Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activation

BBRC Biochemical and Biophysical Research Communications 319 (2004) 980–986 www.elsevier.com/locate/ybbrc

Triptolide (PG-490) induces apoptosis of dendritic cells through sequential p38 MAP kinase phosphorylation and caspase 3 activationq Qiuyan Liu,a,b Taoyong Chen,a Huabiao Chen,a Minghui Zhang,a Nan Li,a Zhanjun Lu,b Pengcheng Ma,c and Xuetao Caoa,* a

Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, PR China b Institute of Immunology, Hebei Medical University, Shijiazhuang 050017, PR China c Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing 210042, PR China Received 3 April 2004 Available online 28 May 2004

Abstract Dendritic cells (DCs) are the most potent antigen-presenting cells that play crucial roles in the regulation of immune response. Triptolide, an active component purified from the medicinal plant Tripterygium wilfordii Hook F., has been demonstrated to act as a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. However, little is known about the effects of triptolide on DCs. The present study shows that triptolide does not affect phenotypic differentiation and LPS-induced maturation of murine DCs. But triptolide can dramatically reduce cell recovery by inducing apoptosis of DCs at concentration as low as 10 ng/ ml, as demonstrated by phosphatidylserine exposure, mitochondria potential decrease, and nuclear DNA condensation. Triptolide induces activation of p38 in DCs, which precedes the activation of caspase 3. SB203580, a specific kinase inhibitor for p38, can block the activation of caspase 3 and inhibit the resultant apoptosis of DCs. Our results suggest that the anti-inflammatory and immunosuppressive activities of triptolide may be due, in part, to its apoptosis-inducing effects on DCs. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Triptolide; Dendritic cells; Apoptosis; MAPK; Caspase

Triptolide, a diterpene triepoxide, is an active component of extracts derived from the medicinal plant Tripterygium wilfordii Hook F. (TWHF), and has antiproliferative and proapoptotic activity in T cells, B cells, monocytes, tumor cells, and even in seminiferous epithelial cells [1–5]. Due to its potent anti-inflammatory and immunosuppressive properties, Tripterygium extracts have been used widely to treat autoimmune diseases including rheumatoid arthritis, immune complex q Abbreviations: DC, dendritic cells; BMDC, bone marrow-derived dendritic cells; DC-LPS, LPS-stimulated dendritic cells; DC-Trip, triptolide-treated dendritic cells; DC-Trip/LPS, trip-treated and LPSstimulated DC; DMSO, dimethyl sulfoxide; ERK, extracellular signalregulated kinase; IL, interleukin; ImDC, immature DC; mDC, mature DC; LPS, lipopolysaccharide; PI, propidium iodide; Trip, triptolide. * Corresponding author. Fax: +86-21-6538-2502. E-mail address: [email protected] (X. Cao).

0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.201

nephritis, and systemic lupus erythematosus (SLE) in China for many years. In organ transplantation, both clinical and experimental studies have demonstrated that Tripterygium extracts effectively prolong allograft survival [6–10]. PG27, a refined extract of Tripterygium that contains PG490 (>97% purity of triptolide) as the active ingredient, is an effective immunosuppressant that prolongs heart and kidney allograft survival in rat transplantation models [6]. PG27 can also effectively prevent graft versus host disease and induce antigenspecific tolerance in murine allogeneic bone marrow transplantation [7]. It has been demonstrated that triptolide exerts potent inhibitory effects on T cell activation and interleukin (IL)-2 gene expression by T cells. Triptolide inhibits transcriptional activation of the IL-2 gene by inhibiting activation of the purine-box regulator of the nuclear factor of activated T cells (NFAT) target

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DNA sequence in the IL-2 enhancer, and also by inhibiting nuclear factor-jB (NF-jB) activation [11]. In contrast, triptolide inhibits T cell activation through calcineurin-independent mechanisms, triggered by cyclosporin A (CsA)-resistant signaling initiated at the costimulatory receptor CD28 [11]. A chloroform– methanol extract of Tripterygium, T2, (5% triptolide) inhibits expression of IL-2 and immunoglobulin by peripheral blood lymphocytes [1,5]. Triptolide can also induce apoptosis of tumor cells, via the p53 pathway and via inhibition of NF-jB and AP-1 transcriptional activity [12–14]. Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APC) that play critical roles in immune regulation, ranging from tolerance induction and the prevention of autoimmunity to the induction of anti-tumor immunity and the protection against infectious agents [15,16]. They originate from bone marrowderived progenitor cells, spread via the bloodstream, and can be found in almost every organ as the sentinels of the immune system. Various experimental models have demonstrated that DCs are critical for the induction of transplant tolerance [17–19]. Donor DCs transplanted en bloc with the allograft migrate to the recipient spleen [20], where they can activate naive T lymphocytes, thereby inducing an immune response via the direct pathway of allorecognition [21]. Several studies showed that preventing the direct pathway of allorecognition by DC depletion resulted in prolonged graft survival [22]. Therefore, DCs might be an important target for immunosuppression to prevent allograft rejection. Despite that the responses of T cells to triptolide treatment are reasonably well characterized, there are few reports concerning the effect of triptolide on other kinds of immune cells, such as DC. To investigate how triptolide affects DC will aid in the full elucidation of the mechanism by which triptolide mediates anti-inflammatory and immunosuppressive effects in vivo. In present study, we investigated the direct effects of triptolide on DC differentiation, maturation, and cell survival. We demonstrate that triptolide induces DC apoptosis, and that p38 MAP kinase and caspase 3 activation are partially responsible for this apoptosis-inducing effect of triptolide. Our results cast new light on the mechanisms underlying the anti-inflammatory and immunosuppressive effects of triptolide.

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described [23]. Triptolide was reconstituted in dimethyl sulfoxide (DMSO) and stock solutions (1 mg/ml) were stored at )20 °C. Triptolide was freshly diluted to the indicated concentrations with culture medium before use. DMSO concentration in experimental conditions never exceeded 0.002% (V/V). Recombinant mouse granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) were purchased from Genzyme (Cambridge, MA). FITC- or PE-labeled antibodies against murine Iab (MHC class II), CD86, CD80, CD40, and isotype Abs were purchased from BD PharMingen (San Diego, CA). Abs against caspase 3, caspase 8, caspase 9, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-ERK (extracellular signal-regulated kinase), anti-phospho-p38, and anti-phospho-SAPK/JNK (c-Jun N-terminal kinase) were obtained from Cell Signaling Tech. (Beverly, MA). The specific p38 kinase inhibitor SB203580, pan-caspase inhibitor zVAD-fmk, and caspase 3 inhibitor DEVD-fmk were obtained from CalBiochem (San Diego, CA). Lipopolysaccharide (LPS) was purchased from Sigma (Escherichia coli, O26:B6, St. Louis, MO). Generation and phenotypic analysis of mouse bone marrow derived DC. Mouse bone-marrow derived DCs were prepared from bone marrow by culture with 10 ng/ml recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF) and 1 ng/ml IL-4, as described previously [24]. On day 6, DCs were harvested as immature DCs (imDC). For preparation of mature DCs (mDCs), imDCs were stimulated with 1 lg/ml LPS for 24 h. Triptolide was supplemented as indicated. For the analysis of the differentiation and maturation of DCs treated with or without triptolide, cells were labeled with FITC-labeled Abs as indicated, and analyzed by FACS assay as described [25]. Apoptosis assay. To determine the externalization of phosphatidylserine on the DC membrane, DCs treated with triptolide in the presence or absence of 100 lM zVAD-fmk (pan-caspase inhibitor), 100 lM DEVD-fmk (caspase 3 inhibitor) or 30 lM SB203580 (p38 inhibitor) were labeled with FITC-Annexin V/PI as instructed (BD PharMingen, San Diego, CA). To examine the alterations in mitochondria potential, cells treated as above were incubated with 1 lM Rhodamine-123 (R123) (Molecular Probes, Eugene, OR) for 20 min at 37 °C and then washed with cold PBS. These cells were analyzed with FCAS and the CellQuest software. To examine the nuclear condensation of apoptotic DCs, cells were harvested at the indicated time points and fixed with 1% paraformaldehyde on ice. Cytospin preparations were made and stained with Hoechst 33258 (Molecular Probes) for 3 min at room temperature. The percentage of apoptotic cells was determined by examining 200 cells and counting the cells that were characterized by condensed or fragmented nuclei under immunofluorescence microscopy. Activation of pro-apoptotic caspases was examined by Western blot for detection of caspase cleavage fragments. Signaling pathway assay and Western blotting. DCs were treated as indicated and total cell lysates were prepared. Equal amounts of proteins were separated by 12% SDS–PAGE and then transferred to nitrocellulose membranes. Activation of the mitogen activated protein kinase (MAPK) signaling pathways was measured by detection of phosphorylated proteins by Western blot assay [26]. Statistical analysis. Comparisons between experimental groups and relevant controls were performed by Student’s t test. A value of p < 0:05 was considered statistically significant.

Results Materials and methods Mice and reagents. Male wild-type C57BL/6 mice, 5–6 weeks of age, were purchased from SIPPR-BK Experimental Animal (Shanghai, China) and housed in a pathogen-free facility for all experiments. Crystalline triptolide (PG490, molecular weight 360, purity 99%) was obtained from the Institute of Dermatology, Chinese Academy of Medical Sciences (Nanjing, PR China), and prepared as previously

Triptolide does not affect phenotype but reduces yield of DC in vitro On day 3, 10 ng/ml triptolide (Trip) was added in DC culture system. On day 6, both untreated and Triptreated cells displayed morphologic and phenotypical

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Triptolide induces apoptosis of mouse bone marrowderived DC at different developmental stages

Fig. 1. Triptolide does not affect the phenotype of DC, but reduces cell yield during DC development. (A) Triptolide (Trip) does not affect the phenotype of DC. C57BL/6 mouse bone marrow cells were cultured with mGM-CSF and mIL-4, and triptolide (10 ng/ml) added on day 3 of culture (Trip-DC). On day 6, 1  106 DCs were stimulated with 1 lg/ ml LPS for 24 h with (DC-Trip10/LPS) or without (DC-LPS) triptolide treatments. DCs were harvested and stained with fluorescence-labeled anti-Iab (MHC-II), anti-CD80, anti-CD86, or anti-CD40 antibody, and analyzed by FACS. Unshaded regions represent isotype controls and shaded regions represent corresponding Abs as indicated. Results were expressed as fluorescence intensity (FI). Numbers above each panel indicated for mean fluorescence intensity. (B) Mouse bone marrow cells were cultured with mGM-CSF and mIL-4, and different concentration triptolide (0, 1, 5, 10, 20, 50, and 100 ng/ml) were added on day 3 of culture. After cultured for 3 days, cells were harvested and counted by trypan blue exclusion or by using the Coulter Counter. Cell survival from at least four independent experiments was expressed as mean ( SD) percentage of control DC. *p < 0:05; **p < 0:01.

characteristics of immature DCs, as demonstrated by low expression of Iab , CD80, CD86, and CD40 (Fig. 1A). When DCs were stimulated with 1 lg/ml LPS for 24 h, both untreated DC and Trip-treated DC display significantly upregulated expression of Iab , CD80, CD86, and CD40, and no differences were observed between two groups (Fig. 1A). These results indicated that triptolide did not affect phenotypic maturation and differentiation of DC. However, yields of viable cell on day 6 of culture were from reduced 20–80% in the presence of different concentrations of triptolide, as assessed by trypan blue exclusion assay (Fig. 1B).

Since 10 ng/ml triptolide significantly decreased the yield of DC, we hypothesized that this effect might be due to triptolide-induced apoptosis of DC. Therefore, DCs, generated following the standard 6-day culture with mGM and mIL-4, were treated with triptolide for 48 h. We found that the Annexin V-positive cells were significantly increased (Fig. 2A). Correspondingly, we observed a decrease in Rho123 uptake by triptolidetreated cells, indicating a reduction in mitochondrial membrane potential (Fig. 2B). As further examination, we examined the nuclear DNA by Hoechst staining, which showed an increased number of cells demonstrating condensed nuclear DNA (Fig. 2C). When day-6 DCs were treated with different concentrations of triptolide (0, 1, 5, 10, 20, 50, and 100 ng/ml, respectively), Annexin V-positive cells were increased in a dose- and time-dependent manner (Fig. 2D). In order to know whether triptolide-induced apoptosis of bone marrow-derived cells in addition to DC, we treated adherent cells on day 3 with 10 ng/ml triptolide and analyzed apoptosis everyday thereafter by Annexin V/PI and Rho123 staining. Triptolide-induced apoptosis of bone marrow-derived cells time-dependently, as evidenced by an increased percentage of Annexin V-positive cells and Rho123low cells (Fig. 3A). To investigate whether triptolide-induced apoptosis of mature DC, we treated day-6 DC with 1 lg/ml LPS for 24 h and then with 10 ng/ml triptolide for additional 48 h. We found that triptolide could induce apoptosis of mature DC, and mature DCs were more sensitive than immature DCs to apoptosis induction by triptolide (Fig. 3B). Therefore, triptolide could induce apoptosis of immature DC (day-6 DC) and mature DC (day-6 DC treated with 1 lg/ml LPS for 24 h) as well as DC progenitors at concentrations as low as 10 ng/ml. Caspase 3 is involved in triptolide-induced DC apoptosis Caspases are involved in the initiation and progression of apoptosis in a variety of cell types [27]. We examined the activation of caspase 3, caspase 8, and caspase 9 in DC after treatments with different concentrations of triptolide. Caspase 3 was activated in DC by P10 ng/ml triptolide, concentrations sufficient to induce apoptosis of DCs, as evidenced by the appearance of the 17 kDa cleaved fragment following triptolide treatments (Fig. 4A). But caspase 8 or caspase 9 was not activated. Pretreatment of immature DC with zVAD-fmk (pan-inhibitor of the caspase family) or DEVD-fmk (specific inhibitor for caspase 3) resulted in a dramatic decrease in the frequency of apoptotic DC induced by triptolide (Fig. 4B). These data sug-

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Fig. 2. Triptolide induces apoptosis of immature DC. (A) On day 6, 1  106 murine DCs were treated with triptolide (10 ng/ml). 48 h later, cells were harvested and analyzed. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. (B) DCs treated as above were labeled with Rhodamine 123 (R123) for the examination of mitochondrial membrane potential. Numerical labels indicated the percentage of cells with decreased membrane potential (M1 for R123low cells). (C) DCs treated as above were stained with Hoechst 33258 for examination of nuclear DNA condensation. The results are representative of three independent experiments. (D) Immature DCs were incubated with different concentrations of triptolide or not for 24 or 48 h. Apoptosis was detected by FACS analysis using Annexin V-FITC/PI. Apoptosis was assessed by counting the percentage of Annexin V-positive cells. Data are shown as means  SD of duplicate cultures. The results are representative of two independent experiments.

Fig. 3. Triptolide induces apoptosis of murine DC at stages of DC development. (A) Triptolide (10 ng/ml, Trip10) induces DC progenitors’ apoptosis. Day 3 DC progenitors were treated with 10 ng/ml triptolide, and cells were harvested on day 4, 5, 6, and 7 of culture for Annexin V/PI (left panel) and R123 labeling (right panel). There are significant differences in percentage of Annexin V-positive cells and R123low cells between triptolide-treated and -untreated DC, p < 0:05. (B) DCs are susceptible to triptolide-induced apoptosis at various stages of DC differentiation and maturation. Day-4 DC, day6 DC, and day-6 DC stimulated with LPS for 24 h were treated with triptolide (10 ng/ml) for 48 h and apoptosis was evaluated by Annexin V/PI staining.

gested that triptolide-induced apoptosis of DC was caspase 3-dependent. Activation of p38 is involved in triptolide-induced DC apoptosis Because MAPK signaling cascade is important in regulating apoptosis [28,29], we examined the effects of

triptolide on these signaling pathways in DC. DCs were treated with triptolide (10 ng/ml) for 0, 30 min, 1, 2, 4, 6, 12, 24, 48, and 60 h, respectively, and total cell lysates were subjected to Western blot for detection of phospho-ERK1/2, phospho-38, and phospho-SAPK/ JNK. The level of phospho-p38 MAP kinase was markedly increased in DC 12 h after exposure to triptolide treatments, which occurred prior to caspase 3

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Discussion

Fig. 4. Activation of caspase 3 is involved in triptolide-induced DC apoptosis. (A) Triptolide treatment induces activation of caspase 3. Triptolide was added at concentrations indicated on day 6, and cells were cultured for additional 48 h. Cells were then collected and total cell lysates were quantified by BCA protein assay kit. Cleavage of caspase 3 (17 kDa fragment), caspase 8, and caspase 9 was detected by Western blot. (B) Inhibition of triptolide-induced apoptosis by caspase inhibitor. On day 6 of culture, and 30 min prior to addition of triptolide (10 ng/ml), pan caspase inhibitor zVAD-fmk (100 lM) or specific caspase 3 inhibitor DEVD-fmk (100 lM) was added to cultures. Cells were harvested 48 h later and then apoptosis was evaluated by Annexin V/PI staining.

cleavage (Fig. 5A) and apoptotic changes of triptolidetreated DC (Fig. 2D). However, no effects on the activation of ERK and JNK pathways by triptolide were observed. In order to determine whether p38 MAP kinase was involved in triptolide-induced DC apoptosis, we pretreated immature DC with the specific p38 MAP kinase inhibitor SB203580 (30 lM) prior to triptolide exposure (10 ng/ml). SB203580 inhibited triptolide-induced DC apoptosis, as quantified by Annexin V/PI staining (Fig. 5B), and the cleavage of caspase 3 (Fig. 5C). However, DEVD-fmk (specific inhibitor for caspase 3) has no effect on p38 MAP kinase activation in response to triptolide treatments (data not shown), indicating that p38 activation is preceding caspase 3 activation. These results suggested that p38 activation is involved in triptolide-induced DC apoptosis, which in turn may be responsible for triptolide-induced caspase 3 cleavage.

Triptolide, an active component of T. wilfordii Hook F. (TWHF), has been widely used in the clinical treatment of autoimmune diseases and prevention of allograft rejection, and even tested in phase I clinical trials for cancer treatment [1–5]. Encouraging data on the potent immunosuppressive effects of triptolide have been obtained; however, mechanistic details remain elusive. In this study, we demonstrate that triptolide can regulate immune response by inducing DC apoptosis. The anti-inflammatory, anti-proliferative, and proapoptotic properties of triptolide are associated with inhibition of NF-jB signaling and inhibition of genes known to regulate cell cycle progression and survival. In human bronchial epithelial cells, triptolide can inhibit constitutive expression of cell cycle regulators and survival genes, including cyclins D1, B1, and A1, cdc-25, bcl-x, and c-jun [30]. It has been reported that triptolide can induce apoptosis in a variety of cell types. Triptolide alone, or in combination with TNF-a or chemotherapy, can induce apoptosis of tumor cells, including human promyelocytic leukemia, T cell lymphoma, and tumor cell lines derived from human hepatocellular carcinoma, nonsmall lung carcinoma (A549), and breast carcinoma (MCF-7) [12–14]. In lung cancer A549 and fibrosarcoma HT1080 cells, triptolide-induced apoptosis involves increased expression of p53, blockade of p21-mediated growth arrest, and inhibition of NF-jB activity. In T cell lines, triptolide-triggered apoptosis is caspase 3-dependent and Fas/Fas ligand-independent [31,32]. We demonstrate that triptolide can induce DC apoptosis, concomitant with the destruction of mitochondrial membrane potential and nuclear condensation, in a time- and dose-dependent manner. Continuous exposure of bone marrow-derived cells, including immature and mature DC, to triptolide causes apoptosis at different stages of DC development, resulting in decreased yields of DC in vitro. Our results suggest that caspase 3 is responsible for triptolide-induced DC apoptosis, supported by observations that triptolide can activate caspase 3 and that specific inhibition of caspase 3 can abrogate the apoptotic effects of triptolide on DC. We could not detect alterations in p53 or p21 expression levels, even when using high levels of triptolide (20 ng/ ml) to induce apoptosis, and the activation of caspase 8 or caspase 9 was also not observed to be induced by triptolide treatment, both of which indicate that caspase 3 may be the responsible apoptosis-inducing factor and that triptolide-induced apoptosis of DCs is through p53independent apoptotic pathway. In RAW264.7 macrophages, triptolide can inhibit MAP kinase phosphatase-1 (MKP-1) induction by LPS, concurrent with activation of p38 and JNK [33]. Exposure of dopaminergic neurons to oxidative stress induces caspase 8- and caspase 9-mediated apoptosis,

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Fig. 5. p38 MAPK is involved in triptolide-induced activation of caspase 3 and apoptosis in DC. (A) Triptolide activates p38 MAPK preceding caspase 3 activation. DCs were treated with triptolide for 0, 6, 12, 24, 48, and 60 h, and then harvested and total cell lysates were prepared. Specific Abs against phospho-p38, p38 (total), phospho-ERK, EKR (total), phospho-SAPK/JNK, SAPK/JNK (total), caspase 3 or actin were used for the Western blot assay. (B) p38 MAPK inhibitor SB203580 inhibits triptolide (10 ng/ml)-induced DC apoptosis. Thirty minutes prior to triptolide treatment (10 ng/ml), DCs were treated with SB203580 (30 lM). Cells were cultured for 48 h and apoptosis wasR evaluated by Annexin V/PI staining. (C) SB203580 inhibits the triptolide-induced activation of caspase 3 in DC. DCs were treated as in (B). Caspase 3 activation was assayed by Western blot and evidenced by the presence of the 17 kDa cleavage fragment.

which is linked to phosphorylation of p38 MAPK [34]. Triptolide alone can dramatically activate p38 in DC 12 h after treatments, preceding the occurrences of decreased mitochondria membrane potential and nuclear condensation. Inhibition of p38 activation by SB203580 blocks both triptolide-induced DC apoptosis and caspase 3 activation, suggesting that the p38 activation may be upstream of triptolide-induced caspase 3 activation and apoptosis in DC. Taken together, our study illustrates the triptolide– p38 activation–caspase 3 cleavage–apoptosis pathway in DC. Although the exact mechanism and importance of triptolide-induced apoptosis in vivo await further investigations, triptolide-induced apoptosis in immature DC, mature DC, and cells in different developmental stages of bone marrow cells may also constitute an important mechanism for triptolide-induced immunosuppression.

Acknowledgments This work was supported by grants from the National Key Basic Research Program of China (2001CB510002) and the National Natural Science Foundation of China (30121002). We sincerely appreciate the excellent technical assistance provided by our technicians Ms. Rui Zhang, Ms. Chunfang Luo, and Ms. Weiqin Ni. We thank Dr. Jane Rayner for critically reading of the manuscript.

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