shown to affect proteasome complex composition and to decrease the level of 26 S proteasome, which is responsible for the degradation of short-lived cellular proteins, including ubiquitin-dependent proteolysis (43Bose S. Brooks P. Mason G.G.F. Rivett A.J. Biochem. J. 2001; 353: 291-297Crossref PubMed Scopus (69) Google Scholar). In monocytes tolerized with 1 ng/ml LPS, IRAK association with MyD88 was induced in a stronger manner by IFNγ than by GM-CSF, which also corresponds to a stronger NF-κB activation. This may thus explain the fact that GM-CSF was only reversing the tolerization, whereas IFNγ induced a 5–10-fold higher TNF production as compared with untreated cells.Similar to a study performed on the THP-1 cell line (23Li L. Cousart S., Hu, J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), we found that endotoxin tolerance, induced by 100 ng/ml as well as 1 ng/ml LPS, resulted in a defect in IRAK kinase activity. Pretreatment of monocytes with IFNγ or GM-CSF enhanced the kinase activity of IRAK. However, the presence of these cytokines did not prevent the inhibition of IRAK kinase activity by LPS tolerization. Nevertheless, monocytes tolerized with 1 ng/ml LPS in the presence of IFNγ or GM-CSF were not impaired for NF-κB activation and TNF production in response to a subsequent LPS stimulation. Thus, IRAK kinase activity seems to be dispensable for NF-κB activation and TNF production in human monocytes. These results are in accordance with previous studies performed on cell lines, showing that IRAK signaling capacity is independent of its kinase activity and that kinase-inactive mutants of IRAK could still activate NF-κB (15Knop J. Martin M.U. FEBS Lett. 1999; 448: 81-85Crossref PubMed Scopus (90) Google Scholar, 16Maschera B. Ray K. Burns K. Volpe F. Biochem. J. 1999; 339: 227-231Crossref PubMed Scopus (79) Google Scholar).In summary, our study shows that IFNγ and GM-CSF prime human monocytes by up-regulating IRAK protein expression and kinase activity and by promoting its association with MyD88 following LPS stimulation. These cytokines can also reverse endotoxin tolerance by inhibiting IRAK degradation and by restoring IRAK/MyD88 association but only when the tolerization has been performed with low doses of LPS. Despite the major progress achieved thanks to antibiotherapy, the frequency and severity of nosocomial infections and the occurrence of sepsis syndrome remain major problems in Western countries (1Randel-Frausto M. Infect. Dis. Clin. North Am. 1999; 13: 299-312Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). There have been many reports on the reduced capacity of circulating leukocytes from septic patients to produce cytokines as compared with cells from healthy controls. This hyporeactivity, also termed deactivation, has been particularly well studied in isolated monocytes and in whole blood assays. Monocytes from septic patients have a diminished capacity to release TNFα,1 IL-1α and -β, IL-6, IL-10, and IL-12 (2Muñoz C. Carlet J. Fitting C. Misset B. Bleriot J.P. Cavaillon J.M. J. Clin. Invest. 1991; 88: 1747-1754Crossref PubMed Scopus (638) Google Scholar, 3Van Deuren M. Van Der Ven-Jongekrijg H. Demacker P.N.M. Baterlink A.K.N. Van Dalen R. Sauerwein R.W. Gallati H. Vannice J. van Der Meer J.W.M. J. Infect. Dis. 1994; 169: 157-161Crossref PubMed Scopus (145) Google Scholar, 4Ertel W. Keel M. Neidhardt R. Steckholzer U. Kremer J.P. Ungethuem U. Trentz O. Blood. 1997; 89: 1612-1620Crossref PubMed Google Scholar); however, this is not the case for IL-1ra (3Van Deuren M. Van Der Ven-Jongekrijg H. Demacker P.N.M. Baterlink A.K.N. Van Dalen R. Sauerwein R.W. Gallati H. Vannice J. van Der Meer J.W.M. J. Infect. Dis. 1994; 169: 157-161Crossref PubMed Scopus (145) Google Scholar). Although a defect in the activation of transcription factor NF-κB has been reported (5Adib-Conquy M. Adrie C. Moine P. Asehnoune K. Fitting C. Pinsky M.R. Dhainaut J.-F. Cavaillon J.-M. Am. J. Respir. Crit. Care Med. 2000; 162: 1877-1883Crossref PubMed Scopus (168) Google Scholar), the precise mechanism leading to this hyporeactivity remains unknown. This deactivation found in leukocytes from septic patients resembles the phenomenon called endotoxin tolerance. Endotoxin tolerance is defined by a reduced capacity of the host (in vivo) or of cultured leukocytes (in vitro) to respond to lipopolysaccharide (LPS) activation following a first exposure to this stimulus. Endotoxin tolerance is thought to be an adaptive response that tends to limit the overwhelming inflammation that occurs during bacterial infection, but it may also favor subsequent infections in survivors of septic shock. It is associated with a decreased production of proinflammatory cytokines and with changes in the cellular levels and composition of NF-κB. Indeed, its has been shown that unlike naive cells, tolerized cells have a predominance of the p50 homodimer of NF-κB (6Ziegler-Heitbrock H.W.L. Wedel A. Schraut W. Ströbel M. Wendelgass P. Sterndorf T. Bäuerle P.A. Haas J.G. Riethmüller G. J. Biol. Chem. 1994; 269: 17001-17004Abstract Full Text PDF PubMed Google Scholar), which in contrast to the p65p50 heterodimer, has a minimal transactivation capacity (7Schmitz M.L. Baeuerle P.A. EMBO J. 1991; 10: 3805-3817Crossref PubMed Scopus (664) Google Scholar, 8Ballard D.W. Dixon E.P. Peffer N.J. Bogerd H. Doerre S. Stein B. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1875-1879Crossref PubMed Scopus (217) Google Scholar). However, other studies have shown that endotoxin tolerance was associated with a depletion of both forms of NF-κB (9Blackwell T.S. Blackwell T.R. Christman J.W. J. Leukocyte Biol. 1997; 62: 885-891Crossref PubMed Scopus (43) Google Scholar). CD14, a glycosylphosphatidylinositol-linked cell surface protein binds specifically the endotoxins (10Wright S. Ramos R. Tobias P. Ulevitch R. Mathison J. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3376) Google Scholar). However, CD14 lacks an intracellular domain, and LPS signal transduction was shown to be mediated by Toll-like receptors (TLR) (11Poltorak A., He, X. Smirnova I. Liu M.Y. Van Huffel C., 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 (6380) Google Scholar, 12Yang R.-B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1098) Google Scholar). TLR family members are transmembrane proteins with an extracellular domain containing leucine-rich repeats and a cytoplasmic domain homologous to that of the IL-1R type I (13Medzhitov R. Preston-Hurlburt P. Janeway C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4378) Google Scholar). Furthermore, it has been reported that TLRs and IL-1R share several signaling molecules including adaptor protein MyD88, interleukin-1 receptor-associated kinase (IRAK), and TNF receptor-activated factor 6 (TRAF6) (14Muzio M. Natoli G. Saccani S. Levrero M. Mantovani A. J. Exp. Med. 1998; 187: 2097-2101Crossref PubMed Scopus (525) Google Scholar). Upon activation, IRAK is recruited to the receptor through MyD88, becomes highly phosphorylated (by autophosphorylation and/or by the action of another kinase), and then relays the signal downstream by interacting with TRAF6. The kinase activity of IRAK does not seem to be essential for signal transduction, bec
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