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The Journal of Immunology, 2006, 177: 665-672.
Copyright © 2006 by The American Association of Immunologists

C/EBPbeta Blocks p65 Phosphorylation and Thereby NF-{kappa}B-Mediated Transcription in TNF-Tolerant Cells1

Andreas Zwergal2,*, Martina Quirling2,*, Bernd Saugel*, Karin C. Huth{dagger}, Carmen Sydlik*, Valeria Poli{ddagger}, Dieter Neumeier*, H. W. Löms Ziegler-Heitbrock§,¶ and Korbinian Brand3,*

* Institute of Clinical Chemistry and Pathobiochemistry, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany; {dagger} Department of Restorative Dentistry and Periodontology, Ludwig-Maximilians-University, Munich, Germany; {ddagger} Department of Genetics, Biology, and Biochemistry, University of Turin, Turin, Italy; § Clinical Cooperation Group Inflammatory Lung Diseases, GSF-Forschungszentrum für Umwelt und Gesundheit-Institute of Inhalation Biology, Gauting, Germany; and Department of Microbiology and Immunology, University of Leicester, Leicester, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-{kappa}B-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPbeta. In this study, we have shown that a {kappa}B motive was sufficient to mediate transcriptional inhibition under TNF tolerance conditions in monocytic cells. Furthermore, in tolerant cells, TNF-induced NF-{kappa}B p65 phosphorylation was markedly decreased, which was accompanied by the formation of C/EBPbeta-p65 complexes. Remarkably, in C/EBPbeta–/– cells incubated under the conditions of TNF tolerance, neither impairment of transcription nor inhibition of p65 phosphorylation was observed. Finally, we showed that C/EBPbeta overexpression reduced p65-mediated transactivation and that association of C/EBPbeta with p65 specifically prevented p65 phosphorylation. Our data demonstrate that C/EBPbeta is an essential signaling component for inhibition of NF-{kappa}B-mediated transcription in TNF-tolerant cells and suggest that this is caused by blockade of p65 phosphorylation. These results define a new molecular mechanism responsible for TNF tolerance in monocytic cells that may contribute to the unresponsiveness seen in patients with sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumor necrosis factor is a master cytokine and major mediator of inflammation, immunity, and apoptosis (1, 2). Inappropriate regulation of TNF production and signaling has been implicated in the pathogenesis of a wide spectrum of human diseases, such as sepsis, rheumatoid arthritis, and tumor cachexia (3, 4, 5, 6). An important mechanism involved in modulation of cellular TNF signaling is the TNF tolerance phenomenon: pre-exposure to TNF reduces sensitivity to subsequent stimulation with this cytokine. Results from in vivo studies demonstrate that TNF pretreatment in several animal species attenuates effects of subsequent TNF stimulation, such as fever, anorexia, and lethality (7, 8, 9, 10). Under the condition of TNF tolerance in SW480 tumor cells a diminished activation of the transcription factor NF-{kappa}B was found (11), whereas in THP-1 monocytic cells an increase of transcriptionally inactive NF-{kappa}B p50 homodimers was detected (12). Another publication describes that short-time TNF preincubation of cultured neurons leads to a reduced association of p65 with the coactivator p300 together with a decrease in transcription of ICAM-1 (13). Finally, in TNF-tolerant monocytic cells, an inhibition of NF-{kappa}B-dependent IL-8 gene expression has been identified at the transcriptional level with potential involvement of C/EBPbeta (14). As can be drawn from these studies, the molecular mechanisms underlying TNF tolerance are still not completely understood.

The NF-{kappa}B system is an integral element of TNF signaling cascades (15, 16). The transcription factor NF-{kappa}B represents a dimeric complex most frequently assembled from the subunits p65 (RelA) and p50 (17, 18, 19). TNF binding to cell surface TNFR1 leads to the recruitment of cytosolic signaling proteins, including TNFR-associated death domain protein, receptor-interacting protein, and TNFR-associated factor 2, which is followed by the activation of the I{kappa}B kinase (IKK)4 complex (15, 20). This high m.w. assembly kinase complex consists of the kinase-active molecules IKK{alpha} and IKKbeta and the regulatory adapter IKK{gamma} (16, 21, 22, 23). The IKK complex phosphorylates the I{kappa}B inhibitor proteins, which trap the NF-{kappa}B dimer in the cytosol in a nonactivated state (17, 18, 24). I{kappa}B is subsequently degraded in a ubiquitin-dependent step by the proteasome, thereby allowing the liberated NF-{kappa}B complex to shuttle into the nucleus and bind to {kappa}B motives, thus initiating target gene expression (17, 18).

Besides regulating NF-{kappa}B localization via I{kappa}B, TNF signaling leads to posttranslational modifications of NF-{kappa}B subunits, such as p65 phosphorylation, suggested to be involved in the regulation of the p65 transactivation potential (15, 23, 25). The TNF-inducible kinases implicated in the phosphorylation of p65 include both IKKs and casein kinase II (25). IKKbeta, for example, phosphorylates p65 on Ser536 within the C-terminal transactivation domain (TAD) 1 (26). It has also been shown that a stimulation with TNF results in a phosphorylation of p65 on Ser529 by casein kinase II (27). The function of NF-{kappa}B dimers is also modulated by complex formation with other transcription factors such as C/EBPbeta (28, 29).

C/EBP proteins are a family of basic leucine zipper transcription factors with six members, including C/EBP{alpha}, -beta, -{delta}, and C/EBP homologous protein, which play pivotal roles in the control of cellular proliferation and differentiation and are involved in metabolic, immune, and inflammatory modulation (29, 30). The activity of C/EBPbeta in the presence of a variety of stimuli is regulated at the expression level, via nuclear export, through posttranslational modifications and by protein interaction with other transcription factors such as NF-{kappa}B (29, 31, 32, 33, 34). C/EBPbeta plays a complex role in the NF-{kappa}B-mediated regulation of promoters, and it has been shown that NF-{kappa}B and C/EBPbeta synergistically activate promoters with C/EBPbeta binding sites, while they inhibit promoters with functional {kappa}B motives (35, 36). In the case of IL-8 or IE1/2 promoters, in which a C/EBPbeta binding site is located directly next to a {kappa}B motive, a finely tuned interaction between NF-{kappa}B and C/EBPbeta seems to decide whether this promoter is activated or not (35, 37).

In earlier experiments, we have shown in TNF-tolerant monocytic cells an inhibition of NF-{kappa}B-dependent IL-8 promoter activity and IL-8 production, but no impairment of NF-{kappa}B activity (gel shift), as well as an increased association of NF-{kappa}B p65 with C/EBPbeta (14). The aim of the present study was to define the molecular mechanisms responsible for the inhibition of NF-{kappa}B-dependent transcription under conditions of TNF tolerance, focusing on the role of p65 and C/EBPbeta. In this study, we identified a {kappa}B motive as the minimal DNA requirement to mediate TNF tolerance, whereas binding of C/EBPbeta to DNA was not necessary. In addition, in TNF-tolerant cells we found a markedly reduced p65 phosphorylation. Remarkably, in the absence of C/EBPbeta, neither transcriptional inhibition nor impairment of p65 phosphorylation was detected under conditions of TNF tolerance. Furthermore, we showed that C/EBPbeta was able to inhibit p65-mediated transactivation and that association of C/EBPbeta with p65 blocked phosphorylation of this NF-{kappa}B subunit.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture conditions and reagents

THP-1 human monocytic cells (obtained from DSMZ) were maintained in suspension in RPMI 1640 (Glutamax1, low endotoxin) containing 7% FCS (low endotoxin), 100 U/ml penicillin, and 100 µg/ml streptomycin (Biochrom) (24). For the experiments, the cells were plated at a density of 2 x 106/well in 6-well culture dishes. HeLa cells (obtained from DSMZ) were cultured in RPMI 1640 containing 7% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. C/EBPbeta wild-type (wt) and C/EBPbeta–/– macrophages were cultured in RPMI 1640 containing 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (38). Human r-{alpha} was obtained from Sigma-Aldrich. Endotoxin contamination was screened by the limulus amebocyte lysate assay (BioWhittaker).

Transfection

For transfection studies, the following luciferase reporter plasmids were used: pXP2-IL-8-Luc wt (133 bp of the IL-8 promoter region), pXP2-IL-8-Luc ({kappa}B mut) (133 bp of IL-8 promoter region with a mutated NF-{kappa}B-binding motive), and pXP2-IL-8-Luc (C/EBPbeta mut) (133 bp of IL-8 promoter region with a mutated C/EBPbeta-binding motive) were a gift from T. Murayama (Kanazawa University, Ishikawa, Japan); pGL2-IL-8-Luc (420 bp of the IL-8 promoter region) was obtained from N. Mackman (The Scripps Research Foundation, La Jolla, CA). We also used the pGL2–3{kappa}B-Luc reporter construct (three {kappa}B-binding motives) (24). For luciferase assays, 1 µg of luciferase reporter plasmid was transiently cotransfected with 0.2 µg of a constitutively active Renilla luciferase control plasmid, pRLtk (Promega). THP-1 cells were transfected using a DEAE-dextran-based protocol (14). After transfection, cells were plated at a density of 2 x 106/well in RPMI 1640 (7% FCS) in a six-well plate and incubated for 72 h (without or with 1 ng/ml TNF pretreatment). After this time, cells were left untreated or restimulated for 5 h with 20 ng/ml TNF. C/EBPbeta (wt and –/–) macrophages were also transfected using a DEAE-dextran-based protocol (14), plated at a density of 2 x 106/well in RPMI 1640 (10% FCS), and preincubated for 32 h without or with TNF (5 ng/ml). Following this, cells were left untreated or restimulated for 5 h with 100 ng/ml TNF. HeLa cells were transfected by a Superfect-based protocol (Qiagen). Subsequent to stimulation, whole cell extracts were harvested, and luciferase activity was determined using the Dual Luciferase Reporter assay system (Promega). Results were expressed as relative luciferase activity (RLA), i.e., firefly relative light units were divided by Renilla relative light units. In the experiments using the Gal-p65/Gal4-Luc system (39), 0.2 µg of Gal4-Luc, 2 µg of Gal-p65 plasmid (both obtained from A. Baldwin, University of North Carolina, Chapel Hill, NC) or Gal-VP-16 expression plasmid (derived from M. Lienhard Schmitz, University of Berne, Berne, Switzerland), and 0.2 µg of Renilla plasmid were transiently transfected. For overexpression experiments, several plasmids were used (1–5 µg) that code for C/EBPbeta wt or a 13-kDa C-terminal C/EBPbeta fragment (truncated; trunc) starting with aa 229 and containing the basic leucine zipper (bZIP) (required for association with p65) and DNA-binding region, but not the TAD (obtained from R. Pope, Northwestern University, Chicago, IL) as well as p65 (obtained from P. Baeuerle, Micromet, Munich, Germany). Furthermore, we applied expression plasmids for HA-tagged p65 536wt or p65 536mut, the latter containing a mutation at position 536 from serine to alanine (obtained from M. Lienhard Schmitz).

SDS-PAGE and Western blot analysis

Cytosolic extracts were isolated, and electrophoresis was performed with 12% polyacrylamide gels (0.1% SDS) (21). The proteins were transferred to nitrocellulose membranes using the Western blot technique. After transfer, the membranes were incubated with Abs against p65, C/EBPbeta (Santa Cruz Biotechnology), phospho-p65 (Ser536) (Cell Signaling Technology), or actin (Sigma-Aldrich). This was followed by the appropriate HRP-conjugated secondary Ab (Dianova). The proteins were visualized on x-ray film using the Chemiluminescent Reagent Plus (PerkinElmer Life Sciences).

Immunoprecipitation (IP)

For association assays, 100 µg of cytosolic extracts was subjected to IP in 0.1 M sodium phosphate buffer (pH 8.1) (leupeptin, antipain, aprotinin, pepstatin A, chymostatin, 0.75 µg/ml each; Sigma-Aldrich). IP was conducted at 4°C for 1 h with 2 µg of anti-C/EBPbeta or 2 µg of anti-p65 (Santa Cruz Biotechnology) and 40 µl of protein A dynabeads (Dynal Biotech). After washing three times with PBS, the precipitated proteins were analyzed by PAGE and Western blot analysis.

In vitro phosphorylation

The substrates (p65, C/EBPbeta) were produced by overexpression in HeLa cells for 24 h. Cytosolic extracts were subjected to IP, which was conducted in TN buffer (200 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM DTT; 0.5 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and leupeptin, antipain, aprotinin, pepstatin A, and chymostatin, 0.75 µg/ml each (Sigma-Aldrich)) at 4°C for 1 h with 2 µg of anti-p65 and 40 µl of 6% protein A-agarose (Roche Diagnostics) (21). Then samples were washed three times with TN buffer and three times with kinase buffer (20 mM HEPES (pH 8.0), 10 mM MgCl2, 100 µM Na3VO4, 20 mM beta-glycerophosphate, 50 mM NaCl, 2 mM DTT; 0.5 µM AESBF and leupeptin, antipain, aprotinin, pepstatin A, and chymostatin, 0.75 µg/ml each (Sigma-Aldrich)). The washed agarose was incubated with cytosolic extracts from tolerance experiments, kinase buffer, and 5 µCi of [{gamma}-32P]ATP (PerkinElmer Life Sciences) at 30°C for 30 min and subsequently washed twice with kinase buffer. Proteins were analyzed on 12% polyacrylamide gels (0.1% SDS), and following Western blot the transferred proteins were visualized by autoradiography. Densitometric analysis was performed for the obtained signals, and the values were corrected for the loading control for each protein.

In vivo phosphorylation

THP-1 cells were plated at a density of 4 x 105 in 24-well plates and left untreated or incubated for 68 h with 1 ng/ml TNF. Subsequently, cells were washed three times with phospho-free medium (Biochrom) and then cultured in phospho-free medium plus [32P]-orthophosphate (PerkinElmer Life Sciences) at 1 µCi/µl for 4 h. Finally, cells were left untreated or stimulated with 20 ng/ml TNF for 15 min. Cells were washed three times again with phospho-free medium, cytosolic extracts were isolated, and p65 was immunoprecipitated and analyzed by PAGE, using the protein A-agarose-based protocol stated above. Following Western blot, radioactively labeled p65 was visualized on x-ray film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-8 promoter- and 3{kappa}B-dependent transcription are inhibited in TNF tolerance

In TNF-tolerant monocytic cells, an inhibition of NF-{kappa}B-dependent IL-8 gene expression has been identified at the transcriptional level, accompanied by an increase of C/EBPbeta-p65 association (14). To further investigate this, monocytic THP-1 cells, which were transfected with several (wt, mutated) IL-8 promoter luciferase constructs, were preincubated with a low-dose of TNF (1 ng/ml) for 72 h to induce tolerance or left untreated (control), and this was followed by stimulation with a higher TNF dose (20 ng/ml). Please note that in the following the low-dose TNF-pretreated cells are designated as tolerant cells. In nontolerant cells, TNF induced a strong increase of IL-8 promoter activity using the wt plasmid, which, as expected, was inhibited in tolerant cells (Fig. 1A, left). The inducibility of this construct was completely abolished when the {kappa}B motive was mutated (Fig. 1A, middle), demonstrating the crucial role of NF-{kappa}B in control of the human IL-8 promoter. The inhibition of IL-8 promotor-dependent transcription was not affected when the C/EBP binding site was mutated (Fig. 1A, right), which suggested that TNF tolerance did not depend on C/EBPbeta binding to DNA. Additional experiments with a 3{kappa}B luciferase plasmid were performed. Similar as we observed for the IL-8 promoter, the TNF-induced luciferase signal decreased significantly in tolerant cells containing the 3{kappa}B plasmid (Fig. 1B). These results demonstrate that TNF tolerance can be mediated solely by a {kappa}B-binding motive as the minimal DNA requirement.


Figure 1
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  		FIGURE 1. IL-8 promoter- and 3{kappa}B-dependent transcription are inhibited in TNF tolerance. A, THP-1 monocytic cells were cotransfected with pXP2-IL-8 luciferase reporter plasmids containing an IL-8 promoter fragment (wt, mutated: {kappa}B or C/EBPbeta site), together with a pRLtk Renilla plasmid. Cells were cultured in medium alone ({square}) or medium with 1 ng/ml TNF (TNF pre; {cjs2113}) for 72 h. Afterward, cells were left untreated or stimulated with 20 ng/ml TNF for 5 h (TNF re). Results are depicted as RLA. Representative data of three independent experiments are shown (mean ± SD). B, A pGL2–3{kappa}B-Luc reporter and the Renilla plasmid were transfected. Cells were exposed to TNF and the data were calculated, as stated above.

 
Inhibition of p65 phosphorylation in TNF-tolerant cells is accompanied by de novo formation of C/EPBbeta-p65 complexes

Next, we investigated the phosphorylation status of the NF-{kappa}B-transactivating subunit p65 under TNF tolerance. To measure in vivo phosphorylation of p65, THP-1 cells were pretreated with low-dose TNF (1 ng/ml) for 68 h, then phospho-labeled for 4 h, and after that stimulated with a high dose of TNF (20 ng/ml) for 15 min. Finally, p65 was immunoprecipitated and separated by SDS-PAGE. These data showed that p65 was strongly phosphorylated in TNF-stimulated nontolerant cells (Fig. 2A, upper two panels). Remarkably, when cells were pre-exposed to low-dose TNF, following restimulation with TNF, a markedly inhibited phospho-labeling of this NF-{kappa}B subunit was observed. To confirm these results, p65 phosphorylation was also monitored by in vitro phosphorylation assays. To generate the substrate, p65 was immunoprecipitated from HeLa cells overexpressing this subunit. Then cytosolic extracts from THP-1 cells were incubated with equal amounts of p65 substrate under kinase assay conditions. Again, the restimulation-induced phosphorylation of p65 was inhibited in tolerant cells (Fig. 2A, lower two panels). To investigate whether phosphorylation of p65 on Ser536, which is located within the TAD-1, is inhibited in tolerant cells, we used a phospho-specific Ab. p65 was strongly phosphorylated on Ser536 in nontolerant cells stimulated with high-dose TNF (Fig. 2B, upper two panels). Similar to the data above, this effect was abolished when we restimulated the TNF-tolerant cells with TNF. Under the same conditions, our studies demonstrated that a significant binding of C/EBPbeta to p65-containing complexes was induced de novo in tolerant cells (Fig. 2B, lower four panels). Investigating the functional relevance of p65 Ser536 by overexpression experiments, we could show a reduced TNF-induced IL-8 promoter and 3{kappa}B-dependent transcription in THP-1 as well as HeLa cells when p65 was mutated at Ser536 (Fig. 2C and data not shown). Taken together, our data demonstrate that TNF-induced p65 phosphorylation is markedly inhibited in TNF-tolerant cells, which is inversely accompanied by the formation of C/EBPbeta-p65 complexes.


Figure 2
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  		FIGURE 2. Inhibition of p65 phosphorylation in TNF-tolerant cells is accompanied by de novo formation of C/EBPbeta-p65 complexes. A, Upper two panels (in vivo), THP-1 cells were incubated with medium or 1 ng/ml TNF (TNF pre) for 68 h and subsequently exposed to phospho-free medium and 1 mCi of 32P-ortho-phosphate for 4 h. Then cells were stimulated with 20 ng/ml TNF for 15 min (TNF re). Cytosolic extracts were subjected to IP with anti-p65, followed by SDS-PAGE, and phospho-labeled p65 was visualized on x-ray films. As a control, the total amount of precipitated p65 was detected by Western blot analysis. A representative film of two independent experiments is shown. Lower two panels (in vitro), HeLa cells were transfected with an expression plasmid for p65 and lysed after 24 h, followed by precipitation of p65 as a substrate. In vitro phosphorylation assays were performed using cytosolic extracts from THP-1 cells that had been pretreated for 72 h with 1 ng/ml TNF (TNF pre), followed by restimulation with 20 ng/ml TNF for 15 min (TNF re). Following this, samples were separated by SDS-PAGE and phospho-p65 was detected on x-ray films. Total precipitated p65 was analyzed by Western blot. Representative data of three independent experiments are shown. B, THP-1 cells were preincubated with 1 ng/ml TNF for 72 h (TNF pre), and following this, stimulated with TNF (20 ng/ml, TNF re) for 15 min. Upper two panels, Cytosolic extracts were analyzed by Western blot for phosphorylation of p65 on Ser536, and protein loading was controlled by detection of actin. Lower four panels, Under the same conditions, C/EBPbeta-p65 complex formation was monitored by subjecting extracts to IP with anti-C/EBPbeta or anti-p65. The precipitates were analyzed by Western blot for the presence of associated p65 or C/EBPbeta, respectively. In addition, the quality of the IP was monitored by Western blot. Representative data of four independent experiments are shown. C, THP-1 or HeLa cells were transfected with the empty control plasmid, p65 536mut, or p65 536wt, together with the pXP2-IL-8-Luc ({square}) or pGL2–3{kappa}B-Luc ({cjs2108}) and incubated with TNF (5 h, 20 ng/ml) before RLA was determined. On the left side of the figure, the values obtained following TNF stimulation in the presence of the control plasmid were defined as 100%, and on the right side the values induced by exposure to TNF in the presence of p65 536wt were set as 100%. Representative experiments performed in duplicate of three independent studies are shown (mean ± SD).

 
Inhibition of IL-8 promoter-mediated transcription in TNF-tolerant cells requires C/EBPbeta

To further investigate the role of C/EBPbeta in TNF tolerance, we used macrophages from mice, which had the C/EBPbeta gene knocked out (38). First, we monitored IL-8 promoter-dependent transcription under tolerance conditions, comparing C/EBPbeta wt and C/EBPbeta–/– cells. For this, C/EBPbeta wt and C/EBPbeta–/– macrophages were transfected with an IL-8 promoter indicator plasmid and then preincubated for 32 h with 5 ng/ml low-dose TNF, followed by restimulation with 100 ng/ml TNF for 5 h. When wt cells were preincubated with TNF, the high-dose TNF-induced IL-8 promoter-dependent transcription (restimulation) was significantly lower compared with the nonpretreated control, indicating tolerance (Fig. 3A, left). In sharp contrast, when preincubated C/EBPbeta–/– cells were restimulated with TNF, the promoter was activated to the same extent as seen in naive knockout cells (Fig. 3A, right). This demonstrates that the transcriptional inhibition in TNF tolerance requires C/EBPbeta. To confirm the specific effect of C/EBPbeta on TNF tolerance, we tried to reinstall TNF tolerance in C/EBPbeta–/– cells by reintroducing the C/EBPbeta molecule. For this purpose, C/EBPbeta–/– cells were used that were transiently or stably transfected with C/EBPbeta (38) and then treated as described above. When such cells were preincubated in medium and then stimulated with TNF, they displayed high IL-8 promoter activity, whereas in cells that had been pretreated with TNF a strongly reduced activity was found following TNF restimulation (Fig. 3B). This pattern was seen in both transiently (Fig. 3B, left) and stably transfected macrophages (Fig. 3B, right). These data demonstrate that tolerance-induced transcriptional inhibition reappears when C/EBPbeta is expressed in C/EBPbeta–/– cells. Using varying amounts of C/EBPbeta expression plasmid, a dose-dependent correlation between C/EBPbeta protein and induction of TNF tolerance was evaluated (data not shown). Similar results as depicted in Fig. 3 were seen when using a 3{kappa}B luciferase reporter plasmid (data not shown). In summary, these experiments show that the C/EBPbeta protein is an essential component of the signaling machinery involved in inhibition of NF-{kappa}B-dependent transcription in TNF-tolerant cells.


Figure 3
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  		FIGURE 3. Inhibition of IL-8 promoter-mediated transcription in tolerant cells requires C/EBPbeta. A, C/EBPbeta wt and C/EBPbeta–/– macrophages were cotransfected with pGL2-IL-8 together with the pRLtk Renilla plasmid. Then cells were cultured in medium alone ({square}) or medium containing 5 ng/ml TNF for 32 h (TNF pre; {cjs2108}). Afterward, the cells were stimulated with 100 ng/ml TNF for 5 h (TNF re). Firefly as well as Renilla relative light units were measured, and results are depicted as RLA. Values obtained for not pretreated, TNF-stimulated cells were defined as 100%. Data of five independent experiments are shown (mean ± SD). B, C/EBPbeta–/– macrophages, which were transiently or stably transfected with a C/EBPbeta expression plasmid, were cotransfected with pGL2-IL-8 together with the Renilla plasmid. Cells were treated and data analyzed as described for A.

 
Inhibition of p65 phosphorylation in TNF-tolerant cells is dependent on the presence of C/EBPbeta

As TNF tolerance disappeared in C/EBPbeta–/– cells, next we investigated whether p65 phosphorylation is altered concomitantly. At first, C/EBPbeta wt and C/EBPbeta–/– cells were treated with TNF, as described in Fig. 3. Cytosolic extracts were exposed to precipitated p65 under in vitro phosphorylation conditions. In C/EBPbeta wt cells, p65 phosphorylation highly increased after high-dose TNF stimulation (Fig. 4, A and B). In tolerant wt cells, p65 phosphorylation was completely blocked when restimulating with TNF. By contrast, in C/EBPbeta–/– cells, pre-exposure to low-dose TNF did not prevent high-dose TNF-induced p65 phosphorylation. Rather, using extracts from C/EBPbeta–/– cells subjected to the tolerance induction protocol and then stimulated with TNF, we could measure the same level of p65 phosphorylation as when we used extracts from stimulated naive knockout cells (Fig. 4A, lower panel, lanes 2 and 4). The data from the last two figures show that no inhibition of transcription and p65 phosphorylation occurred in C/EBPbeta–/– cells under the conditions of TNF tolerance.


Figure 4
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  		FIGURE 4. Inhibition of p65 phosphorylation in TNF-tolerant cells depends on the presence of C/EBPbeta. A, In vitro phosphorylation assays were performed using precipitated p65 as a substrate and cytosolic extracts from C/EBPbeta wt or C/EBPbeta–/– cells that had been either pretreated for 32 h with medium or 5 ng/ml TNF (TNF pre), followed by restimulation with 100 ng/ml TNF for 15 min (TNF re). Following this, samples were separated by SDS-PAGE and phospho-p65 was detected on x-ray films. Total precipitated p65 was analyzed by Western blot analysis. B, Data of three independent experiments were analyzed by densitometry, and ratios of phospho-65 and p65 were calculated (mean ± SD).

 
C/EBPbeta protein is able to prevent p65 transactivation

In the following, we evaluated whether C/EBPbeta can directly modulate the transactivation potential of p65 using a Gal-p65 fusion protein/Gal4-Luc reporter system (39). Gal-p65 binding to the Gal4 motive of the luciferase plasmid depends on the Gal fragment of the fusion protein, whereas Luc gene transcription is mediated by the TAD of the p65 component. Gal-p65, Gal4-Luc, and increasing amounts of C/EBPbeta wt or trunc expression plasmids were transfected into HeLa cells. Overexpression of Gal-p65 induced a marked increase in luciferase activity (Fig. 5A). In these experiments, the transcriptional activity of Gal-p65 almost completely disappeared when C/EBPbeta wt was overexpressed, which was accompanied by increased C/EBPbeta-Gal-p65 complex formation (data not shown). In addition, overexpression of C/EBPbeta trunc also decreased luciferase activity, but the inhibitory effect was less effective compared with C/EBPbeta wt overexpression. In contrast, Gal-VP-16-induced transcriptional activity was not inhibited by C/EBPbeta overexpression, demonstrating specificity of our results (Fig. 5B). These data suggest that C/EBPbeta is able to negatively affect p65-mediated transactivation.


Figure 5
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  		FIGURE 5. Transcriptional activity of p65 is decreased by C/EBPbeta overexpression. A, HeLa cells were cotransfected with the Gal-p65 expression plasmid, the Gal4-Luc reporter, pRLtk Renilla, and increasing amounts of either C/EBPbeta wt expression plasmid (wt) or a vector expressing a trunc form of C/EBPbeta (trunc). The expression level of C/EBPbeta (wt, trunc) was monitored by Western blot (insert: please note that the wt and trunc proteins are detected at different positions on the gel). In addition, cells were transfected solely with the Gal4-Luc plasmid and the pRLtk Renilla plasmid together with the empty control plasmids (Co in A and B). Cells were lysed after 24 h and RLA was measured (n = 3; mean ± SD). B, HeLa cells were transfected with different amounts of C/EBPbeta wt plasmid, respectively, together with the Gal-VP-16 expression plasmid, the Gal4-Luc reporter, and pRLtk Renilla. Cells were lysed after 24 h, and RLA was determined (mean ± SD). A representative experiment (performed in duplicate) of three is shown.

 
C/EBPbeta-p65 association blocks phosphorylation of p65

Finally, we wanted to show that C/EBPbeta-p65 association is able to block p65 phosphorylation. Initially, we established conditions of increased C/EBPbeta-p65 association. Equal amounts of p65 were immunoprecipitated from HeLa cells that had been transfected with p65 expression vector only or with p65 and C/EBPbeta plasmids, respectively. In the first case, only little C/EBPbeta was coimmunoprecipitated (Fig. 6A, middle panel, lanes 1 and 2), but with transfection of C/EBPbeta the amount of coprecipitated C/EBPbeta strongly increased (Fig. 6A, middle panel, lanes 3 and 4). Thus, we generated C/EBPbeta-p65 complexes as a substrate for kinase assays, in which the immunoprecipitate of cells transfected with p65 alone was used as a control. Both substrates were then exposed to cell lysates from either untreated or TNF-stimulated THP-1 cells (20 ng/ml, 15 min) in the presence of [{gamma}-32P]ATP. In these experiments, p65 was readily phosphorylated (Fig. 6A, upper blot, lane 2), but the C/EBPbeta-p65 complex was refractory to phosphorylation (Fig. 6A, upper blot, lane 4). Under these conditions, alterations of p65 phosphorylation could be only caused by C/EBPbeta association because the amount of p65 precipitates was comparable (see Fig. 6A, lower panel: please note that the immunoprecipitate was monitored directly on the kinase assay membrane). The graphical representation of the load-corrected p65 phosphorylation is shown in Fig. 6B. To further investigate whether C/EBPbeta-p65 association can inhibit p65 phosphorylation on Ser536, we used a phospho-specific Ab. HeLa cells were transfected with p65 and increasing amounts of C/EBPbeta under the conditions as described above. In cells that solely overexpressed p65, stimulation with TNF strongly induced Ser536 phospho-labeling (Fig. 6C). However, when C/EBPbeta was additionally overexpressed, an inhibition of TNF-inducible phosphorylation of Ser536 was observed. In conclusion, it can be stated that the association of C/EBPbeta with p65 specifically inhibits the TNF-induced phosphorylation of this NF-{kappa}B subunit.


Figure 6
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  		FIGURE 6. Association of C/EBPbeta with p65 specifically inhibits phosphorylation of p65. A, HeLa cells were transfected with either p65 alone or p65 together with C/EBPbeta (brackets). After 24 h, cytosolic extracts of these cells were prepared and subjected to IP with anti-p65, and equal aliquots were incubated in kinase assays with cytosolic extracts of untreated or TNF-stimulated THP-1 cells (20 ng/ml TNF, 15 min) to monitor p65 phosphorylation. The level of p65 phosphorylation was visualized by autoradiography (upper panel). The presence of associated C/EBPbeta that had been coimmunoprecipitated during IP with anti-p65 (middle panel) as well as p65 loading (lower panel) was monitored on the same membranes. WB, Western blot analysis. B, Data of three independent experiments performed as described in A were analyzed by densitometry, and ratios of phospho-65 and p65 were calculated (mean ± SD). C, HeLa cells were transfected with either p65 alone or p65 together with C/EBPbeta (1 or 5 µg) under the same conditions as described in A. After 24 h, cells were stimulated with TNF (20 ng/ml, 15 min) and cytosolic extracts of these cells were prepared and analyzed by Western blot for phosphorylation of p65 on Ser536. C/EBPbeta overexpression as well as protein loading (actin) are also depicted (Western blot).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TNF plays a pivotal role in coordinating several lines of defense in response to a broad spectrum of pathogens (1, 2). This cytokine activates cells of the innate and adaptive immune system, coordinates apoptosis, initiates expression of acute phase proteins, and changes metabolism toward proteolysis/lipolysis (5, 6, 15, 16). Severe human diseases such as sepsis are accompanied by an overexpression of TNF, conversely associated with hyporeactivity toward this cytokine (2, 3). One of the mechanisms that modulate TNF-induced signaling and subsequent gene expression is TNF tolerance (14).

The present study enlightens molecular mechanisms underlying inhibition of NF-{kappa}B target gene expression observed during TNF tolerance. Monocytic cells were made tolerant toward TNF by long-term preincubation with low-dose TNF. Under these conditions, a {kappa}B-binding motive was shown to represent the minimal DNA requirement to translate the cellular conditions of TNF tolerance to negative transcription. This is consistent with other experiments that found that besides IL-8, several other NF-{kappa}B-dependent genes such as tissue factor, IL-1, or ICAM-1 are also down-regulated in TNF-tolerant cells or under similar conditions (Fig. 1 and data not shown) (13, 14). By use of an IL-8 promoter-dependent reporter containing a mutated C/EBPbeta site, it could also be shown that binding of C/EBPbeta to its cognate DNA site was not necessary for transcriptional inhibition under conditions of TNF tolerance similar as described for the IE1/2 enhancer/promoter (37). It has been demonstrated earlier that neither I{kappa}B proteolysis nor nuclear translocation of NF-{kappa}B and NF-{kappa}B DNA-binding activity was affected in long-term TNF-pretreated monocytic cells (14). This suggests that regulatory mechanisms are involved in this condition that concern the transcriptional activity of the NF-{kappa}B dimer.

It is well known that TNF stimulation induces the phosphorylation of the NF-{kappa}B subunit p65, which is suggested to increase the transactivation potential of p65 (15, 23, 25). Similar to this, in our study, stimulation with high-dose TNF strongly increased p65 phosphorylation in nontolerant cells. However, almost no phosphorylation of p65 was found when we restimulated under the condition of TNF tolerance. These data imply that the reduced transcriptional activity of {kappa}B-dependent promoters under TNF tolerance is due to inhibition of p65 phosphorylation, which may modulate the interaction of this NF-{kappa}B subunit with the coactivator complex (13, 25). In addition, we found that the phosphorylation of Ser536 within the TAD region of p65 was specifically inhibited in TNF-tolerant cells. Ser536 is phosphorylated by the IKKs after TNF stimulation, which leads to an increased {kappa}B-mediated transcription (25, 26). Interestingly, when endogenous p65 out of tolerant cells was used as a substrate for IKKbeta kinase assays, the phosphorylation of p65 was decreased compared with p65 from nontolerant cells (data not shown). This implies that decreased p65 phosphorylation after TNF preincubation is due to a component associated with p65 under these conditions that inhibits interaction between IKKbeta and p65.

In our previous studies and in the experiments described in this work, we found that C/EBPbeta associates with p65-containing complexes in TNF-tolerant cells (14). It has been shown earlier that C/EBPbeta is able to directly associate via the bZIP region with the Rel homology domain and the TAD of p65 (29, 33, 40). It is also known that C/EBPbeta is phosphorylated by a MAPK pathway following TNF stimulation (29, 31). Furthermore, it has been demonstrated that the phosphorylation of a MAPK site adjacent to the bZIP region coincides with a conformational change of C/EBPbeta, allowing direct interaction with distinct mediator complexes determining differential gene activation (41). Therefore, it is likely that preincubation with TNF during tolerance induction initiates similar posttranslational modifications of C/EBPbeta, which may lead to a conformational change and demask the bZIP heterodimerization domain, thereby enabling de novo complex formation of C/EBPbeta with p65 (29, 32, 33). To further evaluate the relevance of this C/EBPbeta-p65 interaction, we used C/EBPbeta–/– macrophage cells (38). Most importantly, in C/EBPbeta–/– cells, no inhibition of NF-{kappa}B-dependent transcription under conditions of TNF tolerance was measured, whereas tolerance was found both in C/EBPbeta wt and C/EBPbeta–/– cells in which C/EBPbeta was retransfected. This proves that the C/EBPbeta protein is an essential component of the signaling machinery involved in TNF tolerance. When the level of phospho-p65 was monitored in C/EBPbeta wt cells, the phosphorylation of this NF-{kappa}B subunit was blocked after preincubation with TNF and restimulation. In contrast, a significant level of p65 phosphorylation was observed under the same tolerance conditions in C/EBPbeta–/– cells. This points out that the absence or presence of C/EBPbeta protein has immediate consequences for p65 phosphorylation.

This was examined in the next step, when we demonstrated that the association of C/EBPbeta with p65 resulted in a markedly reduced phosphorylation of p65, and we also showed that under this condition the phosphorylation of Ser536 within the TAD-1 was blocked. The binding of C/EBPbeta to p65 may mechanically modulate the interaction between this NF-{kappa}B subunit and upstream kinases such as IKK{alpha} and IKKbeta or CKII by masking and therefore blocking specific phosphorylation sites (26, 27). In this context, it is interesting to note that C/EBPbeta is able to form inhibitory boxes (42). Alternatively, additional proteins may be recruited by C/EBPbeta, which negatively interfere with upstream kinase systems. At this point, it should be mentioned that overexpression of C/EBPbeta did not directly affect IKK activity (data not shown). The reduction of p65 phosphorylation in tolerant cells does not alter DNA binding (14), but may rather affect transactivation events, potentially by preventing the conformational changes required for the accessibility of this NF-{kappa}B subunit to the coactivator complex (25, 43). Using a Gal-p65/Gal4-luciferase transcriptional system (39), it could be demonstrated in the present study that p65 transactivation is decreased following C/EBPbeta overexpression. These findings are consistent with the observation that C/EBPbeta overexpression can inhibit effects of TNF stimulation (14). In TNF-preconditioned cells, a lowered phosphorylation of p65 resulted in reduced association with the coactivator p300 together with a decrease in transcription of ICAM-1 (13). In summary, the present study suggests that the association of C/EBPbeta with p65 in tolerant cells results in a blockade of phosphorylation of this NF-{kappa}B subunit, leading to a lowered transcriptional efficiency (Fig. 7).


Figure 7
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  		FIGURE 7. Blockade of NF-{kappa}B p65 phosphorylation by C/EBPbeta under the condition of TNF tolerance. In naive cells, stimulation with TNF induces the activation of the IKK complex, which leads to phosphorylation and degradation of I{kappa}B, releasing the NF-{kappa}B dimer. In addition, the IKK complex together with other kinases phosphorylates the NF-{kappa}B subunit p65, which increases the transactivation potential of this protein. In contrast, in tolerant cells, C/EBPbeta associates with p65, which prevents TNF-induced phosphorylation of p65. This interaction inhibits NF-{kappa}B-mediated transactivation, resulting in reduced gene transcription. The relevant literature is stated in the text.

 
TNF is a master cytokine required to orchestrate the proper immune response (1, 2, 6, 15, 16). What then is the physiological or pathological consequence of TNF tolerance? On the one hand, TNF tolerance may belong to a protective program intended to attenuate the excessive effects of this potent cytokine, thereby helping to control hyperinflammation in the body (3). Similar independent concepts have been described such as anti-TNF immunity, also termed beneficial autoimmunity, to restrain self-destructive immunity (44). In contrast, "too much" TNF tolerance may contribute to a state of anergy of the cell, a condition as seen in immunoparalysis (3). Therefore, sepsis, which is an acute systemic inflammatory process showing hyperinflammation as well as immunoparalysis (3, 45), sometimes even as overlapping phases, may represent a paradigmatical disease in which TNF tolerance may play a fundamental role. In addition, this mechanism may also have relevance to chronic inflammation or malignant processes (4, 5). The response to TNF seems to be a tightrope walk, and it is important to understand the mechanisms for TNF tolerance. This may open up new avenues for diagnostic and therapeutic strategies to treat acute inflammation, e.g., a better understanding of the underlying principles behind hyperinflammation vs immunoparalysis in sepsis would enable specific therapy (46).


   Acknowledgments
 
We thank Dr. Tsugiya Murayama for the pXP2-IL-8-Luc wt, pXP2-IL-8-Luc ({kappa}B mut), and pXP2-IL-8-Luc (C/EBPbeta mut) IL-8 promoter constructs, and Dr. Nigel Mackman for the pGL2-IL-8-Luc IL-8 promoter construct. The C/EBPbeta or p65 expression plasmids are from Dr. Richard Pope or Dr. Patrick Baeuerle, respectively, to whom we are very grateful. Gal-p65 and Gal4-Luc constructs were a gift from Dr. Albert S. Baldwin. The Gal-VP-16 expression plasmid as well as the HA-p65 536wt and 536mut overexpression plasmids were derived from Dr. M. Lienhard Schmitz, to whom we are also very thankful. We also thank Dr. Sharon Page for valuable contributions, and Martina Krautkrämer and Christine Grubmüller for excellent technical support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Stiftung für Pathobiochemie und Molekulare Diagnostik, the Wilhelm Sander-Stiftung, the Deutsche Forschungsgemeinschaft (Zi 288/2-3), and the Medical Faculty of the Technische Universität München. Back

2 A.Z. and M.Q. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Korbinian Brand, Institute of Clinical Chemistry and Pathobiochemistry, Technische Universität München, Klinikum Rechts der Isar, Ismaninger Strasse 22, D-81675 München, Germany. E-mail address: brand{at}klinchem.med.tum.de Back

4 Abbreviations used in this paper: IKK, I{kappa}B kinase; IP, immunoprecipitation; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; RLA, relative luciferase activity; bZIP, basic leucine zipper; TAD, transactivation domain; trunc, truncated; wt, wild type. Back

Received for publication August 31, 2005. Accepted for publication April 14, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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