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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

Tumor necrosis factor (TNF) is a pleiotropic cytokine that plays an important rolein chronic inflammation.1 It is produced by a wide variety of cells of the immunesystem including macrophages and T and B lymphocytes. TNF is a key mediatorof both acquired and natural immunity and appears to contribute to the initiationand progression of many immune-mediated disorders such as rheumatoid arthri-tis2 and Crohn’s disease.3 Antibodies to TNFα have recently been established asan effective therapy for patients with inflammatory bowel diseases, underliningthe importance of this cytokine in the pathogenesis of chronic intestinal inflam-mation. Therefore, understanding the regulation of TNF expression could providenew insights into the complex processes underlying these diseases. In anapproach toward this goal, we have analyzed the transcriptional activation of thehuman TNFα-gene. Using in vivo genomic footprinting in freshly isolated T lym-phocytes, we found altered DMS reactivities at several previously described bind-ing sites for transcription factors upon stimulation of the cells with antibodies toCD3/CD28 or PMA/ionomycin. On the basis of the footprinting data, we providea model of TNF promoter regulation in T lymphocytes. In further studies wefound that TNFa promoter activity was suppressed by the drug methotrexate in adose-dependent manner, providing a possible molecular mechanism for the ben-eficial effect of methotrexate in patients with Crohn’s disease.

MATERIALS AND METHODS

Isolation of Primary Human CD4+ T Lymphocytes

Human peripheral blood mononuclear cells (PBMC) were isolated fromhealthy volunteers using Ficoll-Hypaque gradients. PBMC were then further

311

aAddress for corresponding author: Markus F. Neurath, M.D., Laboratory ofImmunology, I. Medical Clinic, University Mainz, Langenbeckstr. 1, 55131 Mainz,Germany. Telephone: 0049-6131-173382; Fax: 0049-6131-175508.

purified using immunomagnetic beads (Dynal, Oslo, Norway) to isolate CD4+ Tlymphocytes. The cells were cultured at a density of 106/ml culture medium inhumidified atmosphere with 5% CO2 at 37°C.

In Vivo Footprinting of Primary T Lymphocytes

In vivo footprinting of the human TNFα promoter was performed as previ-ously described.4

DEAE Transfection of THP-1 Cells and Reporter Gene Analysis

The TNF promoter was cloned as a 1.2-kb fragment into the SmaI site of thepromoterless pXP1 luciferase reporter gene vector. Ten micorgrams of this pXP1-TNF-Luc vector were transfected into 107 THP-1 cells using the DEAE transfec-tion protocol. After 24 hours the cells were stimulated with 1 µg/ ml ionomycin.The stimulation was allowed to proceed for 18 hours before the cells were har-vested and analyzed for luciferase activity using a commercially available system(Promega) according to the manufacturer’s recommendations.

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, protections were observed at binding sites for EGR-1,NFATp, ATF-2/ c-jun, and ETS-1. FIGURE 1 shows a model of TNF promoteractivation in primary T lymphocytes based on the in vivo footprinting results.

Methotrexate, a drug used for the treatment of chronic intestinal inflammation,has been shown to inhibit TNFα protein secretion from peripheral blood mononu-clear cells, as measured by ELISA. However, the molecular mechanism leadingto this effect is unknown. To examine whether this downregulation could be dueto decreased TNFα promoter activity we have performed transient transfectionexperiments in THP-1 cells. For this purpose the cells were transfected with aluciferase reporter gene vector under the control of the human TNFα promoter.The cells were stimulated 24 hours after transfection, and methotrexate wasadded at various concentrations. After a further 18 hours, cells were harvestedand assayed for luciferase activity. FIGURE 2 demonstrates that induction of TNFpromoter activity by ionomycin was strongly inhibited by methotrexate in a dose-dependent manner.

312 ANNALS NEW YORK ACADEMY OF SCIENCES

DISCUSSION

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-kB. However, only lim-ited 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 TNFα promoter in transient transfection experimentsproviding a potential molecular mechanism for the drug’s physiologic effects.Subsequent studies will have to show if methotrexate acts by interfering with thecoordinate binding of transcription factors to the TNFα promoter and thusdirectly inhibits promoter activation.

BECKER et al.: TNFα PROMOTER 313

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 induces transcriptional activity. Methotrexate potentially interferes with this acti-vation pathway, at least partly by downregulation of TNF promoter activity.

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, interleukin-

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

4. MUELLER, P. R. 1989. In vivo footprinting of a muscle-specific enhancer by ligation-mediated 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.

314 ANNALS NEW YORK ACADEMY OF SCIENCES

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.