ENHANCER BINDING PROTEIN IS INVOLVED IN THE EXPRESSION OF THE TUMOUR NECROSIS FACTOR GENE IN HUMAN MONOCYTES

CCAAT/ENHANCER BINDING PROTEIN IS INVOLVED IN THE EXPRESSION OF THE TUMOUR NECROSIS FACTOR GENE IN HUMAN MONOCYTES Angela Wedel,

Gabi Sulski, H. W. Löms Ziegler-Heitbrock

Within the human TNF promoter we have identified two sites at positions 2189 and 2101 that show C/EBP specific binding of nuclear proteins from cells of the human monocytic line Mono Mac 6. Supershift analysis with anti C/EBP antibodies revealed that the complexes formed consist of both C/EBPα and C/EBPβ. When studying reporter constructs with a 59deletion series of the TNF promoter in cotransfection experiments with a C/EBPβ expression plasmid, a construct with the 21064 TNF fragment gave 26-fold transactivation, the 2630 fragment showed 23-fold transactivation and the 2107 fragment (containing the 2101 C/EBP binding motif) still gave 16-fold transactivation. Mutagenesis of the 2101 site in the 2630 construct resulted in a reduction of C/EBP driven transactivation from 26-fold to 7-fold. Finally, when Mono Mac 6 cells were transfected with these constructs, stimulation by LPS induced a 19-fold transactivation in the 2630 wild type construct, while the 2630 construct carrying the 2101 mutation was transactivated only 4-fold. Hence, the data indicate that the 2101 C/EBP motif is crucial for TNF gene expression in human monocytes. © 1996 Academic Press Limited

While originally discovered based on its anti-tumour activity, TNF has turned out to be a major proinflammatory cytokine that activates a broad array of cells including leukocytes and endothelial cells.1–4 The main producer cells of TNF are the leukocytes, and among these the monocytes/macrophages are the major source of this cytokine. Expression of TNF in these cells is rapidly induced by cytokines and by LPS from Gram negative bacteria.5 LPS binds to the CD14 cell surface receptor6 followed by rapid mobilization of the transcription factor NF-κB.7 It has been directly demonstrated in the murine system, and indirectly in the human system, that members of the NF-κB/Rel family are important for TNF gene expression in monocytes/macrophages.8–10 Another major family of transcription factors present in leukocytes is the family of the C/EBP proteins that are predominantly found in B cells and in monocytes/macrophages (for review see Refs 11, 12). These proteins form homo- and heterodimers through their leucine zipper region,13 and they bind

From the Institute for Immunology, University of Munich, Goethestr. 31, 80336 München, Germany Correspondence to: H. W. L. Ziegler-Heitbrock Received 11 September 1995; accepted for publication 25 November 1995 © 1996 Academic Press Limited 1043-4666/96/05033517 $18.00/0 KEY WORDS: C/EBP/monocytes/TNF/LPS/transactivation CYTOKINE, Vol. 8, No. 5 (May), 1996: pp 335–341

to DNA sequences with the consensus T(T/G)NNGNAA(T/G).14 A role for C/EBP in cytokine gene expression has been shown, for instance, for IL-6 and IL-8.15,16 Also for the TNF gene a role for C/EBP was suggested17 but the binding motif and its exact role has not been defined. We now show that the 2101 motif TGGAGAAAC specifically binds C/EBP and that mutagenesis of this motif blunts LPS-induced gene expression in human monocytes.

RESULTS The human TNF promoter contains two C/EBP binding motifs In a search for C/EBP binding sites, which are consistent with the C/EBP consensus T (T/G) NNG NAA (T/G), we identified four potential motifs at 2438, 2189, 2124, 2101 on the non-coding strand (Table 1). We then studied binding of nuclear extracts from Mono Mac 6 cells to these four motifs in the human TNF promoter. Among these motifs only those at positions 2101 and 2189 gave binding with such extracts (Fig. 1). There was no difference in binding activity for extracts from unstimulated and LPS stimulated Mono Mac 6 cells. To further substantiate the binding properties of the four motifs, we performed competition studies with a prototypic, radiolabelled C/EBP motif from the 23.5 335

336 / Wedel et al.

CYTOKINE, Vol. 8, No. 5 (May 1996: 335–341)

TABLE 1.

Sequence motifs used for gel shift analysis

C/EBP consensus: C/EBP prototype*: 2438 TNF: 2189 TNF: 2124 TNF: 2101 TNF: 2101 mTNF:

59 – TTNN GNAAT – 39 G G 59-caTGGGAAGA TTGAGCAAT CTAAGAGact-39 59-tcgaGGGGC TTCA GAAAG CTGAGTcga-39 59-agcttGGGA TTTG GAAAG TTACAgatc-39 59-agctTCATC TGGAGGAAG CGGTAGatc-39 59-tcgaCTTGG TGGAGAAAC CCATGgatc-39 59-tcgaCTTGG TGGATAATC TTATGgatc-39

SP1#:

59-tcgACTGGGCGGAGTTAGGGGCGGATGaagct-39

Genomic sequences are written in capital letters. *Taken from the 23.5 kb enhancer of the mouse albumin gene.18 #Taken from the SV40 early promoter.19

kb enhancer of the mouse albumin gene with the four motifs from the TNF promoter as competitor. Figure 2 illustrates that competition was achieved only with the 2189 and 2101 motifs, while the 2438 and 2124 sites were inactive. For definition of the types of C/EBP proteins, that are present in Mono Mac 6 cells and bind to the TNF promoter, supershift analyses were performed. In these studies anti C/EBPδ had no effect on mobility in the gelshift with the 2101 motif but anti C/EBPα eliminated part of the binding proteins i.e. the upper band (Fig. 3). Furthermore, anti C/EBPβ removed more of the lower, high mobility band. This indicates that the upper band preferentially contains C/EBPα and the lower band preferentially C/EBβ. A mixture of anti

C/EBPα and β eliminated almost the entire binding activity. Similar data were obtained when using the 2189 motif (data not shown). Hence, these results suggest that the human TNF promotor contains two sites at positions 2101 and 2189, which bind C/EBP proteins present in human monocytic cells.

Figure 1. Binding of nuclear extracts to potential C/EBP motifs in the human TNF promoter.

Figure 2. Competition analysis with potential C/EBP motifs in the human TNF promoter.

Nuclear extracts from Mono Mac 6 cells, incubated for 1 h with or without LPS, were admixed with radiolabelled oligonucleotides encompassing four potential C/EBP motifs. One of three experiments.

Nuclear extracts from Mono Mac 6 cells were admixed with increasing amounts of unlabelled competitor oligonucleotides. To this the radiolabelled C/EBP motif from the mouse albumin gene was added. One of three experiments.

Transactivation of the human TNF promoter by C/EBP In an approach to elucidate the possible role of these C/EBPs in TNF gene expression, we cotransfected K562 cells with a deletion series of TNF promoter-luciferase reportergene constructs together with a C/EBPβ expression plasmid or an empty expression plasmid. Figure 4 demonstrates that the 21064 TNF promoter reporter construct was transactivated 26-fold by C/EBPβ, and that even in the shortest construct (pX-107)—which still contains the 2101 C/EBP bind-

C/EBP in TNF expression / 337

motif by replacing the central G and the last A by a T each (cf. Table 1). As shown in Figure 5, the mutation abrogated binding of the C/EBP proteins, since the mutated motif was unable to compete for binding of C/EBP proteins from Mono Mac 6 to the wildtype motif. Transfection of the 2630 construct carrying this mutation in the 2101 motif together with the C/EBPβ expression plasmid in K562 gave only a very low level of transactivation (7-fold) compared to the wildtype reporter plasmid (26-fold) (Fig. 6). Hence, it appears that among the two binding sites for C/EBP, the 2101 site is of crucial importance, when using an expression plasmid driven C/EBPβ transcription factor. C/EBP is involved in LPS stimulated TNF expression in human monocytic cells

Figure 3. Super shift analysis of the nuclear proteins binding to the 2101 C/EBP motif from the human TNF promoter. Nuclear extracts from Mono Mac 6 were incubated with radiolabelled 2101 C/EBP motif followed by incubation with different antisera for 30 min at 4°C. One of three experiments.

ing site—transactivation was still 16-fold. By comparison, the pT81·luc plasmid carrying the minimal promoter of the thymidinekinase gene was transactivated only 3-fold. Mutagenesis of the 2101 C/EBP motif Since the shortest deletion construct pX-107, containing the 2101 site was still functionally active in the cotransfection studies (Fig. 4), we mutagenized this

Figure 4.

Finally, we tested the involvement of C/EBP proteins in the LPS-induced transactivation of the TNF gene. For this purpose, Mono Mac 6 cells were transfected with the pX-630 or the pX-630 (2101 m) construct, and cells were then stimulated with LPS. The results show that LPS induces a 19-fold transactivation of the wildtype construct, while the mutated reporter construct gave only a 4-fold increase in luciferase activity (Fig. 7). These data show that the 2101 motif is crucially involved in LPS-induced TNF gene expression in human monocytes.

DISCUSSION The C/EBP molecules form a family of transcription factors of growing importance. These molecules are composed of three modules which are the c-terminal leucine-zipper, the basic DNA binding domain and the transactivating region (for review see Ref. 12). The activity of C/EBP homo- or heterodimers appears to be

Transactivation of the human TNF promoter by recombinant C/EBP.

K562 cells were cotransfected with TNF promoter luciferase reporter constructs and C/EBP expression plasmid or control plasmid. After overnight culture luciferase activity was determined in a luminometer. Given is the average of fold induction by C/EBP expression plasmid compared to empty plasmid 6 SD of four experiments. Difference between pX-107 and pT81·luc is significant at P , 0.05.

338 / Wedel et al.

CYTOKINE, Vol. 8, No. 5 (May 1996: 335–341)

Figure 5. Competition analysis with wild type and mutated 2101 C/EBP motif from the human TNF promoter. Nuclear extracts were admixed with increasing amounts of unlabelled wildtype or unlabelled mutated 2101 C/EBP motif (2101 m). To this radiolabelled 2101 motif was added. One of four experiments.

Figure 6.

regulated by phosphorylation at different sites.20 Within the transactivating region phosporylation of a negative element will result in derepression and enhanced transactivation.21 C/EBP proteins can transactivate a variety of genes including cytokines (for review see Ref. 12). Also for the human TNF gene a role was suggested based on the presence of a sequence at 2189 which fits the consensus motif.14 In addition Pope et al.17 have shown that C/EBP expression can transactivate a reporter plasmid directed by the human TNF promoter, but the site of C/EBP binding was not defined. We have performed a search of the 59 region up to 21064 of the human TNF gene, and we identified four potential sites that might be recognized by C/EBP. Of these, only two proved to be valid sites as evidenced by direct binding and by competition analysis. In the gel shift analyses two complexes of different mobility were shown to bind to these sites. Supershift revealed that anti C/EBPα primarily removed the upper, low mobility complex, while anti C/EBPβ treatment resulted in disappearance of the high mobility complex. These results suggest that the upper complex consists of C/EBPα homodimers and the lower of C/EBPβ homodimers, while there is no direct evidence for C/EBP α-β heterodimers.

Transactivation of the wildtype and mutated TNF promoter by recombinant C/EBP.

K562 cells were cotransfected with C/EBP expression plasmid or empty plasmid and either wild type or mutated TNF promoter luciferase reporter construct. The mutation introduced eliminates C/EBP binding at the 2101 site as demonstrated in Fig. 5. After overnight culture luciferase activity was determined in a luminometer. Given is the average of fold induction by C/EBP expression plasmid compared to empty plasmid 6 SD of three experiments.

Figure 7. Effect of mutation of the 2101 C/EBP motif on LPS-induced transactivation of the human TNF promoter. Mono Mac 6 cells were transfected by DEAE Dextran with wildtype and mutated promoter reporter gene constructs as indicated, and after overnight culture cells were stimulated with or without LPS for 4 h. Luciferase activity of cell lysates was determined in a luminometer. Given is average 6 SD from five experiments.

C/EBP in TNF expression / 339

It is very possible that the C/EBP complexes bound to the human TNF promoter are more complex and contain additional transcription factors. Along these lines, a specific interaction has been demonstrated for C/EBP and the transcription factor NF-κB.16,22–24 In our studies, supershift analysis with antibodies directed against NF-κB p50, p65, c-rel and p52 did, however, not affect the proteins binding to the 2101 site (data not shown) suggesting that NF-κB/Rel-proteins are not part of this complex. Cotransfection of a 59-deletion series of the TNF promoter luciferase constructs with a C/EBP β expression plasmid demonstrated still efficient transactivation of the 2107 construct. This suggests that the 2101 C/EBP motif is sufficient for C/EBP driven transactivation. In fact, mutagenesis of the 2101 site within a much larger context (2630) essentially abolished C/EBP driven transactivation. Furthermore, LPS-stimulated transactivation of the 2630 construct was similarly ablated by mutation of the 2101 site. These results demonstrate that the 2101 C/EBP site is instrumental for the expression of the human TNF gene. While LPS appears to affect TNF gene expression by an effect that involves C/EBP, we found no evidence of LPS increasing the binding of C/EBP proteins in monocyte nuclear extracts to the 2101 or 2189 motif. Still, LPS might increase the transactivation potential of C/EBP by inducing phosphorylation of this transcription factor. In studies with a C/EBP specific reportergene transfected Mono Mac 6 cells we did not, however, find any evidence for LPS-induced transactivation (data not shown). Alternately, LPS may act through other transcription factors that bind to a separate site and that interact with C/EBP. The transcription factor NF-κB is one such candidate, which is efficiently mobilized by LPS.7,25 The most prominent NF-κB binding site is at 2605 in the human TNF promoter.10 In fact, LPS-induced transactivation requires further upstream elements, since the 2107 construct cannot be efficiently stimulated by LPS (data not shown). There are some additional transcription factors that have been implicated in the regulation of the human TNF gene. In T cells, transcription factors ets and NFAT have been implicated in TNF gene expression.26–28 In monocytes, a contribution by EGR-1 has been discussed29 and evidence from several studies indicates that c-jun is involved in controlling expression of TNF in these cells.30–32 These factors may also interact with the C/EBP to control tissue specific TNF gene expression in human monocytes and macrophages. Taken together, the exact mode of action of LPS in C/EBP-mediated transactivation of the human TNF promoter is still unclear. Nevertheless, as has been demonstrated herein, it is evident that C/EBP proteins

are instrumental in controlling expression at the TNF gene in human monocytes.

MATERIALS AND METHODS Cell lines The haemapoietic stem cell line K56233 provided by B. Read (Kensington, MD) was cultured in RPMI 1640 medium with 10% FCS (fetal calf serum). The monocytic cell line Mono Mac 6 was established in this laboratory34 and was maintained in LPS-free RPMI 1640 with 10% FCS and supplements as given.

Reporter gene analysis Mono Mac 6 cells were transfected with luciferase reporter constructs (5 µg of plasmid/107 cells) according to Shakov et al.8 using DEAE-dextran (62.5–125 µg/ml). The cells were cultured for 24 h (1.25 3 106 cells/ml); after 20 h half of the cells were stimulated with LPS (1 µg/ml) for 4 h. Cell lysates were prepared as described,35 and the protein concentration was determined according to the method of Bradford (Biorad, Munich, Germany). The luciferase activity was measured in a luminometer (model LB9501 from Berthold, Wildbad, Germany) using luciferase reagent (E1501 from Promega, Madison, USA). Constructs employed contained a 59-deletion series of the TNF 59- and promoter region which was cloned into the pXP2 luciferase plasmid (kindly provided by S. Nordeen, Denver, CO, USA). The TNF fragments inserted are Pstl/Hgal- for pX-1064, EcoRI/Hgal- for pX-630 and Sacl/Hgal fragment for pX-107. These were derived from a plasmid clone kindly provided by E. Weiβ (Munich, Germany).36 pT81·luc (kindly provided by S. Nordeen, Denver, CO, USA), which was used as reference plasmid, contains the minimal promoter of the HSV thymidinekinase gene.37 For construction of the mutant pX-630(2101 m) the Sacl/Styl fragment of pX-630 (59-GAGCT9CATGGGTTTCTCCAC9CAAGG-39) was substituted with a chemically synthezised mutated Sacl/Styl-fragment (59-GAGCT9CATAAGATTATCCAC9CAAGG-39). As shown in Table 1 the base exchanges affect the central G and the second A of a potential C/EBP binding site with the antisense strand fitting the consensus sequence (T(T/G)NNGNAA(T/G)). Co-transfection of K562 cells was performed by electroporation with a single pulse at 260 V and 960 µF using 0.4 cm cuvettes and the Gene Pulser from Biorad (Munich, Germany). 5 3 106 cells (in 800 µl HBS 5 HEPES-buffered saline) were cotransfected with 50 µg of reporter plasmid and 50 µg of either the expression plasmid pMSV·C/EBPβ or of the empty reference plasmid pMSV. After 10 min of rest at 4°C the cells were cultured at 5 3 105 cells/ml for 24 h. Cell lysates were prepared and luciferase activity determined as described above. pMSV·C/EBPβ (kindly provided by S. L. McKnight, Tularik Inc., South San Francisco CA, USA) directed expression of the mouse C/EBPβ gene under the control of the MSV LTR; p·MSV was constructed by deleting the C/EBP gene.

340 / Wedel et al.

Gelshift and -supershift analysis Nuclear extracts from untreated cells or from cells stimulated with LPS (1 µg/ml) for 1 h were prepared according to Dignam et al.38 The protein concentration was determined as described above, and 3–6 µg of protein was incubated with 30 000–50 000 cpm of the given double stranded oligonucleotide, which had been labelled by fill-in reaction with (α32P) dATP (Amersham, Braunschweig, Germany). The binding reaction was performed for 20 min at 21°C in the presence of 0.5 µg of poly(dl/dC) in 10 mM TrisHCl, pH7.5; 50 mM KCl; 5 mM MgCl2; 1 mM EDTA; 1 mM DTT; 1 µg/µl BSA; 20% glycerol; 0.5% aprotinin (Sigma, Munich, Germany) and 0.1 mM PMSF. The complexes were separated by gel electrophoresis on a 4% polyacrylamide gel and run with 1 3 TBE (Tris-buffer containing borate and EDTA). For competition studies the competitor oligonucleotide was added together with the labelled oligonucleotide to the binding reaction. The sequences of the oligonucleotides used are given in Table 1 with one strand of the filled-in double stranded oligonucleotide. The C/EBP oligonucleotide used as a prototype encompasses the palindromic C/EBP binding-site of the mouse serum albumin gene. The oligonucleotides 2438, 2189, 2124 and 2101 TNF contain potential C/EBP-sites of the human TNF gene, which are localized to the antisense strand. The oligonucleotide 2101 m TNF contains the same mutations as the reporter construct pX-630 (2101 m) affecting the potential C/EBP binding site (see above). The SP1 oligonucleotide, containing two SP1-binding sites of SV40, was used as unrelated competitor. For supershift analysis, 1 or 2 µl of different polyclonal antibodies (from Santa Cruz Biotechnology, CA, USA: antiC/EBPα (sc-61X), anti-C/EBPβ (sc-150X) or anti C/EBPδ (sc-151X)) or control pre-immune rabbit serum were added after the binding reaction, and the samples were incubated another 30 min at 4°C.

Acknowledgement This work was supported by a grant from Deutsche Forschungsgemeinschaft and by the Fritz Thyssen Stiftung. The authors thank A. Leutz, Berlin, for the helpful discussion.

REFERENCES 1. Old LJ (1985) Tumor necrosis factor (TNF). Science 230:630–632. 2. Beutler B, Cerami A (1987) Cachectin: more than a tumor necrosis factor. The New England Journal of Medicine 316:379–385. 3. Lee SH, Crocker PR, Westaby S, Key N, Mason DY, Gordon S, Weatherall DJ (1988) Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters. J Exp Med 168:1193–1198. 4. Jäättelä M (1991) Biology of disease. Biologic activities and mechanisms of action of tumor necrosis factor-alpha/cachectin. Lab Invest 64:724–742. 5. Ziegler-Heitbrock HWL (1989) The biology of the monocyte system. European Journal of Cell Biology 49:1–12.

CYTOKINE, Vol. 8, No. 5 (May 1996: 335–341) 6. Wright SD, Ramos RA, Robias PS, Ulevitch RJ, Mathison JC (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249:1431–1433. 7. Müller JM, Ziegler-Heitbrock HWL, Bäuerle P (1993) Nuclear factor kappa B, a mediator of lipopolysaccharide effects. Immunobiol 187:233–256. 8. Shakov AN, Collart MA, Vassalli P, Nedospasov SA, Jongeneel CV (1990) κB-type enhancers are involved in LPSmediated transcriptional activation of the TNF-α gene in primary macrophages. J Exp Med 171:35–47. 9. Drouet C, Shakhov AN, Jongeneel CV (1991) Enhancers and transcription factors controlling the inducibility of the TNF-α promoter in primary macrophages. J Immunol 147:1694–1700. 10. Ziegler-Heitbrock HWL, Sternsdorf T, Liese J, Belohradsky B, Weber C, Wedel A, Schreck R, Bäuerle P, Ströbel M (1993) Pyrrolidine dithiocarbamate inhibits NF-κB mobilization and TNF production in human monocytes. J Immunol 151:6986–6993. 11. Hurst HC (1994) Transcription factors 1: bZlP proteins. Protein Profile 1:123–168. 12. Wedel A, Ziegler-Heitbrock HWL (1995) The C/EBP family of transcription factors. Immunobiology 193:171–185. 13. Landschulz WH, Johnson PF, McKnight SL (1988) The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759–1764. 14. Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T (1990) A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. Embo J 9:1897–1906. 15. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S (1993) Transcription factors NFIL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 90:10193–10197. 16. Stein B, Cogswell PC, Baldwin A (1993) Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZlP interaction. Mol Cell Biol 13:3964–3974. 17. Pope RM, Leutz A, Ness SA (1994) C/EBP beta regulation of the tumor necrosis factor alpha gene. J Clin Invest 94:1449–1455. 18. Liu J-K, Bergman Y, Zaret KS (1988) The mouse albumin promoter and a distal upstream site are simultaneously DNAse-1 hypersensitive in liver chromatin and bind similar liver-abundant factors in vitro. Genes Dev 2:528–541. 19. Dynan WS, Tjian R (1983) The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell 35:79–87. 20. Trautwein C, Caelles C, van der Geer P, Hunter T, Karin M, Chojkier M (1993) Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain. Nat 364:544–547. 21. Kowenz-Leutz E, Twamley G, Ansieau S, Leutz A (1994) Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression. Genes Dev 8:2781–2791. 22. LeClair KP, Blanar MA, Sharp PA (1992) The p50 subunit of NF-kappa B associates with the NF-IL 6 transcription factor. Proc Natl Acad Sci USA 89:8145–8149. 23. Merola M, Blanchard B, Tovey MG (1995) submitted. 24. Ray A, Hannink M, Ray BK (1995) Concerted participation of NF-κB and C/EBP heteromer in lipopolysaccharide induction of serum amyloid a gene expression in liver. The Journal of Biological Chemistry 270:7365–7374. 25. Haas JG, Bäuerle PA, Riethmüller G, Ziegler-Heitbrock HWL (1990) Molecular mechanisms in down-regulation of tumor necrosis factor expression. Proc Natl Acad Sci USA 87:9563–9567. 26. Krämer B, Wiegmann K, Krönke M (1995) Regulation of the human TNF promoter by the transcription factor Ets. The Journal of Biological Chemistry 270:6577–6583. 27. McCaffrey PG, Goldfeld AE, Rao A (1994) The role of NFATp in cyclosporin A-sensitive tumor necrosis factor-α gene transcription. The Journal of Biological Chemistry 269:30445–30450.

C/EBP in TNF expression / 341 28. Goldfeld AE, Tsai E, Kincaid R, Belshaw PJ, Schrieber SL, Strominger JL, Rao A (1994) Calcineurin mediates human tumor necrosis factor α gene induction in stimulated T and B cells. J Exp Med 180:763–768. 29. Krämer B, Meichle A, Hensel G, Charnay P, Krönke M (1994) Characterization of an Krox-24/Egr-1-responsive element in the human tumor necrosis factor promoter. Biochem Biophys Acta 1219:413–421. 30. Economou JS, Rhoades K, Essner R, McBride WH, Gasson JC, Morton DL (1989) Genetic analysis of the human tumor necrosis factor α/cachectin promoter region in a macrophage cell line. J Exp Med 170:321–326. 31. Leitman DC, Ribeiro RCJ, Mackow ER, Baxter JD, West BL (1991) Identification of a tumor necrosis factor-responsive element in the tumor necrosis factor α gene. The Journal of Biological Chemistry 266:9343–9346. 32. Rhoades KL, Golub SH, Economou JS (1992) The regulation of the human tumor necrosis factor α promoter region in macrophage, T cell, and B cell lines. The Journal of Biological Chemistry 267:22102–22107. 33. Lozzio CB, Lozzio BB, Yang W-K, Ichiki AT, Bamberger

EG (1976) Absence of thymus-derived lymphocyte markers in myelogenous leukemia (PH1) cell line K-562. Cancer Res 36:4657. 34. Ziegler-Heitbrock HWL, Thiel E, Fütterer A, Herzog V, Wirtz A, Riethmüller G (1988) Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. Int J Cancer 41:456–461. 35. DeWet JR, Wood KV, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737. 36. Messer G, Spengler U, Jung MC, Honold G, Blömer K, Pape GR, Riethmüller G, Weiss EH (1991) Polymorphic structure of the tumor necrosis factor (TNF) Locus: An Ncol polymorphism in the first intron of the human TNF-β gene correlates with a variant amino acid in position 26 and a reduced level of TNF-β production. J Exp Med 173:209–219. 37. Nordeen SK (1988) Luciferase reporter gene vectors for analysis of promoters and enhancers. Bio Techniques 6:454–457. 38. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489.