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. 2004 Dec;143(7):919-27.
doi: 10.1038/sj.bjp.0706000. Epub 2004 Oct 25.

Plant alkaloid tetrandrine downregulates IkappaBalpha kinases-IkappaBalpha-NF-kappaB signaling pathway in human peripheral blood T cell

Ling-Jun Ho  1 , Ting-Yi Juan, Ping Chao, Wan-Lin Wu, Deh-Ming Chang, Sun-Yran Chang, Jenn-Haung Lai
Affiliations

Plant alkaloid tetrandrine downregulates IkappaBalpha kinases-IkappaBalpha-NF-kappaB signaling pathway in human peripheral blood T cell

Ling-Jun Ho et al. Br J Pharmacol. 2004 Dec.

Abstract

Plant alkaloid tetrandrine (Tet), purified from Chinese herb Han-Fang Chi, is a potent immunomodulator used to treat rheumatic disorders, silicosis and hypertension in mainland China. We previously demonstrated that Tet effectively suppresses cytokine production and proliferation of CD28-costimulated T cells. In the present study, we investigated the possible involvement of nuclear factor kappa B (NF-kappaB) transcription factors, critical in CD28 costimulation, in Tet-mediated immunosuppression in human peripheral blood T cells. We showed that Tet inhibited NF-kappaB DNA-binding activities induced by various stimuli, including CD28 costimulation. At equal molar concentrations, Tet was as strong as methotrexate in suppressing CD28-costimulated NF-kappaB activities. Since Tet itself did not affect NF-kappaB binding to its corresponding DNA sequence, the results suggested that Tet might regulate NF-kappaB upstream signaling molecules. Further studies demonstrated that Tet could prevent the degradation of IkappaBalpha and inhibit nuclear translocation of p65 by blocking IkappaBalpha kinases alpha and beta activities. In addition, the activation of mitogen-activated protein kinases such as c-jun N-terminal kinase, p38 and extracellular signal-regulated kinase and activator protein-1 DNA-binding activity were all downregulated by Tet. Transfection assays performed in purified human peripheral blood T cells also confirmed the inhibition of NF-kappaB transcriptional activity by Tet. When four Tet analogues were readily compared, dauricine appeared to preserve the most potent inhibition on CD28-costimulated but not on H(2)O(2)-induced NF-kappaB DNA-binding activities. Our results provide the molecular basis of immunomodulation of Tet for being a potential disease-modifying antirheumatic drug in the therapy of autoimmune disorders like rheumatoid arthritis.

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Figures

Figure 1
Figure 1
Tet dose-dependently inhibited various stimuli-induced NF-κB DNA-binding activities. Human peripheral blood T cells at 2 × 106 ml−1 were pretreated with various concentrations of Tet for 2 h and then stimulated or not with PMA + ionomycin (a) for 2 h, PHA (b) for 3 h or ConA (c) for 6 h. The nuclear extracts were prepared and analyzed by EMSA as described in Methods. The results shown are representative of at least three independent experiments using different donor T cells.
Figure 2
Figure 2
Tet inhibited CD28-costimulated NF-κB DNA-binding activities but had no effect on direct binding of NF-κB to its consensus sequence. In (a), the purified T cells were treated or not with Tet at indicated concentrations for 2 h and then stimulated with anti-CD3+anti-CD28 mAbs for 6 h and the EMSA was performed as in Figure 1. In (b), after stimulated or not with anti-CD3+anti-CD28 mAbs, the T-cell nuclear extracts were preincubated in the presence or absence of anti-p65, anti-p50, anti-IκBα or anti-IκBβ polyclonal antibodies for 30 min. Then, the radiolabeled κB oligonucleotides were added into the mixture and the samples were analyzed by EMSA. S stands for supershifted bands. In (c), the T cells were treated with Tet for various time points before (−) or after (+) addition of anti-CD3+anti-CD28 mAbs. The nuclear extracts were analyzed by EMSA. In (d), after treatment, the CD28-costimulated T-cell nuclear extracts were incubated or not with various concentrations of Tet and then analyzed with EMSA.
Figure 3
Figure 3
Suppressive potency of Tet and Western DMARDs, methotrexate and sulfasalazine. The purified T cells were treated in the presence or absence of Tet, methotrexate or sulfasalazine at the indicated concentrations for 2 h and then stimulated with anti-CD3+anti-CD28 mAbs for 6 h. The EMSA analysis was performed as described in Methods.
Figure 4
Figure 4
Tet prevented IκBα degradation, nuclear translocation of p65 and blocked IKKα and IKKβ kinases activities. In (a), human peripheral blood T cells at 2 × 106 ml−1 were pretreated or not with Tet for various time points and then stimulated with PMA+ionomycin for various time points. After washing, cell pellets were collected and the total cell lysates or nuclear extracts were prepared and analyzed for determining the protein levels of IκBα and p65, respectively, in Western blotting assays as described in Methods. In (b), T cells were pretreated or not with various concentrations of Tet and then stimulated with PMA+ionomycin. After washing, cell pellets were collected and the total cell lysates were immunoprecipitated with anti-IKKα or anti-IKKβ antibodies. After sequential washing, GST-IκBα and 10 μCi of [γ-32P]ATP were added. After kinase reaction, the reaction mixture was analyzed. For determination of the IKKα and IKKβ protein level, Western blotting assays were performed. In (c), the fold induction was presented as a comparison with the intensity determined in the medium control.
Figure 5
Figure 5
Tet downregulated MAP kinase activities as well as AP-1 DNA-binding activity. In (a), human peripheral blood T cells were pretreated or not with various concentrations of Tet for 2 h and then stimulated with PMA+ionomycin for 30 min. After washing, cell pellets were collected and the total cell lysates were immunoprecipitated with anti-JNK, anti-p38 or anti-ERK antibodies. After sequential washing, the substrates (GST-c-Jun for JNK and myelin basic protein for both p38 and ERK) and 10 μCi of [γ-32P]ATP were added. After the kinase reaction, the reaction mixture was analyzed. For determination of the JNK protein level, Western blotting assays were performed. In (b), T cells at 2 × 106 ml−1 were pretreated with various concentrations of Tet for 2 h and then stimulated or not with anti-CD3+anti-CD28 mAbs for 6 h. The nuclear extracts were prepared and analyzed by EMSA as described to determine the AP-1 DNA-binding activities.
Figure 6
Figure 6
Tet suppressed transcriptional activity of NF-κB in human peripheral blood T cells. Human peripheral blood T cells at 1 × 106 ml−1 were mixed together with pNF-κB-Luc reporter plasmids and the transfection reagent. The transfection was performed using an Amaxa Nucleofector according to the manufacturer's instructions. After electroporation, the cells were equally divided and pretreated in the presence or absence of Tet at various dosages for 2 h. After stimulation with PMA+ionomycin (a) or anti-CD3+anti-CD28 mAbs (b) for another 22 h, cells were collected and the total cell lysates were analyzed for luciferase activities. The representative data out of at least three independent experiments with similar results are shown.
Figure 7
Figure 7
Dauricine, among Tet analogues, preserved the most potent capacity in inhibiting CD28-costimulated NF-κB DNA-binding activities. The structures of Tet analogues are shown in (a). In (b), T cells were pretreated with various concentrations of Tet and its analogues, including hernandezine (Her), berbamine (Ber) and dauricine (Dau) for 2 h, and then stimulated with anti-CD3+anti-CD28 mAbs for 6 h. The NF-κB DNA-binding activities were performed as described in Figure 1. In (c), the experiments were performed exactly as in (b), except that the stimuli anti-CD3+anti-CD28 mAbs was replaced with an oxidant H2O2. The representative results of at least three independent experiments are shown.
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