Transcriptional Inhibition of Interleukin-8 Expression in Tumor Necrosis Factor-tolerant Cells

Abstract

There is some evidence that the potent cytokine tumor necrosis factor (TNF) is able to induce tolerance after repeated stimulation of cells. To investigate the molecular mechanisms mediating this phenomenon, the expression of interleukin-8 (IL-8), which is regulated by transcription factors NF-κB and C/EBPβ, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNF-induced (restimulation with a high dose) IL-8 promoter-dependent transcription as well as IL-8 production. Under these conditions neither activation of NF-κB nor IκB proteolysis was affected after TNF re-stimulation, albeit a slightly reduced IκB-α level was found in the TNF pretreated but not re-stimulated sample. Remarkably, in tolerant cells an increased binding of C/EBPβ to its IL-8 promoter-specific DNA motif as well as an elevated association of C/EBPβ protein with p65-containing NF-κB complexes was observed. Finally, overexpression of C/EBPβ, but not p65 or Oct-1, markedly prevented TNF-induced IL-8 promoter-dependent transcription. Taken together, these data indicate that the expression of IL-8 is inhibited at the transcriptional level in TNF-tolerant cells and C/EBPβ is involved under these conditions in mediating the negative-regulatory effects, a mechanism that may play a role in inflammatory processes such as sepsis. There is some evidence that the potent cytokine tumor necrosis factor (TNF) is able to induce tolerance after repeated stimulation of cells. To investigate the molecular mechanisms mediating this phenomenon, the expression of interleukin-8 (IL-8), which is regulated by transcription factors NF-κB and C/EBPβ, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNF-induced (restimulation with a high dose) IL-8 promoter-dependent transcription as well as IL-8 production. Under these conditions neither activation of NF-κB nor IκB proteolysis was affected after TNF re-stimulation, albeit a slightly reduced IκB-α level was found in the TNF pretreated but not re-stimulated sample. Remarkably, in tolerant cells an increased binding of C/EBPβ to its IL-8 promoter-specific DNA motif as well as an elevated association of C/EBPβ protein with p65-containing NF-κB complexes was observed. Finally, overexpression of C/EBPβ, but not p65 or Oct-1, markedly prevented TNF-induced IL-8 promoter-dependent transcription. Taken together, these data indicate that the expression of IL-8 is inhibited at the transcriptional level in TNF-tolerant cells and C/EBPβ is involved under these conditions in mediating the negative-regulatory effects, a mechanism that may play a role in inflammatory processes such as sepsis. The phenomenon that pre-exposure to a certain substance induces reduced sensitivity to subsequent challenge with the same stimulus is termed tolerance (1Ziegler-Heitbrock H.W.L. J. Inflamm. 1995; 45: 13-26Google Scholar). This concept may be considered protective under acute inflammatory conditions like sepsis in prevention of the deleterious effects that would likely result from persistent cytokine signaling (1Ziegler-Heitbrock H.W.L. J. Inflamm. 1995; 45: 13-26Google Scholar). There is some evidence from in vivo studies that the potent cytokine TNF 1The abbreviations used are: TNF, tumor necrosis factor; IL-8, interleukin-8; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation; CMV, cytomegalovirus. (2Bazzoni F. Beutler B. N. Engl. J. Med. 1996; 334: 1717-1725Google Scholar) is able to induce tolerance-like conditions. For example, pretreatment with TNF in several animal species selectively affects certain TNF effects such as fever, anorexia, and lethality (3Goldbach J.-M. Roth J. Störr B. Zeisberger E. Am. J. Physiol. 1996; 270: R749-R754Google Scholar, 4Socher S.H. Friedman A. Martinez D. J. Exp. Med. 1988; 167: 1957-1962Google Scholar, 5Fraker D.L. Stovroff M.C. Merino M.J. Norton J.A. J. Exp. Med. 1988; 168: 95-105Google Scholar) and partially affects gastrointestinal toxicity (6Patton J.S. Peters P.M. McCabe J. Crase D. Hansen S. Chen A.B. Liggitt D. J. Clin. Invest. 1987; 80: 1587-1596Google Scholar). Cyclooxygenase inhibitors prevent the induction of tolerance to the toxic effect of TNF (7Takahashi N. Brouckaert P. Fiers W. J. Immunotherapy. 1993; 14: 16-21Google Scholar). In addition, some form of cross-tolerance has been described, e.g. pre-exposure to TNF also modulates certain effects of lipopolysaccharide including fever, hypophagia, and lethality (3Goldbach J.-M. Roth J. Störr B. Zeisberger E. Am. J. Physiol. 1996; 270: R749-R754Google Scholar, 5Fraker D.L. Stovroff M.C. Merino M.J. Norton J.A. J. Exp. Med. 1988; 168: 95-105Google Scholar, 8Porter M.H. Arnold M. Langhans W. Am. J. Physiol. 1998; 274: R741-R745Google Scholar). The molecular mechanisms underlying TNF tolerance are only poorly understood (4Socher S.H. Friedman A. Martinez D. J. Exp. Med. 1988; 167: 1957-1962Google Scholar, 9Takahashi N. Brouckaert P. Fiers W. Am. J. Physiol. 1995; 269: R398-R405Google Scholar, 10Takahashi N. Brouckaert P. Bemelmans M.H.A. Buurman W.A. Fiers W. Cytokine. 1994; 6: 235-242Google Scholar). Interestingly, it has been described that long term (11Laegreid A. Thommesen L. Gullstein Jahr T. Sundan A. Espevik T. J. Biol. Chem. 1995; 270: 25418-25425Google Scholar) as well as short term (12Ferlito M. Romanenko O.G. Ashton S. Squadrito F. Halushka P.V. Cook J.A. J. Leukocyte Biol. 2001; 70: 821-829Google Scholar, 13Poppers D.M. Schwenger P. Vilcek J. J. Biol. Chem. 2000; 275: 29587-29593Google Scholar) pretreatment with TNF reduced subsequent TNF-induced activation of transcription factor NF-κB and/or proteolysis of its inhibitor IκB-α. TNF is a potent activator of NF-κB, which is a dimeric complex most frequently assembled from the subunits RelA (p65) and p50 (14Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Google Scholar, 15Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Google Scholar, 16Wulczyn F.G. Krappmann D. Scheidereit C. J. Mol. Med. 1996; 74: 749-769Google Scholar). Activation of this transcription factor by TNF is initiated mainly by binding to cell surface TNF receptor 1 (17Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Google Scholar). Subsequent signaling occurs through the recruitment of cytosolic signaling proteins including TNF receptor-associated death domain protein, receptor-interacting protein, and TNF receptor-associated factor 2, eventually leading to the activation of the IκB kinase complex (17Baud V. Karin M. Trends Cell Biol. 2001; 11: 372-377Google Scholar, 18Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 19Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Google Scholar). This high molecular weight assembly kinase phosphorylates the IκB inhibitor proteins (18Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 20Israel A. Trends Cell Biol. 2000; 10: 129-133Google Scholar), which trap the NF-κB dimer in the cytosol in a non-activated state (14Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Google Scholar, 16Wulczyn F.G. Krappmann D. Scheidereit C. J. Mol. Med. 1996; 74: 749-769Google Scholar). IκB is subsequently degraded in an ubiquitin-dependent step by the proteasome, thereby allowing the liberated NF-κB dimer to translocate to the nuclear compartment (14Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Google Scholar, 15Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Google Scholar, 16Wulczyn F.G. Krappmann D. Scheidereit C. J. Mol. Med. 1996; 74: 749-769Google Scholar). Within the nucleus, NF-κB is involved in the coordinated expression of numerous target genes, including the potent cytokine and chemokine interleukin-8 (IL-8) (14Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Google Scholar, 15Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Google Scholar, 16Wulczyn F.G. Krappmann D. Scheidereit C. J. Mol. Med. 1996; 74: 749-769Google Scholar, 21Matsushima K. Morishita K. Yoshimura T. Lavu S. Kobayashi Y. Lew W. Appella E. Kung H.F. Leonard E.J. Oppenheim J.J. J. Exp. Med. 1988; 167: 1883-1893Google Scholar, 22Roebuck K.A. J. Interferon Cytokine Res. 1999; 19: 429-438Google Scholar). IL-8, a member of the CXC family of chemokines, has been implicated in a variety of inflammatory diseases (21Matsushima K. Morishita K. Yoshimura T. Lavu S. Kobayashi Y. Lew W. Appella E. Kung H.F. Leonard E.J. Oppenheim J.J. J. Exp. Med. 1988; 167: 1883-1893Google Scholar, 22Roebuck K.A. J. Interferon Cytokine Res. 1999; 19: 429-438Google Scholar). Gene transcription is one major point of control at which expression of IL-8 is regulated (22Roebuck K.A. J. Interferon Cytokine Res. 1999; 19: 429-438Google Scholar, 23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Scholar). Functional studies indicate that IL-8 transcriptional responses to mediators such as TNF are rapid and require only 100 nucleotides of 5′-flanking DNA upstream of the TATA box (22Roebuck K.A. J. Interferon Cytokine Res. 1999; 19: 429-438Google Scholar, 23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Scholar). Within this region DNA binding sites for the inducible transcription factors NF-κB and C/EBPβ (24Landschulz W.H. Johnson P.F. Adashi E.Y. Graves B.J. McKnight S.L. Genes Dev. 1988; 2: 786-800Google Scholar, 25Wedel A. Ziegler-Heitbrock H.W. Immunobiol. 1995; 193: 171-185Google Scholar) were found located next to each other (see Fig. 1; Ref. 23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Scholar). Transcription factors from these families bind the IL-8 promoter as dimers, and several distinct subunit combinations have been identified (23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Scholar, 26Kunsch C. Rosen C.A. Mol. Cell. Biol. 1993; 13: 6137-6146Google Scholar). C/EBPβ physically interacts with NF-κB, and functional cooperativity among the factors appears to be critical for optimal IL-8 promoter activity in different cell types (27Stein B. Cogswell P.C. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Google Scholar, 28Stein B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar). IL-8 transcription appears to be activated by a promoter recruitment mechanism where inducible transcription factors are required for binding of TATA box proteins and formation of a stable preinitiation complex (22Roebuck K.A. J. Interferon Cytokine Res. 1999; 19: 429-438Google Scholar). In addition, the POU-homeodomain transcription factor Oct-1, which binds to an overlapping sequence within the C/EBPβ site, appears to be involved in regulation of basal transcriptional activity of the IL-8 promoter (29Wu G.D. Lai E.J. Huang N. Wen X. J. Biol. Chem. 1997; 272: 2396-2403Google Scholar). The aim of this study was to investigate the molecular mechanisms underlying TNF tolerance. To initiate tolerance-like conditions, monocytic cells were pretreated with low TNF concentrations for 72 h and then re-stimulated with a high TNF dose. Under these conditions the expression of the IL-8 gene was monitored as a read-out, and the roles of NF-κB, C/EBPβ, and Oct-1 were examined. Cell Culture Conditions and Reagents—THP-1 human monocytic cells (DSMZ, Braunschweig, Germany) were maintained in suspension in RPMI 1640 (Glutamax-1, low endotoxin) containing 7% fetal calf serum (low endotoxin), 100 units/ml penicillin, and 100 μg/ml streptomycin (Biochrom, Berlin, Germany) (30Fischer C. Page S. Weber M. Eisele T. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar). For the the cells were at a of well in TNF was from cells were in fetal calf 100 units/ml penicillin, 100 μg/ml was by the toxicity of the cell conditions was monitored by cell and the of studies of the IL-8 promoter a was (30Fischer C. Page S. Weber M. Eisele T. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar, S. C. B. M. U. G. M. Ziegler-Heitbrock H.W.L. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar). This was with of a control cells a (30Fischer C. Page S. Weber M. Eisele T. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar, S. C. B. M. U. G. M. Ziegler-Heitbrock H.W.L. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar). cells were at a of of RPMI with 7% fetal calf serum in a and for 72 h or with TNF this the cells were or for h with TNF at Subsequent to stimulation the cells were and activity was the are as which that were by of of IL-8 protein was by were and as described S. C. B. M. U. G. M. Ziegler-Heitbrock H.W.L. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar, K. Page S. G. A. Page M. C. P.A. Neumeier D. J. Clin. Invest. 1996; Scholar). The of the IL-8 (IL-8) as well as NF-κB (IL-8) are in Fig. 1 B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar, G.D. Lai E.J. Huang N. Wen X. J. Biol. Chem. 1997; 272: 2396-2403Google Scholar). were as a and with the of DNA In some the (IL-8) was which the sequence of C/EBPβ (IL-8) and NF-κB Oct-1 and binding was with and kinase proteins were with the for at in of binding as described B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar). were in were and by and nuclear were with of Germany) of in binding at for 1 h The were and In were with a of NF-κB (IL-8), C/EBPβ (IL-8), and Oct-1 as well as and and nuclear were as described (30Fischer C. Page S. Weber M. Eisele T. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar), and was with The proteins were to the the were with TNF receptor-associated factors 1 and 2, receptor-interacting protein, IκB or This was by the The proteins were the were to (30Fischer C. Page S. Weber M. Eisele T. Neumeier D. Brand K. J. Biol. Chem. 1999; 274: Scholar) in 1 μg/ml was at with of or and of protein with the proteins were by and used C/EBPβ, Oct-1 and p65 (14Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Google Scholar, S. B. Cytokine. 2000; Scholar, P.C. E. J. 1997; Scholar). containing was used as a were with of and 1 of cells cells were or for h with TNF at by and activity was Pretreatment with TNF TNF-induced IL-8 Transcription as as IL-8 the study to with a low of TNF for 72 h to induce a tolerance-like as described (11Laegreid A. Thommesen L. Gullstein Jahr T. Sundan A. Espevik T. J. Biol. Chem. 1995; 270: 25418-25425Google Scholar) and then with a and for IL-8 promoter-dependent transcription as well as IL-8 that in the the low cells are as were with monocytic cells to a in which TNF its effect IL-8 promoter-dependent transcription by In these studies a in transcriptional activity in a from to 100 The was for most of the with 1 TNF or for 72 h as TNF in the 2, and by with a for h as TNF in 2, and Pretreatment with TNF to a in IL-8 promoter-dependent transcriptional activity cells were re-stimulated with the TNF with cells that were with with the found a of the of the IL-8 protein by in tolerant cells effects were cells were under the same conditions not activation as well as IκB proteolysis are not cells were with or containing 1 TNF for 72 h by stimulation with TNF TNF for nuclear were to an containing the NF-κB of the IL-8 indicate NF-κB The a control in which a of was to the were with the of the re-stimulated cells TNF p65 and Oct-1 as well as C/EBPβ, and the the complexes in the by with an as well as nuclear were by for the of the proteins are for each which were repeated at receptor-interacting IκB binding activity of C/EBPβ as well as association of C/EBPβ protein with p65 are increased in TNF-tolerant cells. monocytic cells were with or with 1 TNF for 72 h and then or with TNF TNF for nuclear were by a C/EBPβ (IL-8) or binding to these is by for C/EBPβ (IL-8) or an were nuclear of the re-stimulated cells TNF C/EBPβ, Oct-1, and The the of the by with C/EBPβ or Oct-1 The same was in studies with a of C/EBPβ (IL-8), or Oct-1 nuclear were and to with or The proteins were then by for the of p65 or C/EBPβ The an a control was under the same conditions, and the protein was by were for each of Oct-1 TNF-induced IL-8 promoter-dependent monocytic cells were with or with 1 TNF for 72 h and then or with TNF TNF for were by as well as a Oct-1 sequence as indicate Oct-1 of was used for studies nuclear of the re-stimulated cells TNF cells were with and an overexpression for Oct-1 or the cells were or with TNF for h as well as were are as activity of Activation of NF-κB as as IκB the IL-8 gene is regulated by NF-κB (22Roebuck K.A. J. Interferon Cytokine Res. 1999; 19: 429-438Google Scholar, 23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Scholar), it was next to low TNF pretreatment the activation of monocytic cells were re-stimulated with TNF for and were an the motif of the human IL-8 promoter (IL-8), Fig. with TNF a NF-κB binding activity in cells or a slightly increased NF-κB binding activity in low TNF pretreated cells NF-κB binding was as well as Under the same conditions, TNF-induced IκB-α proteolysis was not affected after re-stimulation, by albeit a slightly reduced IκB-α level was found in the but not re-stimulated stimulation with TNF was by an in the nuclear of p65 and p50 The of several upstream signaling involved in TNF-induced NF-κB activation such as TNF receptor-associated factor 1 and 2, receptor-interacting protein, and IκB as well as were not The expression of TNF receptor 1 as well as TNF receptor-associated factor were not affected under these conditions as by not C/EBPβ DNA as as of C/EBPβ with p65 under TNF other transcription factors NF-κB are involved in the regulation of the IL-8 including C/EBPβ B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar). To this transcription factor is involved in TNF were an the C/EBPβ binding of the IL-8 promoter (IL-8), Fig. in cells that were pretreated with TNF or for 72 h and then re-stimulated with a for Under these conditions an elevated binding of nuclear proteins to the C/EBPβ was in TNF-tolerant cells as well as the of C/EBPβ but also a of Oct-1 in these In the same nuclear also the binding of nuclear proteins to the sequence to and which was not under these conditions studies a functional and association NF-κB and family proteins (27Stein B. Cogswell P.C. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Google Scholar, 28Stein B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar, S. J. Biol. Chem. 2001; Scholar). in the to by studies C/EBPβ with NF-κB complexes in cells that were pretreated with TNF and then re-stimulated with this cytokine as described C/EBPβ was in nuclear and p65 was in the by or Remarkably, these an increased association of C/EBPβ protein with p65-containing NF-κB complexes in TNF-tolerant cells which was most after TNF To the of the the was by an In addition, control were or not of these proteins in the nuclear of TNF was was with an not of C/EBPβ TNF-induced IL-8 C/EBPβ is involved in the regulation of TNF-induced IL-8 promoter-dependent transcription cells were with the control or expression for C/EBPβ or a these that the of C/EBPβ inhibited TNF-induced IL-8 promoter-dependent effect was found the protein was control with cells C/EBPβ, an elevated DNA binding of this transcription factor the C/EBPβ (IL-8) as well as the (IL-8) (see TNF stimulation of these a the (IL-8) which binding of C/EBPβ and NF-κB data not p65 overexpression a effect of p65 IL-8 promoter-dependent but in to the studies with C/EBPβ, effect TNF-induced transcriptional activity was found The of the proteins was also monitored by not of Oct-1 TNF-induced IL-8 transcription factor Oct-1 has been to be involved in the regulation of the IL-8 gene (29Wu G.D. Lai E.J. Huang N. Wen X. J. Biol. Chem. 1997; 272: 2396-2403Google Scholar). DNA binding activity was an an Oct-1 In these also an increased binding of nuclear proteins to the Oct-1 in TNF-tolerant cells of the was by and data not Oct-1 was in effect TNF-induced activation of IL-8 promoter-dependent transcription was studies the of TNF tolerance in vivo and that this phenomenon may play an role in such as and sepsis (4Socher S.H. Friedman A. Martinez D. J. Exp. Med. 1988; 167: 1957-1962Google Scholar, 5Fraker D.L. Stovroff M.C. Merino M.J. Norton J.A. J. Exp. Med. 1988; 168: 95-105Google Scholar, 8Porter M.H. Arnold M. Langhans W. Am. J. Physiol. 1998; 274: R741-R745Google Scholar, 9Takahashi N. Brouckaert P. Fiers W. Am. J. Physiol. 1995; 269: R398-R405Google Scholar). the underlying molecular and mechanisms have not been well The study that long term with low TNF doses a tolerant state in monocytic cells. Under these conditions, cells were re-stimulated with a subsequent high of TNF, a of IL-8 promoter-dependent transcription as well as protein of IL-8, which is regulated by NF-κB transcription factors 22Roebuck K.A. J. Interferon Cytokine Res. 1999; 19: 429-438Google and 23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Fig. The that in TNF-tolerant cells neither the activation of NF-κB nor IκB proteolysis was affected after TNF transcriptional also be that in but not a slightly reduced IκB-α level was found that may be to a low level of signaling with IκB-α under the conditions of TNF as described D.M. Schwenger P. Vilcek J. J. Biol. Chem. 2000; 275: 29587-29593Google Scholar). In an were in which long term TNF pretreatment inhibited TNF-induced NF-κB activation by in a human cell (11Laegreid A. Thommesen L. Gullstein Jahr T. Sundan A. Espevik T. J. Biol. Chem. 1995; 270: 25418-25425Google Scholar), cell Interestingly, in a study it was that TNF and modulates in by a mechanism D. J. J. J. Scholar). is of that short term pretreatment of cells appears to to a different form of TNF tolerance under these conditions an of NF-κB activation as well as IκB was found (12Ferlito M. Romanenko O.G. Ashton S. Squadrito F. Halushka P.V. Cook J.A. J. Leukocyte Biol. 2001; 70: 821-829Google Scholar) that in with reduced expression of TNF not Interestingly, several mechanisms have also been found in cells in which transcriptional mechanisms of L. J. Res. 2001; Scholar, H.W.L. A. W. M. P. T. P.A. G. J. Biol. Chem. 1994; 269: Scholar, J. S. N. J. Clin. Invest. 1998; Scholar) as well as of NF-κB (11Laegreid A. Thommesen L. Gullstein Jahr T. Sundan A. Espevik T. J. Biol. Chem. 1995; 270: 25418-25425Google Scholar, M. J. Biol. Chem. Scholar) are for this Remarkably, under TNF tolerance conditions low an increased DNA binding of C/EBPβ to its IL-8 promoter-specific motif C/EBPβ is a transcriptional protein that is involved in and regulation of a variety of including IL-8 (23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Scholar, W.H. Johnson P.F. Adashi E.Y. Graves B.J. McKnight S.L. Genes Dev. 1988; 2: 786-800Google Scholar, 25Wedel A. Ziegler-Heitbrock H.W. Immunobiol. 1995; 193: 171-185Google B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar). was also S. J. Biol. Chem. 2001; Scholar) that binding of proteins and NF-κB to DNA may the effect of by a stable In addition, in studies an increased association of C/EBPβ protein with p65-containing NF-κB complexes in the of tolerant which was most after TNF In this it be that functional and NF-κB subunits and family as well as other proteins have been (27Stein B. Cogswell P.C. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Google Scholar, 28Stein B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar, P.A. U. S. A. Scholar, W. D. T. Cell. 1993; 74: Scholar) that are by a the study was able to functional that overexpression of C/EBPβ, but not inhibited TNF-induced IL-8 promoter-dependent transcription. were in a which that C/EBPβ overexpression transcriptional activity an IL-8 promoter B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar). Taken C/EBPβ as a in mediating the effects IL-8 expression in TNF-tolerant cells. also found an increased binding activity of the transcription factor Oct-1 in cells. has been that Oct-1 basal transcriptional activity of the IL-8 promoter by binding to an overlapping that of C/EBPβ (29Wu G.D. Lai E.J. Huang N. Wen X. J. Biol. Chem. 1997; 272: 2396-2403Google Scholar) and as a transcriptional for a of other U. S. A. Scholar, S. J. 1995; Scholar, M. W. Cell. 1990; Scholar). overexpression of Oct-1 effect TNF-induced IL-8 promoter-dependent that Oct-1 is not involved in regulation under TNF tolerance conditions. The role of C/EBPβ in with NF-κB and binding sites appears to be In NF-κB and with binding sites but with binding sites (27Stein B. Cogswell P.C. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Google Scholar). an for the family proteins have been to activation of the gene D. J. 1990; Scholar) and activity S. J. Biol. Chem. 2001; Scholar) as well as expression of the NF-κB target gene A. K. Mol. Cell. Biol. 2001; Scholar). In the of the IL-8 in which the binding is to the NF-κB (23Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1990; 265: 21128-21133Google Scholar), to the S. J. Biol. Chem. 2001; Scholar), a NF-κB and appears to the IL-8 promoter is activated or inhibited B. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 7191-7198Google Scholar). in TNF-tolerant cells an increased binding of C/EBPβ to its IL-8 promoter motif as well as association of this protein with The found these transcription proteins may be for the effects of C/EBPβ it has been (27Stein B. Cogswell P.C. Baldwin A.S. Mol. Cell. Biol. 1993; 13: 3964-3974Google Scholar) that the of NF-κB to with a critical of the basal transcriptional and/or that C/EBPβ and NF-κB form a transcription factor complex with reduced transcriptional activity at In the study a molecular mechanism IL-8 gene expression in TNF-tolerant cells. of tolerance to control deleterious effects of TNF may play a role in of in and and may and in processes such as of and Germany) for the of the C/EBPβ, Oct-1, or p65 The IL-8 promoter was from to are also for with the receptor studies as well as and for