Regulation of P100 (NFKB2)

Regulation of p100 (NFKB2) expression in human monocytes in response to inﬂammatory mediators and lymphokines H de Wit1, WHA Dokter2, SB Koopmans1, C Lummen1, M van der Leij1, JW Smit1 and E Vellenga1

1Department of Internal Medicine, Division of Hematology, and 2Biomedical Technology Center, University of Groningen, The Netherlands

The transcription factor NF-␬B plays an important role in the expression in human monocytes is regulated and how this cor- regulated expression of cytokines in human monocytes. A p100 relates with the well defined effects of up- and down-regu- subunit of NF-␬B has I␬B-like properties by sequestering the p65 transactivating subunit in the cytosol of cells. In transient lation of cytokine gene expression in monocytes. To study transfection assays we demonstrated that p100 has an inhibi- these aspects in more detail, we analyzed the regulation of tory effect on the NF-␬B-dependent IL-6 promoter activity. In p100 in response to lipopolysaccharide (LPS) and different view of this finding, we studied the regulation of the p100 sub- cytokines. unit in human monocytes in response to LPS, the inflammatory Transient transfection assays demonstrated that a p100 ␤ ␣ cytokines IL-1 and TNF- and lymphokines. The results dem- expression vector inhibits NF-␬B-driven IL-6 promoter onstrate that LPS, IL-1␤, and TNF-␣ induce p100 expression at mRNA and protein level while IFN-␥, IL-3 and IL-4/IL-10 have activity. In addition it was shown that p100 in monocytes is no effect. The induction of p100 expression was shown to be regulated at transcriptional and post-transcriptional levels in mediated by a two-fold increase in the p100 transcription rate response to LPS, IL-1 and TNF. In contrast, IL-3, IL-4/IL-10 and and a two-fold increase in p100 mRNA stability. Furthermore IFN-␥ failed to induce p100 expression. the p100 mediated upregulation was dependent on a tyrosine kinase dependent pathway rather than the protein kinase C pathway. NF-␬B is a complex of either p50 homodimers or a p50/p65 heterodimer. The latter is known to strongly autoregu- late p100 transcription. We therefore examined the composition Materials and methods of NF-␬B induced by LPS vs the different lymphokines. LPS- induced NF-␬B showed a distinct p65 supershift whereas the composition of NF-␬B induced by different lymphokines did not show a change in p65. We conclude that the p100 subunit of the Cell culture and isolation of monocytes transcription factor NF-␬B is induced by different inflammatory mediators while lymphokines fail to induce p100 expression which may be caused by the induction of NF-␬B predominantly Peripheral blood cells were obtained from volunteer platelet consisting of p50 homodimers. donors, and mononuclear cell suspensions were prepared by Keywords: p100; NFKB2; monocytes; inflammation; lymphokines Ficoll–Hypaque density-gradient centrifugation. T lympho- cytes were removed by E-rosetting with 2-aminoethyl-isothio- uronium-bromide-treated sheep red blood cells. Monocytes Introduction were further enriched by plastic adherence for 1 h at 37°C and demonstrated a purity Ͼ95% detected by FACS analysis The transcription factor nuclear factor-␬B (NF-␬B) is involved with an anti-CD-14 monoclonal antibody (Becton Dickinson, in the transcriptional regulation of several cytokine genes. Sunnyville, CA, USA) and staining. Monocytes were cultured More specific, the transcriptional regulation of genes encoding in RPMI-1640 medium containing 10% FBS and antibiotics. for interleukin-1 (IL-1), IL-6, IL-8, and granulocyte-colony sti- TF-1 cells were cultured in RPMI-1640 medium (Flow, Rock- mulating factor (G-CSF) in human monocytes strongly depend ville, MD, USA) containing 10-ng/ml IL-3 (Genetics Institute, on the activation of NF-␬B.1–7 This underscores the impor- Cambridge, MA, USA), 10% fetal bovine serum (FBS; tance of this transcription factor in hematopoiesis and Hyclone, Logan, UT, USA) and antibiotics. inflammation. NF-␬B is composed of different subunits bound Monocytes were stimulated with 1 ␮g/ml lipopolysaccha- to an inhibitor protein (I␬B) which dissociates upon activation ride (LPS) from Pseudomonas aeruginosa (Sigma, St Louis, finally leading to the nuclear translocation of NF-␬B.8–10 MO, USA), interferon-␥ (100 U/ml; Boehringer Mannheim, The subunits of NF-␬B are encoded by the genes NFKB1 Mannheim, Germany), tumor necrosis factor-␣ (500 U/ml; a (p105), NFKB2 (p100), RelA (p65), RelB and c-rel.11–18 gift from Dr GR Adolf, Ernst, Boehringer Ingelheim, Vienna, Recently it has been shown in T cell lines that p100 has a Austria), IL-1␤ (100 U/ml; Immunex; Seattle, WA, USA), IL- strong I␬B-like activity by sequestering the RelA subunit of NF- 3 (10 ng/ml; Genetics Institute, Cambridge, MA, USA), IL-4 ␬B in the cytosol of cells.19 Transfection experiments with (15 ng/ml; a gift from Dr P Trotta, Schering-Plough, Bloom- expression vectors for RelA and p100 confirmed these results field, NJ, USA), IL-10 (5 ng/ml; Schering-Plough, Kenilworth, by demonstrating that p100 inhibited the RelA-mediated tran- NJ, USA). Polyclonal anti-serum against IL-1␣, IL-1␤ (gifts scriptional activity of reporter genes.20,21 In view of these data from Dr S Gillis, Immunex, Seattle, WA, USA), and TNF-␣ (a it is conceivable that I␬B and p100 might have important gift from Dr GR Adolf) was used at a dilution of 1:1000. These functions in negative control mechanisms. dilutions completely neutralize the activity of IL-1 (200 U/ml) Limited information is available regarding how the p100 and TNF (200 U/ml) tested in in vitro culture assays. Okadaic acid (Sigma), cycloheximide (Sigma), actinomycin-D (Boehringer Mannheim), genistein (Sigma), chelerithrin (Sigma) and staurosporin (Sigma) were used at concentrations Correspondence: E Vellenga, Department of Internal Medicine, ␮ ␮ ␮ ␮ Division of Hematology, University Hospital, Hanzeplein 1, 9713 GZ of 30 ng/ml, 10 g/ml, 10 g/ml, 40 M,1 M and 20 mM, Groningen, The Netherlands; Fax: 31 50 3614862 respectively. The viability of monocytes after stimulation was Received 11 June 1997; accepted 11 November 1997 Ͼ99% in all experiments. P100 (NFKB2) regulation in human monocytes H de Wit et al 364 Expression plasmids and transfection procedures Run on transcription assay

A p100 expression vector was constructed by cloning a 2.9 kb The transcription rate of the NFKB2 gene was studied as HindIII–XbaI p100 cDNA (a gift from Dr G Nabel, Ann Arbor, described previously.23 In short, nuclei of 5 × 107 cells were MI, USA) containing the entire p100 coding sequence into isolated and resuspended in transcription buffer containing ␬ pcDNA3 (InVitroGen, San Diego, CA, USA). An I B 32P-UTP (3000 Ci/mmol; Amersham) and incubated at 26°C expression vector was kindly provided by Dr A Baldwin for 20 min. Transcription was terminated and nuclear RNA ␬ (Chapel Hill, NC, USA). An NF- B-driven IL-6 promoter was isolated and hybridized to linearized pGEM and to lin- luciferase reporter plasmid was a gift from Dr S Akira earized plasmids containing the cDNA inserts for the p100, IL- (Osaka, Japan). 1 and GAPDH genes. Hybridization, washing, exposure and ␬ TF-1 cells were co-transfected with the NF- B driven quantification of transcription rates was performed as luciferase reporter plasmid in the absence and presence of described above. pcDNA3, a p100 expression vector, and an I␬B expression vector. Transfection was performed by electroporation at 240 V and 960 ␮F and subsequently cells were cultured in RPMI-1640 medium plus 10% FBS in the absence or presence Electrophoretic mobility shift assay (EMSA) of Okadaic acid or IL-1. After 24 h luciferase activity in cell extracts was measured using a luminometer. Monocytes were stimulated and nuclear extracts were prepared according to the mini-scale procedure as described.23,24 Nuclear extracts were divided in small aliquots and stored RNA preparation and Northern blot analysis at −80°C. A double-stranded synthetic oligonucleotide probe contain- Total cellular RNA was isolated using the guanidium ing the NF-␬B(5′-AGCTGCGGGGATTTTCCCTG-3′) was used isothiocyanate/cesium chloride method. Fifteen microgram in a gel retardation assay. Fifty nanograms of HPLC-purified samples of total cellular RNA were size fractionated on 1.1% single-stranded oligonucleotide were labeled with T4-poly- agarose gels with 2.2 M formaldehyde and blotted onto nylon nucleotide kinase and ␥32P ATP (3000 Ci/mmol; Amersham), membranes (Hybond N+; Amersham, Bucks, UK). A separated from non-incorporated radiolabel by sephadex G50 HindIII/XbaI excised p100 cDNA probe (a gift of Dr G Nabel) chromatography, ethanol precipitated, dried, and dissolved 32 was labeled with P dCTP (3000 Ci/mmol; Amersham) using in 20 ␮lof10mM Tris-HCl (pH 7.5), 50 mM NaCl, 22 the hexanucleotide primer technique. Hybridization was 10 mM MgCl ,1mM EDTA and 1 mM DTT, containing a four- ° 2 performed at 65 C for 18 h in 0.5 M Na2HPO4, pH 7.2, 1 mM fold excess of the opposite strand. Annealing of the two EDTA, 7% SDS. Membranes were washed in 2 × SSC, 0.1% strands was performed by heating the mixture for 2 min at SDS (65°C, 30 min), 1 × SSC, 0.1% SDS (65°C, 30 min) and 90°C and slow cooling to room temperature. finally in 0.3 × SSC, 0.1% SDS (65°C, 30 min). Membranes Ten micrograms of nuclear extract and 0.1 ng double- were then exposed to X-ray films (Kodak X-Omat XAR) at stranded labeled oligonucleotide were incubated in 20 mM − ° 80 C using an intensifying screen. Quantification of mRNA HEPS (pH 7.9), 60 mM KCl, 0.06 mM EDTA, 0.6 mM DTT, levels was performed by densitometry using a gel scan laser 2mM spermidine, 10% glycerol, supplemented with 2 ␮g densitometer (Pharmacia LKB, Uppsala, Sweden). poly(dI-dC). The binding reaction was performed at 26°C for 25 min. Supershift experiments were performed by incubating the nuclear extracts with polyclonal antibodies against p50, p52 and p65 subunits of NF-␬B (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The specificity of NF-␬B DNA-binding was tested by competition experiments using a 100-fold excess of unlabeled self and non-self oligonucleotide. The samples were loaded on pre-run (30 min, 100 V) 4% (30:1) polyacrylamide gels and run for 1 h at 150 V in 0.5 × TBE at room temperature. Gels were dried and exposed to Kodak XAR films (Rochester, NY, USA) at −80°C with an intensifying screen.

Western blot analysis

Monocytes were stimulated and equal amounts of cytosol extracts were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Cytosol extracts Figure 1 Transfection experiments in TF-1 cells with an NF-␬B- were prepared according to the miniscale procedure.24 Blots driven IL-6 promoter luciferase reporter plasmid co-transfected with were stained with Ponceau S to control protein loading in ␬ either a pcDNA3 plasmid, a p100 expression vector or an I B each lane. Immunoreactive proteins were detected using the expression vector. Transfected cells were stimulated with medium or ECL chemiluminescent detection system (Amersham) and a with Okadaic acid (30 ng/ml) and after 24 h luciferase activity was determined. The histogram shows the relative luciferase activity p52 rabbit polyclonal antibody (Santa Cruz Biotechnology). presented as the mean and s.d. from four independent transfection Shown are the p100 proteins (Figures 2 and 5). The p52 pro- experiments. tein could not be demonstrated in cytosol extracts. P100 (NFKB2) regulation in human monocytes H de Wit et al 365 Statistical analysis LPS-induced p100 expression in human monocytes

Statistical analysis was performed using the Student’s t-test for To study the regulation of p100 expression in monocytes, cells paired observations. were stimulated with LPS (1 ␮g/ml) and at different points of time total cellular RNA was isolated and analyzed for the expression of NF-␬B p100 mRNA. Unstimulated cells show a Results basal expression of p100 mRNA which could be strongly induced by LPS stimulation (Figure 2a). Optimal p100 mRNA ␬ NF- B-dependent IL-6 promoter activity is inhibited expression was noticed after 3 hours of stimulation with LPS. by p100 Comparable results were obtained at protein level as depicted in Figure 2b. TF-1 cells were co-transfected with an NF-␬B driven IL-6 promoter luciferase reporter plasmid in conjunction with an empty expression vector (pcDNA3), a p100 expression vector, or an I␬B expression vector. After transfection, cells were stimulated with medium (control) or Okadaic acid(30 ng/ml) which is known to be a strong inducer of IL-6 mRNA and NF- ␬B activity in monocytic cells.25 As shown in Figure 1, Oka- daic acid strongly induces the NF-␬B-induced luciferase activity in TF-1 cells. Cells co-transfected with a p100 expression vector show a signiﬁcantly decreased luciferase activity (100% vs 24.2% ± 6.4%; X ± s.d., n = 4; P Ͻ 0.01) which was comparable to inhibitory effects of co-transfection with an I␬B expression vector (100% vs 23.1% ± 6.9%; X ± s.d., n = 4; P Ͻ 0.01). Moreover, co-transfection with both p100 and I␬B expression vectors seemed to have an additive inhibitory effect on the NF-␬B-dependent IL-6 promoter activity compared to p100 and I␬B alone (9.7% ± 3.0% vs 24.2% and 23.1%, respectively; X ± s.d., n = 4; P Ͻ 0.05). Similar results were observed after stimulation with IL-1 (data not shown). This demonstrates that p100 had an I␬B-like activity and is able to inhibit NF-␬B-dependent IL-6 promoter activity. In view of these results we focused on regulation of p100 expression in monocytes.

Figure 3 Effects of protein synthesis inhibition and inhibition of second messenger pathways on the LPS-induced p100 mRNA expression. (a) Monocytes were treated with medium (control), cycloheximide (CHX; 10 ␮g/ml) for 3 h, LPS (1 ␮g/ml) for 3 h, and pretreated with cycloheximide for 30 min followed by LPS stimulation for 3 h. (b) Monocytes were treated with medium (control), LPS (1 ␮g/ml) for 3 h and pre-treated with genistein (40 ␮M) and staurosporin (20 mM) followed by LPS for 3 h. Total cellular RNA was analyzed for Figure 2 Kinetics of LPS-induced p100. Monocytes were treated p100 mRNA expression by Northern blotting. Hybridization with a with LPS (1 ␮g/ml) for the indicated periods of time. Northern (a) and 28S probe was assessed to control RNA loading in each lane. Quanti- Western blotting (b) were used to detect p100 mRNA and protein, ﬁcation of mRNA levels was performed by densitometric scanning. respectively. The experiment shown is representative of three experi- The experiments shown are representative of two independent experiments performed. ments. P100 (NFKB2) regulation in human monocytes H de Wit et al 366 Signalling pathways involved in the p100 regulation superinduction of p100 mRNA in response to cycloheximide stimulation. LPS treatment in the presence of cycloheximide To study whether the LPS-induced p100 mRNA expression further induced p100 mRNA, indicating that p100 induction was dependent on protein synthesis, monocytes were pre- is independent of protein synthesis. Next, we studied the role treated with cycloheximide (10 ␮g/ml) for 30 min followed by of the protein kinase C (PKC) and the tyrosine kinase-depen- stimulation with LPS (1 ␮g/ml) for 3 h. Figure 3a demonstrates dent second messenger pathways. Monocytes were pre-

Figure 4 (a) Nuclear run on experiment to determine the p100 transcription rate in response to LPS. Monocytes were treated with medium (control) and with LPS (1 ␮g/ml) for 3 h. After transcription was blocked, RNA was isolated and hybridized to plasmids containing no insert (pGEM), p100, IL-1, and GAPDH cDNA inserts. Relative mRNA levels were determined by densitometric scanning and normalization of the signal to the respective GAPDH signals. (b) Experiments to determine the half-life of p100 mRNA. Monocytes were treated with medium (control) and with LPS (1 ␮g/ml) for 3 h. Actinomycin-D (10 ␮g/ml) was added to inhibit transcription. At various time points after the addition of actinomycin-D p100 mRNA was detected by Northern blotting. The graphic analysis of p100 mRNA half-life was determined by densitometric scanning and normalization to 28S signals. The experiments shown are representative of two independent experiments. P100 (NFKB2) regulation in human monocytes H de Wit et al 367 treated with staurosporin (20 mM) and with genistein (40 ␮M) with LPS resulted in half-life of approximately 120 min. We for 30 min to inhibit the PKC and tyrosine kinase-dependent conclude that LPS induces p100 expression in monocytes by pathway, respectively, followed by LPS stimulation for 3 h increasing the p100 transcription rate and p100 mRNA (Figure 3b). Staurosporin and chelerithrin (data not shown) stability. failed to inhibit the LPS-induced p100 mRNA expression although the PKC-dependent pathway was effectively blocked since the LPS-induced IL-1 mRNA expression could be Modulatory effects of lymphokines and inﬂammatory inhibited (data not shown). In contrast, a reduced LPS-depen- cytokines on the p100 expression dent p100 mRNA expression (65%) was noticed in monocytes that were pretreated with genistein. This suggests a role for IL-1 and TNF are both strong inducers of an inﬂammatory the tyrosine kinase-dependent signaling pathway in the LPS- reponse that is proceeded by a transient induction of NF-␬B induced p100 expression while the PKC pathway seems not binding activity.3,26,27 To study the effects on p100 mRNA to be involved. expression, monocytes were stimulated with TNF-␣ Next nuclear run on experiments were performed to study (500 U/ml) and IL-1␤ (100 U/ml) at different points of time. the p100 transcription rate. Figure 4a shows a representative The results presented in Figures 5a and 6a show a time-depen- experiment with a basal transcription of the p100 gene in dent increase of p100 mRNA expression with optimal unstimulated monocytes. Stimulation with LPS for 3 h resulted expression at 1 h and 3 h of stimulation respectively. Compa- in a two-fold increase in the transcription rate of the p100 rable results were obtained at protein level (Figure 5c). Next gene. Potential regulation of stability of p100 mRNA was stud- we excluded the possibility that the LPS-mediated effects are ied by inhibiting transcription with actinomycin-D after cell related to the release of IL-1 and TNF since anti-TNF and anti- stimulation. The half-life of p100 mRNA of unstimulated IL-1 did not suppress the LPS-mediated p100 upregulation. In monocytes was approximately 60 min (Figure 4b). Stimulation contrast, stimulation with IFN-␥ (100 U/ml), IL-3 (10 ng/ml),

Figure 5 Effects of TNF-␣ on the p100 expression. (a) Monocytes were treated with TNF-␣ (500 U/ml) for the indicated periods of time. (b) Treatment of monocytes with anti-TNF to control the speciﬁcity of the LPS-induced p100 mRNA expression. p100 mRNA was determined by Northern blotting. (c) Western blot to determine p100 protein levels in cytosol extracts of monocytes treated with TNF-␣. The experiment shown is representative of two independent experiments. P100 (NFKB2) regulation in human monocytes H de Wit et al 368 Discussion

The expression of cytokines such as IL-1, IL-6, TNF, and other inflammatory mediators by human monocytes strongly depends on the activation of the transcription factor NF-␬B. Feedback mechanisms are necessary to limit the cytokine production and thus control the inflammatory response. A feas- ible negative regulator of the transcription factor NK-␬B might be the p100 subunit which is able to sequester the transactivating p65 subunit in the cytosol of cells. In the present study we analyzed the function and regulation of the p100 subunit of the transcription factor NF-␬B especially in human monocytes. First it was shown by transient transfection experiments that p100 is able to inhibit the NF-␬B-dependent IL-6 promoter activity. In these experiments the degree of suppression was comparable to the inhibition induced by I␬B. Moreover, co-transfection of both expression vectors resulted in a further inhibition suggesting a specific function of either protein. In monocytes p100 is selectively induced, in a protein-independent way, by LPS, IL-1 and TNF but not by the lymphokines IFN-␥, IL-3 and IL-4/IL-10. Both transcriptional and post- transcriptional mechanisms control expression of p100, in part regulated by tyrosine kinase second messenger pathways. Apart from the involvement of protein tyrosine kinase pathways, Shapira et al29 demonstrated the involvement of a PKC- dependent pathway in monocytes. However, a PKC-dependent pathway seems not to be involved in the control of p100 mRNA expression since staurosporin and chelerythrin chloride failed to have an inhibitory effect. Studies with reporter assays in which p100 promoter deletions were used have shown that different ␬B-binding sites are important in the regulation of the gene.30 In view of these data it would be expected that all cytokines which are capable of inducing NF-␬B would upregulate the expression of the p100 gene. However, the results demonstrate that IFN-␥ and IL-3 did not modulate p100 mRNA expression although they did induce NF-␬B. The discrepancy between the effects of IFN-␥ and IL-3 vs LPS, IL-1 and TNF might be related to the fact that the composition of NF-␬B was different. In supershift experiments we demonstrated that LPS induced a distinct p65 supershift while the p65 supershift in extracts of monocytes stimulated with IFN-␥ and IL-3 was similar to that in unstimulated cells. These data suggest that the LPS-induced NF-␬B ␤ Figure 6 Effects of IL-1 on p100 mRNA expression. Monocytes consists of p65/p50 heterodimer whereas IFN-␥ and IL-3 were treated with IL-1␤ (100 U/ml) for the indicated periods of time (a) and treatment with anti-IL-1 was assessed to control the specificity induce p50 homodimers. These results are in line with data 19 of LPS-induced p100 mRNA (b). Northern blotting was used to detect obtained by Sun et al demonstrating that p65 markedly p100 mRNA levels. The experiments shown are representative of two induced p100 protein while no effect was observed of p50. independent experiments. Finally the results demonstrate that activation of NF-␬Bis associated with the induction of proteins that act as repressors. and IL-4 (15 ng/ml)/IL-10 (5 ng/ml) did not change the p100 Interestingly, I␬B and p100 induction follow different time mRNA levels (Figure 7). Similarly, priming of monocytes with kinetics. I␬B protein is induced within 30 min after stimulation these cytokines followed by LPS stimulation did not affect whereas p100 protein expression is optimal after 3 h of stimu- p100 mRNA expression. lation,9 which is preceded by a decline in IL-1 and IL-6 mRNA Next we questioned whether a difference in NF-␬B compo- expression.31 Optimal expression of IL-1, G-CSF and IL-6 sition might be related to the diversity in effects since all the mRNA in monocytes is observed after 1–2 h of activation.32 It activators are capable of inducing NF-␬B DNA binding is still unknown what the relative roles for both repressors are activity.28 Supershift experiments were performed using anti- in feedback mechanisms of NF-␬B activity. However, it might bodies against the p50 and p65 subunits of NF-␬B. Figure 8 be speculated that the degree and type of repression depends shows a basal DNA binding of NF-␬B in unstimulated cells on the second messenger pathway that is triggered. In this which can be enhanced by LPS, IL-3 and IFN-␥ stimulation. regard it is of interest to note the difference in binding; I␬B No clear difference was noticed with regard to the p50 super- binds to p50/p65 heterodimers whereas p100 binds the p65 shift. In contrast, the LPS-induced NF-␬B shows a distinct p65 subunit.19,33 Further studies are needed to elucidate the com- supershift as compared to unstimulated cells which was not plex interactions and feedback mechanisms that control NF- observed in cells stimulated with the other activators. ␬B-mediated responses. P100 (NFKB2) regulation in human monocytes H de Wit et al 369

Figure 7 Effects of lymphokines on the p100 mRNA expression. Monocytes were treated with medium (control), LPS (1 ␮g/ml) for 3 h, and with the lymphokines IFN-␥ (100 U/ml), IL-3 (10 ng/ml), and IL-4 (15 ng/ml)/IL-10 (5 ng/ml) for 6 h. Furthermore, monocytes were primed with the different lymphokines for 3 h followed by LPS stimulation for 2 h. Northern blotting was used to determine p100 mRNA levels. The experiments shown are representative of three independent experiments.

Figure 8 Analysis of the composition of the NF-␬B complex in response to LPS and lymphokines. Monocytes were stimulated with medium (control), LPS (1 ␮g/ml), IFN-␥ (100 U/ml), IL-3 (10 ng/ml), and with IL-4 (15 ng/ml)/IL-10 (5 ng/ml) for 2 h. Nuclear extracts were used in a supershift experiment with antibodies against the p50 and the p65 subunits of NF-␬B.

Acknowledgements Dr S Gillis for providing IL-1␤ and the antisera against IL-1. The p100 cDNA probe was kindly provided by Dr G Nabel. This study was supported by a grant from the Jan Kornelis de The IL-6 promoter luciferase plasmid was a gift from Dr S Cock Foundation. We thank Dr GR Ernst for providing TNF- Akira. ␣ and anti-TNF, Dr P Trotta for providing IL-4 and IL-10, and P100 (NFKB2) regulation in human monocytes H de Wit et al 370 References P, Bravo R. Rel B, a new Rel family transcription activator that can interact with p50-NF-␬B. Mol Cell Biol 1992; 12: 674–684. 18 Brownell E, Mittereder N, Rice N. A human rel proto-oncogene 1 Hiscott J, Marios J, Garoufalis J, d’Addario M, Roulston A, Kwan cDNA containing an Alu fragment as a potential coding exon. Oncogene 1989; 4: 935–942. I Pepin N, Lacoste J, Nguyen H, Bensi G, Fenton M. Characteriz- ´ ation of a functional NF-␬B site in the human interleukin-1␤ pro- 19 Sun SC, Ganchi PA, Beraud C, Ballard DW, Greene WC. 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