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Activation and Methotrexate-MediatedSuppression of the TNFα Promoter in T Cells and Macrophages

CHRISTOPH BECKER, KARINA BARBULESCU, KAI

HILDNER, KARL-HERMANN MEYER ZUM

BÜSCHENFELDE, AND MARKUS F. NEURATHa

Laboratory of Immunology, I. Medical Clinic, University ofMainz, 55131 Mainz, Germany

INTRODUCTION

Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays an importantrole in chronic inflammation.1 It is produced by a wide variety of cells of theimmune system including macrophages and T and B lymphocytes. TNF is a keymediator of both acquired and natural immunity and appears to contribute to theinitiation and progression of many immune-mediated disorders such as rheuma-toid arthritis2 and Crohn’s disease.3 Antibodies to TNFa have recently been estab-lished as an effective therapy for patients with inflammatory bowel diseases,underlining the importance of this cytokine in the pathogenesis of chronic intesti-nal inflammation. Therefore, understanding the regulation of TNF expressioncould provide new insights into the complex processes underlying these diseases.In an approach toward this goal, we have analyzed the transcriptional activationof the human TNFa-gene. Using in vivo genomic footprinting in freshly isolatedT lymphocytes, we found altered DMS reactivities at several previouslydescribed binding sites for transcription factors upon stimulation of the cells withantibodies to CD3/CD28 or PMA/ionomycin. On the basis of the footprintingdata, we provide a model of TNF promoter regulation in T lymphocytes. In fur-ther studies we found that TNFα promoter activity was suppressed by the drugmethotrexate in a dose-dependent manner, providing a possible molecular mech-anism for the beneficial effect of methotrexate in patients with Crohn’s disease.

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aCorresponding author: Markus F. Neurath, M.D., Laboratory of Immunology, I.Medical Clinic, University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany. Telephone:0049-6131-173382; Fax: 0049-6131-172489.

MATERIALS AND METHODS

Isolation of primary human CD4+ T lymphocytes: Human peripheral bloodmononuclear cells (PBMC) were isolated from healthy volunteers using Ficoll-Hypaque gradients. PBMC were then further purified using immunomagneticbeads (Dynal, Oslo, Norway) to isolate CD4+ T lymphocytes. The cells were cul-tured at a density of 106/ml culture medium in humidified atmosphere with 5%CO2 at 37°C.

In vivo footprinting of primary T lymphocytes: In vivo footprinting of thehuman TNFα promoter was performed as previously described.4

DEAE transfection of THP-1 cells and reporter gene analysis: The TNF pro-moter was cloned as a 1.2-kb fragment into the SmaI site of the promoterlesspXP1 luciferase reporter gene vector. Ten micrograms of this pXP1-TNF-Lucvector were transfected into 107 THP-1 cells using the DEAE transfection proto-col. After 24 hours the cells were stimulated with 1 µg/ml ionomycin. The stim-ulation was allowed to proceed for 18 hours before the cells were harvested andanalyzed for luciferase activity using a commercially available system (Promega)according to the manufacturers recommendations.

220 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Model of transcriptional activation of the human TNFα promoter in Tlymphocytes based on in vivo footprinting results. Stimulation of T lymphocytesinduces binding of the indicated transcription factors to the TNF promoter and subse-quently to transcriptional activity. Methotrexate potentially interferes with this activationpathway, at least partly by downregulation of TNF promoter activity.

RESULTS

Recent studies on transcriptional regulation of the TNF gene have demon-strated the capability of various transcription factors to bind to the promoterregion in vitro.5 However, it is clear that gene regulation in vivo is complicated bymany additional factors such as the nucleosomal context. To overcome this prob-lem, we have performed in vivo genomic footprinting of the TNFα promoter inprimary T lymphocytes. Altered DMS reactivities at various sites with previouslydescribed regulatory function were observed in particular within 200 bp upstreamof the transcriptional start site. Accordingly, upon stimulation with αCD3/αCD28or PMA/ionomycin, protectins were observed at binding sites for EGR-1, NFATp,ATF-2/c-jun, and ETS-1. FIGURE 1 shows a model of TNF promoter activation inprimary T lymphocytes based on the in vivo footprinting results. Methotrexate, adrug used for the treatment of chronic intestinal inflammation, has been shown toinhibit TNFα protein secretion from peripheral blood mononuclear cells, as mea-sured by ELISA. However, the molecular mechanism leading to this effect isunknown. To examine whether this downregulation could be due to decreasedTNFα promoter activity, we have performed transient transfection experiments in

BECKER et al.: SUPPRESSION OF TNFα PROMOTER 221

FIGURE 2. Activity of the TNF promoter in methotrexate-treated THP-1 cells. THP-1 cells were transfected with the pXP1-TNF-Luc vector containing the human TNF pro-moter upstream of a luciferase reporter gene. Cells were stimulated with ionomycin 24hours after transfection, and methotrexate (MTX) was added at the indicated concentra-tions. Luciferase activity was assessed after an additional 18 hours. Two representativeexperiments are shown. Data are expressed as fold induction of each stimulation condi-tion compared to the unstimulated control.

THP-1 cells. For this purpose the cells were transfected with a luciferase reportergene vector under the control of the human TNFα promoter. The cells were stim-ulated 24 hours after transfection and methotrexate was added at various concen-trations. After a further 18 hours, cells were harvested and assayed for luciferaseactivity. FIGURE 2 demonstrates that induction of TNF promoter activity by iono-mycin was strongly inhibited by methotrexate in a dose-dependent manner.

Previous studies on transcriptional regulation of TNF gene expression haveidentified various regulatory target sites for transcription factors in the TNF pro-moter including for ATF-2/c-jun, ETS-1, NFATp, and NF-κB. However, onlylimited information on their contribution to TNF promoter regulation in vivo inprimary lymphocytes has been available. In the present study, we have analyzedin vivo and in vitro protein/DNA interactions at the human TNF promoter in pri-mary T lymphocytes.

In summary, the in vivo footprinting data demonstrated that the TNF promoterin vivo in primary lymphocytes is regulated by the coordinate binding of tran-scription factors representative of different families. Methotrexate, a drug suc-cessfully used for treatment of chronic intestinal inflammation, stronglysuppressed induction of the TNFa promoter in transient transfection experiments,providing a potential molecular mechanism for the drug’s physiologic effects.Subsequent studies will have to show whether methotrexate acts by interferingwith the coordinate binding of transcription factors to the TNFα promoter andthus directly inhibits promoter activation.

REFERENCES

1. DI, G. N. 1997. Expression of TNF-alpha by human plasma cells in chronic inflam-mation. J. Leukoc. Biol. 61: 667.

2. RIDDERSTAD, A. 1991. Cytokines in rheumatoid arthritis. Ann. Med. 23: 219.3. REIMUND, J. M. 1996. Increased production of tumour necrosis factor-alpha inter-

leukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies frompatients with Crohn’s disease. Gut 39: 684.

4. MUELLER, P. R. 1989. In vivo footprinting of a muscle specific enhancer by ligationmediated PCR. Science 246: 780.

5. TSAI, E. Y. 1996. Tumor necrosis factor alpha gene regulation in activated T cellsinvolves ATF-2/Jun and NFATp. Mol. Cell Biol. 16: 459.

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