Leflunomide Suppresses TNF-Induced Cellular Responses: Effects on NF-κB, Activator Protein-1, c-Jun N-Terminal Protein Kinase, and Apoptosis

Materials

Leflunomide (A77 1726), a generous gift from Dr. Robert R. Bartlett (Hoechst AG, Weisbaden, Germany), was made up as 5 mM solution in water. Penicillin, streptomycin, RPMI 1640 medium, and FCS were obtained from Life Technologies (Grand Island, NY). Glycine, NaCl, and BSA were obtained from Sigma (St. Louis, MO). Bacteria-derived human rTNF, purified to homogeneity with a sp. act. of 5 × 10⁷ U/mg, was kindly provided by Genentech (South San Francisco, CA). Abs against I-κBα, JNK1, cyclin D1, c-Jun, c-Fos, and p50 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Poly(ADP-ribose) polymerase (PARP) Ab was purchased from PharMingen (San Diego, CA). Phospho-I-κBα (Ser³²) Ab was purchased from New England BioLabs (Beverly, MA). Anti-IKKα or anti-IKKβ Abs were kindly provided by Imgenex (San Diego, CA). Expression plasmids encoding FLAG-tagged NF-κB-inducing kinase (NIK) (23) were kindly provided by Dr. David Wallach (Weizmann Institute of Science, Rehovot, Israel). The expression plasmid encoding myc-tagged TNFR-associated factor 2 (TRAF2) has been previously described (24).

Cell lines

HeLa (human epithelial cells) and T-Jurkat (T cells) were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml).

Isolation of PBLs

Freshly drawn human blood was incubated with 2.5% gelatin in saline (1:1 ratio) for 30 min at 37°C. The supernatant was layered on Histopaque 1077 (from Sigma) and centrifuged at 1500 rpm for 30 min at room temperature. The cells were then collected from the top layer of Histopaque, diluted with Dulbecco’s PBS, and centrifuged at 2000 rpm for 10 min. To get rid of mixed reticulocytes, pellet was suspended in 0.2% NaCl for 1 min, immediately diluted with equal volume of 1.6% NaCl, and centrifuged at 1000 rpm. To remove macrophages by adherence, the pellet was suspended in RPMI 1640 medium supplemented with 10% FBS and cultured for 2 h at 37°C, CO₂ incubator in a petri dish. Then the lymphocytes were harvested from the medium by centrifugation at 1000 rpm.

NF-κB activation assays

To determine NF-κB activation, EMSA were conducted essentially as described (25). Briefly, nuclear extracts prepared from TNF-treated cells (2 × 10⁶/ml) were incubated with ³²P end-labeled 45-mer double-stranded NF-κB oligonucleotide (4 μg protein with 16 fmol DNA) from the HIV long terminal repeat, 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′ (underline indicates NF-κB binding sites) for 15 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5′-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3′, was used to examine the specificity of binding of NF-κB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. The dried gels were visualized, and radioactive bands were quantitated by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.

NF-κB-dependent reporter gene transcription

The effect of leflunomide on TNF-, TRAF-2-, NIK-, and p65 (transactivation subunit of NF-κB)-induced NF-κB-dependent reporter gene transcription was measured as previously described (24). Briefly, HeLa cells (0.1 × 10⁶ cells/well) were plated in six-well plates, pretreated with 10 μM leflunomide for 2 h, and then transfected by the calcium phosphate method with medium (1 ml) containing plasmid DNAs for TRAF2, NIK, or p65 (1 μg each) along with 0.5 μg NF-κB promoter DNA linked to heat-stable secretory alkaline phosphatase (SEAP) gene. The total final amount of DNA was maintained at 3 μg by the addition of the control plasmid pCMVFLAG1 DNA.

To examine TNF-induced reporter gene expression, we transfected cells with the SEAP expression plasmid for 10 h before treating them with TNF (1 nM). Treatment with leflunomide was continued during the transfection reaction. Twenty-four hours after transfection, cell culture-conditioned medium was harvested and analyzed (25 μl) for alkaline phosphatase activity essentially as described by the protocol of Clontech (Palo Alto, CA). The activity of SEAP was assayed on a 96-well fluorescent plate reader (Fluoroscan II; Labsystems, Chicago, IL) with excitation set at 360 nm and emission at 460 nm. This reporter system was specific because TNF-induced NF-κB SEAP activity was inhibited by overexpression of either an I-κBα mutant lacking Ser^32/36, a kinase-inactive NIK, or a dominant-negative TRAF2 mutant (24).

I-κBα phosphorylation

To determine the effect of leflunomide on I-κBα phosphorylation, cytoplasmic extracts were prepared from cells (2 × 10⁶/ml) treated with leflunomide (10 μM) for 2 h, then with N-acetyl leucyl leucyl nonleucinal (ALLN) (100 μg/ml) for an additional 1 h, and then with TNF (1 nM) for 15 min, resolved on 10% SDS-PAGE, and then analyzed by Western blot using Abs against either I-κBα or phosphorylated I-κBα (26). After electrophoresis, the proteins were detected by chemiluminescence (ECL; Amersham, Arlington Heights, IL).

I-κBα kinase assay

The IKK assays were performed as described (27). Briefly, IKK signalosomes were precipitated by treating 300 μg cytoplasmic extracts with 1μg anti-IKKα Ab (IMG-136) overnight at 4°C, followed by treatment with 20 μl protein A/G-Sepharose (Pierce, Rockford, IL). After 2 h, the beads were washed three times with lysis buffer and three times with the kinase assay buffer, and then resuspended in 20 μl of kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl₂, 2 mM DTT, 20 μCi γ-ATP, 10 mM unlabeled ATP, and 2 μg of substrate GST-I-κBα 1–54(1–54). After incubation at 30°C for 30 min, the reaction was terminated by boiling with 5 μl of 5× SDS sample buffer for 5 min. Finally, the protein was resolved on 10% polyacrylamide gel under reducing conditions, the gel was dried, and the radioactive bands were visualized by PhosphorImager. To determine the total amounts of IKKα and IKKβ in each sample, 60 μg of the cytoplasmic protein was resolved on 7.5% acrylamide gels, electrotransferred to a nitrocellulose membrane, blocked the membrane with 5% nonfat milk protein for 1 h, and then incubated with either anti-IKKα or anti-IKKβ (IMG-129) Abs (at 1/500 dilution) for 1 h. The membrane was then washed and reacted with HRP-conjugated secondary anti-mouse IgG Ab, and finally detected by chemiluminescence (ECL; Amersham).

AP-1 activation assay

The activation of AP-1 was determined as described (28). Briefly, 6 μg of nuclear extract prepared as indicated above was incubated with 16 fmol of the ³²P end-labeled AP-1 consensus oligonucleotide 5′-CGCTTGATGACTCAGCCGGAA-3′ (bold indicates AP-1 binding site) for 15 min at 37°C and analyzed by using 6% native polyacrylamide gel. The specificity of binding was examined by supershift with anti-c-fos and anti-c-jun Abs and by competition with unlabeled oligonucleotide. Visualization and quantitation of radioactive bands were done as indicated above.

c-Jun kinase assay

The c-Jun kinase assay was performed by a modified method, as described earlier (29). Briefly, after treatment of cells with TNF for 10 min, cell extracts were prepared, immunoprecipitated with anti-JNK Ab, collected the immune complexes by incubation with protein A/G-Sepharose beads, and performed the in vitro kinase assays using GST-Jun 1–79(1–79) as a substrate and [γ-³²P]ATP. Reactions were stopped by the addition of SDS sample buffer and subjected the samples to SDS-PAGE. GST-Jun 1–79(1–79) was visualized by staining with Coomassie blue, and the dried gel was analyzed by a PhosphorImager (Molecular Dynamics).

Cytotoxicity assay

TNF-induced cytotoxicity was measured by the MTT assay (28). Briefly, 5000 cells/well were incubated in the presence or absence of the indicated test sample in a final volume of 0.1 ml for 72 h at 37°C. Thereafter, 0.025 ml of MTT solution (5 mg/ml in PBS) was added to each well. After a 2-h incubation at 37°C, 0.1 ml of the extraction buffer (20% SDS, 50% dimethyl formamide) was added. After an overnight incubation at 37°C, the OD at 590 nm were measured using a 96-well multiscanner autoreader (Dynatech MR 5000), with the extraction buffer as a blank.

Immunoblot analysis of PARP degradation

TNF-induced apoptosis was examined by proteolytic cleavage of PARP (30). Briefly, cells (2 × 10⁶/ml) were treated with different concentrations of leflunomide at 37°C for 2 h and then stimulated with 1 nM TNF with cycloheximide (2 μg/ml) for 2 h at 37°C. The cells were then washed and extracted by treatment for 30 min on ice with 0.05 ml buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF, 0.5 μg/ml benzamidine, 1 mM DTT, and 1 mM sodium vanadate. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 μg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with mouse anti-PARP Ab, and then detected by chemiluminescence (ECL; Amersham). Apoptosis was represented by the cleavage of 116-kDa PARP into a 85-kDa product.

Determination of lipid peroxidation

TNF-induced lipid peroxidation was determined by detection of thiobarbituric acid-reactive malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters, as described (26). Results were normalized with the amount of MDA equivalents/mg of protein and expressed as a percentage of thiobarbituric acid-reactive substances above control values. Untreated cells showed 0.571 ± 0.126 nmol of MDA equivalents/mg of protein.

Measurement of ROI

The production of ROI upon treatment of cells with TNF was determined by flow cytometry, as described (26).

Radiolabeling of TNF and receptor-binding assay

Human TNF was iodinated with [¹²⁵I]Na by the Iodogen method, purified, and examined for cell surface receptors, as described previously (30).

Leflunomide represses TNF-induced NF-κB-dependent reporter gene expression

Previously, we have shown that leflunomide blocks TNF-induced NF-κB activation, I-κBα degradation, and NF-κB-dependent reporter gene expression (11). TNF-induced NF-κB activation is mediated through sequential interaction of the TNFR (TNFR1) with TNFR-associated death domain, TRAF2, NIK, and IKK-β, resulting in phosphorylation of I-κBa (31, 32). To delineate the site of action of leflunomide in the TNF-signaling pathway leading to NF-κB activation, HeLa cells were transfected with TNFR1, TRAF2, NIK, and p65 plasmids, and then NF-κB-dependent SEAP expression monitored in leflunomide-untreated and -treated cells. Due to higher transfection efficiency, HeLa cells were used. As shown in Fig. 1⇓, TNFR1, TRAF2, NIK, and p65 plasmids induced gene expression, and leflunomide suppressed TNFR1-, TRAF2-, and NIK-induced expression, but had little effect on p65-induced NF-κB reporter expression. RANK, another NF-κB-inducing receptor, which is a member of the TNFR1 family, was minimally affected by leflunomide, indicating the specificity. Specificity of the assay results is also indicated by suppression of TNF-induced NF-κB reporter activity by the dominant-negative I-κBα plasmid. Thus, leflunomide must act at a step downstream from IKK-β. Because NIK is known to activate IKK-β, which in turn phosphorylates I-κBα, it appears that leflunomide blocked the activity of IKK-β, a kinase that phosphorylates I-κBα directly.

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FIGURE 1.

Leflunomide inhibits NF-κB-dependent reporter gene expression induced by and activated by TNF, TNFR1, TRAF2, and NIK. Leflunomide-treated HeLa cells were transiently transfected with indicated plasmids along with the NF-κB-containing plasmid linked to the SEAP gene. Where indicated, cells were exposed to 1 nM TNF also. After 24 h in culture, cell supernatants were collected, and 25 μl of it was assayed for secreted alkaline phosphatase activity, as described in Materials and Methods. Results are expressed as fold activity over the nontransfected control. The specificity of the assay was examined by suppression of TNF-induced NF-κB reporter activity by I-κBα dominant-negative mutant plasmid.

Leflunomide inhibits TNF-dependent phosphorylation I-κBα

To determine whether inhibition of TNF-induced I-κBα degradation by leflunomide was due to suppression of I-κBα phosphorylation, cells were treated with the proteosome inhibitor ALLN (33) for 1 h, and then the hyperphosphorylated form of I-κBα was assayed by Western blot using Abs that detect only the serine-phosphorylated form of I-κBα. The results clearly show that TNF induced the phosphorylation of I-κBα and leflunomide suppressed it (Fig. 2⇓A, upper panel). The hyperphosphorylated form of I-κBα also appeared as a slow-migrating band on SDS-PAGE (Fig. 2⇓A, lower panel). The lack of a slow-migrating band in leflunomide-treated cells indicates that leflunomide blocked TNF-induced I-κBα phosphorylation.

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FIGURE 2.

Leflunomide inhibits TNF-induced phosphorylation of I-κBα. A, Jurkat cells (2 × 10⁶/ml) were incubated first with leflunomide (10 μM) for 2 h and then with ALLN (100 μg/ml) for an additional 1 h before treatment with TNF (1 nM) for 15 min, and then analyzed by Western blot using Abs against either phosphorylated I-κBα (upper panel) or I-κBα (lower panel). S, Indicates slow-migrating band, and N is normal-migrating band. B, Jurkat cells (5 × 10⁶ cells), untreated or pretreated with 10 μM leflunomide for 2 h, were activated with 0.1 nM TNF for different times, thereafter prepared the cytoplasmic extracts and assayed for IKK by the immune complex kinase assay (upper panel) and for IKKα (middle panel) and IKKβ (lower panel) protein by the Western blot analysis, as described in Materials and Methods. C, Effect of leflunomide on TNF-induced activation of NF-κB in lymphocytes. Human lymphocytes were isolated from fresh blood and 2 × 10⁶cells/ml were preincubated for 2 h at 37°C with 10 or 25 μM leflunomide, followed by TNF (0.1 nM) for 30 min, and then tested for NF-κB activation, as described in Materials and Methods.

Leflunomide inhibits TNF-induced IKK-β activation

Because TNF-induced phosphorylation of I-κBα is mediated through IKK-β, these results suggest that leflunomide must inhibit IKK-β activation. Therefore, we investigated the effect of leflunomide on TNF-induced IKK-β activation. As shown in Fig. 2⇑B (upper panel), in the immune complex kinase assays, TNF activated IKK-β in a time-dependent manner and leflunomide treatment completely suppressed the activation. Under these conditions, leflunomide had no effect on the IKK-α (middle panel) and IKK-β (lower panel) protein levels.

Leflunomide inhibits TNF-induced NF-κB activation in normal cells

All the experiments described above were performed with the Jurkat cell lines. Previously, we have shown that leflunomide also blocks TNF-induced NF-κB activation in U-937 cells (myeloid) and epithelial (HeLa) and glioma (H4) cells (11). Whether leflunomide also affects NF-κB in normal cells was examined. As shown in Fig. 2⇑C, TNF activated NF-κB by 4-fold in normal human PBLs and the pretreatment with leflunomide abolished the activation in a dose-dependent manner. These results suggest that the suppressive effect of leflunomide is not restricted to tumor cells.

Leflunomide inhibits TNF-induced AP-1 activation

Previously, we have shown that leflunomide has no effect on the constitutive levels of AP-1 (11). TNF is also one of the most potent activators of AP-1 (34). TNF induced AP-1 expression by 5-fold in Jurkat cells at 1 nM concentration (Fig. 3⇓A). The activation of AP-1 was completely inhibited by leflunomide in a concentration-dependent manner, with maximum suppression occurring at 5 μM (Fig. 3⇓A). Supershift analysis with specific Abs against c-fos and c-jun indicates that TNF-induced AP-1 consisted of fos and jun (Fig. 3⇓B). The lack of supershift by unrelated Abs and disappearance of the AP-1 band by competition with unlabeled oligonucleotide indicate the inhibition was specific (Fig. 3⇓B).

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FIGURE 3.

Leflunomide inhibits TNF-dependent AP-1 activation. A, Jurkat cells (2 × 10⁶) were pretreated with the indicated concentrations of leflunomide for 2 h at 37°C. Then cells were stimulated with 1 nM TNF for 1 h and assayed for AP-1, as described in Materials and Methods. B, Supershift and specificity of AP-1 activation. Nuclear extracts were prepared from untreated or TNF (1 nM)-treated Jurkat cells (2 × 10⁶/ml), incubated for 15 min with either different Abs or unlabeled AP-1 oligo, and then assayed for AP-1 by EMSA, as described in Materials and Methods. C, Leflunomide inhibits TNF-induced c-Jun-kinase activation. Jurkat cells were pretreated with different concentrations of leflunomide, as indicated, for 2 h and then stimulated with 1 nM TNF at 37°C for 10 min. Thereafter, the cells were washed, pellets were extracted, and c-Jun-kinase activation was detected from the extract, as described in Materials and Methods.

Leflunomide inhibits TNF-induced JNK activation

Previously, we have shown that leflunomide blocks TNF-induced mitogen-activated protein/extracellular signal-related kinase kinase activation (11). JNK, the downstream kinase, is known to be required for the activation of AP-1 (35, 36, 37). Whether JNK is also modulated by leflunomide was examined. Jurkat cells were pretreated with different concentrations of leflunomide for 2 h and then stimulated with TNF (1 nM) for 10 min; activation of JNK was then measured. TNF activated JNK by about 13-fold, an activation that gradually decreased with increasing concentrations of leflunomide. A 10 μM concentration of leflunomide inhibited most of the JNK induced by TNF (Fig. 3⇑C). Thus, it is possible that leflunomide blocks TNF-induced AP-1 activation through suppression of JNK activation.

Leflunomide blocks TNF-induced cytotoxicity and caspase activation

Among the cytokines, TNF is one of the most potent inducers of apoptosis (for references, see Ref. 38). Whether leflunomide modulates TNF-induced apoptosis was also investigated. Jurkat cells were treated with various concentrations of leflunomide either in the absence or presence of TNF and then examined for cytotoxicity by the MTT method (Fig. 4⇓A). TNF was cytotoxic to Jurkat cells, and leflunomide abolished TNF-induced cytotoxicity in a dose-dependent manner, reaching complete inhibition at 10 μM leflunomide. We also examined the cytotoxic effect of various concentrations of TNF either in the absence or presence of leflunomide (Fig. 4⇓B). The cytotoxic effects of TNF in Jurkat cells were dose dependent, with almost 60% killing occurring at 0.5 nM concentration of the cytokine. This cytotoxicity was nearly completely inhibited at all TNF doses by treatment of cells with 10 μM leflunomide.

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FIGURE 4.

Leflunomide blocks TNF-induced cytotoxicity and PARP degradation. A, Jurkat cells were treated with different concentrations of leflunomide for 2 h at 37°C and then incubated with 1 nM TNF for 72 h at 37°C, in a CO₂ incubator. Thereafter, cell viability was examined by the MTT method, as described in Materials and Methods, and expressed as mean OD of triplicate assays. B, Jurkat cells were treated with 10 μM leflunomide for 2 h at 37°C and then incubated with different concentrations of TNF for 72 h at 37°C in a CO₂ incubator. Thereafter, cell viability was examined by the MTT method. C, Jurkat cells were incubated with different concentrations of leflunomide for 2 h at 37°C, CO₂ incubator and then treated with 2 μg/ml cycloheximide and 1 nM TNF for 2 h at 37°C and washed. The pellet was extracted, and Western blot conducted using anti-PARP mAb. The bands were located at 116 and 80 kDa.

Because the cytotoxic effects of TNF are mediated through the activation of caspases, we also examined the effect of leflunomide on TNF-induced caspase activation. Activated caspase-2, -3, and -7 are known to cleave PARP protein. As shown in Fig. 4⇑C, TNF induced complete cleavage of PARP, and this cleavage was inhibited in a dose-dependent manner by treatment of cells with leflunomide, with maximum effect at 10 μM concentration. Thus, leflunomide also blocked TNF-induced apoptosis.

Leflunomide blocks TNF-induced ROI generation and lipid peroxidation

Previous reports have shown that TNF activates NF-κB, AP-1, JNK, and apoptosis through generation of ROI (27, 39, 40, 41). Whether leflunomide suppresses TNF signaling through suppression of ROI generation was examined by flow cytometry. As shown in Fig. 5⇓A, TNF induced ROI generation in a time-dependent manner, and this was suppressed on pretreatment of cells with leflunomide. Because lipid peroxidation has also been implicated in TNF signaling (42), we also examined the effect of leflunomide on TNF-induced lipid peroxidation. Results in Fig. 5⇓B show that TNF induced lipid peroxidation in Jurkat cells, and it was completely suppressed by leflunomide. Thus, it is quite likely that leflunomide blocks TNF signaling through suppression of ROI generation and lipid peroxidation. Although earliest time we examined ROI generation is at 1 h, whereas NF-κB and AP-1 activation can be seen at 15 min, it is possible that low levels of ROI generated at early times are either not detectable or other mechanisms are involved in TNF signaling.

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FIGURE 5.

Leflunomide inhibits TNF-induced ROI generation (A) and lipid peroxidation (B). For A, Jurkat cells (5 × 10⁵/ml) were treated with 10 μM leflunomide for 2 h and then exposed to TNF (0.1 nM) for indicated times at 37°C in a CO₂ incubator. ROI production was then determined by the flow cytometry method, as described in Materials and Methods. The results shown are representative of two independent experiments. For B, Jurkat cells (3 × 10⁶ in 1 ml) were pretreated with different concentrations of leflunomide for 2 h and then incubated with 1 nM TNF for 1 h and assayed for lipid peroxidation, as described in Materials and Methods.

Uridine reverses the suppressive effects of leflunomide on TNF signaling

The antiproliferative effects of leflunomide on B and T cells can be reversed by uridine, suggesting the critical role of DHODH (12, 15). To determine whether uridine reverses the suppression of TNF-induced NF-κB activation in Jurkat cells, we pretreated cells with leflunomide in the presence of various concentrations of uridine and then treated them with TNF. As shown in Fig. 6⇓A, TNF-induced NF-κB activation was suppressed by leflunomide, and uridine reversed the suppression in a dose-dependent manner. Uridine or leflunomide by themselves did not activate NF-κB, and uridine alone had minimal effect on TNF-mediated NF-κB activation. Thus, leflunomide mediated its effects on TNF-induced NF-κB activation by interfering with the pyrimidine biosynthesis pathway. We also examined the effect of uridine on the leflunomide-induced suppression of TNF-mediated cytotoxicity. As shown in Fig. 6⇓B, 50 μM uridine completely reversed the effects of leflunomide on the cytotoxicity caused by TNF. Thus, pyrimidine biosynthesis plays a critical role in the TNF signaling.

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FIGURE 6.

Uridine reverses leflunomide-mediated inhibition of NF-κB activation and cytotoxicity induced by TNF. A, Jurkat cells (2 × 10⁶/ml) were pretreated with different concentrations of uridine for 1 h and then treated with 10 μM leflunomide for 2 h at 37°C. Thereafter, cells were activated with TNF (100 pM) for 30 min, and nuclear extracts prepared and analyzed by EMSA. B, Jurkat cells (5 × 10³/0.1 ml) were pretreated with 50 μM uridine for 1 h and then treated with 10 μM leflunomide for 2 h at 37°C. Thereafter, cells were incubated with different concentrations of TNF for 72 h and examined for cell viability by the MTT method, as described in Materials and Methods.

Leflunomide has no effect on TNFR

Because leflunomide blocked a variety of signals activated by TNF, it is possible that leflunomide down-regulated TNFR. Therefore, we examined the effect of leflunomide on TNFR by receptor-binding assays. TNF bound to Jurkat cells, and this binding was completely unaffected by pretreatment of cells with leflunomide (data not shown), suggesting that the effects of leflunomide were not due to down-regulation of TNFR.