Li H et al / Acta Pharmacol Sin 2002 Sep; 23 (9): 775-781

Triptolide inhibits proinflammatory factor-induced over-expression of class II MHC and B7 molecules in renal tubular epithelial cells

LI Heng, LIU Zhi-Hong¹, DAI Chun-Sun, LIU Dong, LI Lei-Shi

Research Institute of Nephrology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China

¹ Correspondence to Prof LIU Zhi-Hong. Phn 86-25-480-9112. Fax 86-25-458-1545. E-mail zhihong@public1.ptt.js.cn

Received 2001-10-29 Accepted 2002-06-18

KEY WORDS triptolide; epithelial cells; major histocompatibility complex; antigens CD80; antigens CD86; intercellular adhesion molecule-1

ABSTRACT

AIM: To investigate the effects of triptolide on proinflammatory factor-induced over-expression of class II major histocompatibility complex (class II MHC), B7-1, B7-2, and intercellular adhesion molecule-1 (ICAM-1) in human proximal tubular epithelial (HKC) cells. METHODS: HKC cells were exposed to both interferon ¦Ã (IFN-¦Ã, 200 mg/L) and tumor necrosis factor a (TNF-¦Á, 20 ¦Ìg/L) and cultured in media containing different concentrations of triptolide for 24 h. Class II MHC, B7-1, B7-2, and ICAM-1 levels in HKC cells were evaluated by flow cytometry. ICAM-1 mRNA level was measured by semi-quantitative RT-PCR method. RESULTS: HKC cells expressed quite high level of ICAM-1 and very low levels of class II MHC, B7-1, and B7-2 molecules. Class II MHC, B7-1, B7-2, and ICAM-1 levels in HKC cells were significantly increased by the costimulation of IFN-¦Ã and TNF-¦Á. Triptolide inhibited the over-expression of class II MHC, B7-1, and B7-2 molecules in a concentration-dependent manner. The up-regulations of ICAM-1 molecules and ICAM-1 mRNA level were not altered by triptolide. Conclusion: Triptolide can significantly inhibit proinflammatory factors induced over-expression of class II MHC, B7-1, and B7-2 costimulatory factors in tubular epithelial cells. These results may contribute to the therapeutic effects of triptolide in some renal diseases.

INTRODUCTION

Renal tubular epithelial cell is one of the major cell kinds in tubulointerstitium, and always thought to be the "victim" of tubulointersitial injuries, but recent works suggested that renal tubular epithelial cells played an important role in renal inflammatory damages. Renal tubular epithelial cells have been proved to up-regulate class II major histocompatibility complex (class II MHC) molecules in inflammatory renal diseases and in vitro, when stimulated by IFN-¦Ã and lipopolysaccharide (LPS)^[1-3]. Moreover, in vitro and in vivo studies demonstrated that renal tubular epithelial cells could be induced to express costimulatory molecules, such as B7-1 and ICAM-1, under distinct experimental conditions^[2-4]. The relevance of these cell-surface markers to immune-mediated events in the kidney has been further supported by their increased expression on renal tubular epithelial cells in vivo, in settings such as chronic-¦Ãraft-versus-host disease, allograft rejection, and lupus nephritis^[1,2,5,6]. These results suggest that renal tubular epithelial cells may play an important role in initiating or propagating renal damage through interactions with T lymphocytes.

Tripterygium wilfordii Hook f (TWHf), a member of celastraceae plant family, has been used as anti-inflammatory agent in traditional Chinese medicine for centuries^[7]. The extract of the herb was proved to have potent immunosuppressive activity in vivo and in vitro^[8,9]. Studies have shown that TWHf extract is effective in the therapy of rheumatoid arthritis, systemic lupus erythematosus, glomerular nephritis, and acute rejection after transplantation^[10,11]. Hundreds of patients who suffered in inflammatory renal diseases had taken the TWHf extract therapy and got obvious improvement in our institute. Among the large number of components that have been isolated from the extract of TWHf, triptolide is one of the major active components with most potent immunosuppressive activity^[12]. To further understand the mechanism of the immunosuppressive activity of triptolide, the effects of triptolide on the regulation of class II MHC, B7-1, B7-2, and ICAM-1 molecules in human proximal tubular epithelial cells were investigated in this study.

MATERIALS AND METHODS

Reagents Triptolide (purity >99 %) was obtained from Institute of Dermatology, Chinese Academy of Medical Sciences (Nanjing, China)^[13]. Human IFN-¦Ã was purchased from Jingmei Biotech Co Ltd. (Shenzhen, China). Human recombinant TNF-¦Á was purchased from Promega (Madison, WI, USA). Monoclonal mouse anti-human HLA-DP, DQ, and DR antigen antibody (Clone: CR3/43) which was able to anti HLA-DP, DQ, and DR at the same time, monoclonal mouse anti-human ICAM-1 (CD54) antibody (Clone: 6.5B5) and fluorescein isothiocyanate (FITC) labeled rabbit anti-mouse immunoglobulins were purchased from DAKO (Glostrup, Denmark). Monoclonal mouse anti-human CD80 (B7-1, Clone: BB1) and CD86 (B7-2, Clone: FUN-1) antibody were got from PharMingen (San Diego, CA, USA).

Cell culture and treatment Human renal proximal tubular epithelial (HKC) cells was a gift from Prof ZHENG Fa-Lei (Peking Union Medical College, Beijing, China)^[14]. Cells in passages 10-15 were cultured, and treated in RPMI-1640 (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10 % heat-inactivated fetal calf serum (FCS). Cells at 80 %-90 % confluent were cultured in serum free medium for 24 h before treated with IFN-¦Ã (200 ¦Ìg/L), TNF-¦Á (20 ¦Ìg/L), and different concentrations of triptolide (0.4, 2.0, 10 ¦Ìg/L) for another 24 h. Cells cultured in RPMI-1640/10 % FCS or treated with IFN-¦Ã and TNF-¦Á only served as controls. Human spleen cells were primary cultured from donor spleen and served as positive control for measurement of class II MHC, B7-1, B7-2, and ICAM-1 molecules by flow cytometry. In brief, donor spleen tissue was sliced into 1 cm³ cubes, and squeezed through a 200-hole/cm² steal mesh. Single nuclear cells were collected by centrifugation and cultured in RPMI-1640 containing 10 % FCS and lipopolysaccharide (LPS, 10 mg/L) for 72 h.

Flow cytometry HKC cells (1×10⁶ ) were harvested by versene solution (0.02 %), washed twice in 1 mL PBS (0.01 mol/L, pH 7.4) solution, fixed in 0.1 % paraformaldehyde for 15 min, and then washed and incubated in 200 mL PBS containing 0.5 % bovine serum albumin (BSA) and 0.02 % Na₃N for 40 min at 4 ¡æ with proper antibodies (mouse anti-human HLA-DP, DQ, DR, ICAM-1, CD80, and CD86 antibody). Then the cells were washed and incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG in dark at 4 ¡æ for 30 min. The antibodies were diluted to recommend concentrations according to the manufactures' instructions. Background fluorescence was adjusted with cells labeled with mouse control antibodies as the first antibody. LPS-activated human spleen cells served as positive controls. Analyses were done on a Coulter flow-cytometer (Miami, Florida, USA).

RT-PCR Total cellular RNA was isolated using Trizol reagent (Sangon, Shanghai, China) in accordance with the manufacturer's instructions. After assessing purity of final products by A value ratios at 260/280 nm, RNA samples were reverse-transcribed into cDNA by a reverse-transcription kit (Promega). The human ICAM-1 primers were 5'-TATGGCAACGACTCC TTCT-3' (sense) and 5'-CATTCAGCGTCACCTTGG-3' (antisense). A pair of porcine ¦Â-actin primers was used at the same time as control: 5'-CTACAA-TGAGCTGCGTGTGG-3' (sense) and 5'-TAGCTCTTCTTCAGGGAGGA-3' (antisense). After amplification, PCR products were electrophoresed through a 2 % agarose gel with ethidium bromide (0.5 mg/L) to visualize the DNA bands. The expected lengths of PCR products were 237 bp (ICAM-1) and 450 bp (¦Â-actin). The results were analyzed by BF-300 image system and its software provided by Sixing Biotech (Shanghai, China).

Statistical analysis Data were expressed as mean±SD. Analysis of variance (ANOVA) and F test were used to evaluate the statistical significance. P<0.05 was considered statistically significant.

RESULTS

IFN-¦Ã and TNF-¦Á induced over-expression of class II MHC, B7-1, B7-2, and ICAM-1 in HKC cells The LPS-stimulated spleen cells expressed high levels of class II MHC, B7-1, B7-2, and ICAM-1 molecules when analyzed by flow cytometry. HKC cells cultured in RPMI-1640/10 % FCS expressed quite high levels of ICAM-1 and very low levels of class II MHC, B7-1, and B7-2 molecules. When stimulated with IFN-¦Ã (200 ¦Ìg/L) and TNF-¦Á (20 mg/L) together for 24 h, the expression of class II MHC, B7-1, B7-2, and ICAM-1 in HKC cells are all significantly increased (Tab 1, Fig 1). The expression of ICAM-1 mRNA also increased significantly by the costimulation of IFN-¦Ã and TNF-¦Á (Fig 2).

Tab 1. Effect of triptolide (Trip) on the expression (mean fluorescence intensity) of class II MHC, B7-1, B7-2, and ICAM-1 in human tubular epithelial cells. n=3. Mean±SD. ^cP<0.01 vs control. ^eP<0.05, ^fP<0.01 vs IFN-¦Ã+TNF-¦Á group.

Group	Class II MHC	B7-1	B7-2	ICAM-1
Control	1.7¡À0.4	0.93¡À0.15	0.92¡À0.24	99¡À18
IFN-g (200 mg/L) +TNF-a (20 mg/L)	7.3¡À1.5c	1.69¡À0.18c	2.3¡À0.3c	155¡À24c
IFN-g +TNF-a+Trip (0.4 mg/L)	5.1¡À1.0	1.7¡À0.3	1.9¡À0.4	161¡À21
IFN-g +TNF-a+Trip (2 mg/L)	4.9¡À1.0e	1.42¡À0.25	1.58¡À0.16f	166¡À31
IFN-g +TNF-a+Trip (10 mg/L)	2.94¡À0.23f	1.34¡À0.19e	1.45¡À0.28f	154¡À31

Fig 1. IFN-¦Ã and TNF-¦Á induced up-regulation of the expression of class II MHC, B7-1, B7-2, and ICAM-1 in HKC cells. A: class II MHC; B: B7-1; C: B7-2; D: ICAM-1. Line 1 (black): Negative control; Line 2 (blue): Cells without IFN-¦Ã and TNF-¦Á treatment; Line 3 (green): Cells treated with IFN-¦Ã (200 ¦Ìg/L)and TNF-¦Á (20 ¦Ìg/L).

Fig 2. ICAM-1 mRNA expression in HKC cells. A: ICAM-1 mRNA were reverse-transcribed, amplified with PCR, and electrophoresed through a 2 % agarose gel. B: The result was analyzed by BF-300 Image System. Lane 1: Control; Lane 2: Treated with IFN-¦Ã (200 ¦Ìg/L) and TNF-¦Á (20 ¦Ìg/L); Lane 3: Treated with triptolide (2 ¦Ìg/L), IFN-¦Ã, and TNF-¦Á; Lane 4: Treated with triptolide (10 ¦Ìg/L), IFN-¦Ã, and TNF-¦Á. n=4. MeanSD. ^bP<0.05 vs control.

Triptolide inhibited the up-regulation of class II MHC, B7-1, and B7-2 Triptolide inhibited the over-expression of class II MHC, B7-1, and B7-2 molecules in a dose-dependent manner. But triptolide has no significant influence on the proinflammatory factors induced up-regulation of ICAM-1 molecules and ICAM-1 mRNA level. (Tab 1, Fig 2, 3)

Fig 3. The modulatory effects of triptolide on the expression of class II MHC, B7-1, B7-2, and ICAM-1 in HKC cells. A: class II MHC; B: B7-1; C: B7-2; D: ICAM-1. Line 1 (red): Treated with triptolide (10 ¦Ìg/L), IFN-¦Ã, and TNF-¦Á; Line 2 (green): Treated with IFN-¦Ã and TNF-¦Á.

DISCUSSION

Tripterygium wilfordii Hook f (TWHf) has been used in traditional Chinese medicine for centuries. A large number of compounds have been identified from the extract of TWHf, including diterpenoids (such as triptolide and tripdiolide), alkaloids, glycosides, ¦Â-sitosterol, and triptoquinones^[15]. Previous studies have shown that triptolide (PG490) is the most efficient constituents of TWHf with potent immunsuppessive and anti-inflammatory abilities. Some other ingredients of TWHf, such as tripdiolide and tripcholorolide, also have been proved to have immunosuppressive effect by some experimental systems, but their effects seems not as strong as triptolide's^[16]. Although triptolide is often used as a replacer of corticoid in the patients who can not stand corticoid-therapy, our clinical experience and some experimental results showed that the effects of triptolide were different from some other immunosuppressants. Triptolide is useful in some patients who suffered in IgA nephropathy to whom corticoid was not effective^[17], and is more effective in preventing T cell proliferation and interferon-gamma production than is FK506^[18]. Lots of works have been done to investigate the mechanisms of Triptolide's potent immunosuppressive effects. Studies have shown that triptolide inhibits mitogen-induced lymphocyte proliferation, mixed lymphocyte reaction, generation of cytotoxic T cells and induces apoptotic death of T lymphocyte^[13,19,20]. Trip-tolide can also markedly increase cAMP/PKA activity in T lymphocyte^[21], and is a potent inhibitor of NF-¦ÊB activation in T lymphocyte^[22]. However, the previous works on triptolide focused on its effects on lymphocytes, the effects of triptolide on the immune abilities of renal tubular epithelial cells are still not understood. In this study, the impact of triptolide on class II MHC, B7-1, B7-2, and ICAM-1 expression on renal tubular epithelial cells was investigated. Class II MHC plays a major role in the antigen-specific T-cell activation by presenting antigen and peptide antigen to the T-cell receptor (TCR) on T lymphocyte^[23]. But this trimolecular interaction is insufficient for complete triggering of T-cell activation. Costimulatory molecules, such as B7-1, B7-2, and CD28 (one of B7-1 and B7-2's counter receptors) contribute to T-cell activation by increasing or stabilizing adhesion between T cell and antigen presenting cell and by transmitting signals that augment TCR-mediated activation signals^[24]. CD28 is expressed almost exclusively on T cells, but B7-1 and B7-2 are expressed or could be induced to express on a variety of cell types, including renal tubular epithelial cells in this study. Renal tubular epithelial cells which have been induced to express class II MHC and B7 costimulatory factors have been proved to have T-cell-activating functions^[2]. ICAM-1 is another type of costimulatory factor, and is abundantly expressed on tubular epithelial cells. ICAM-1 by itself does not elicit an inflammatory response and more likely functions as an inflammation-enhancing molecule^[25]. In this study, we found triptolide could significantly inhibit proinflammatory factors induced up-regulation of class II MHC, B7-1, and B7-2 on tubular epithelial cells, but had no significant influence on ICAM-1 expression. These results suggest that inhibition of class II MHC and B7 costimulatory factors expressions in tubular epithelial cells, and further inhibition of the antigen-presenting activity of tubular epithelial cells may also contribute to the immunosuppressive effects of triptolide.

The effect of IFN-¦Ã or TNF-¦Á alone on the expression of those factors on HKC cells was observed too. We found that IFN-¦Ã could up-regulate the expression of class II MHC and ICAM-1 but not B7-1 and B7-2, and that TNF-¦Á could up-regulate the expression of B7-1, B7-2, and ICAM-1 but not class II MHC (data not shown). But the results show that triptolide can inhibit the over-expressions of class II MHC, B7-1, and B7-2 but not ICAM-1. The molecular mechanisms underlying these manifestations are still not clear now.

REFERENCES

• 1 Wuthrich BP, Yui MA, Mazoujian G, Nabavi N, Glimcher LH, Kelly VE. Enhanced MHC class II expression in renal proximal tubules precedes loss of renal function in MRL-lpr mice with lupus nephritis. Am J Pathol 1989; 134: 45-51.
• 2 Banu N, Meyers CM. IFN-¦Ã and LPS differentially modulate class II MHC and B7-1 expression on murine renal tubular epithelial cells. Kidney Int 1999; 55: 2250-63.
• 3 Gonzalez-Posada JM, Garcia-Castro MC, Tamajon LP, Torres A, Hernandez D, Losada M, et al. HLA-DR class II and ICAM-1 expression on tubular cells taken by fine-needle aspiration biopsy in renal allograft dysfunction. Nephrol Dialysis Transplant 1996; 11: 148-52.
• 4 Jevnikar AM, Wuthrich RP, Takei F, Xu HW, Brennan DC, Glimcher LH, et al. Differing regulation and function of ICAM-1 and class II antigens on renal tubular cells. Kidney Int 1990; 38: 417-25.
• 5 Huan HD, Liu ZH, Zhou H, Wang QW, Ji SM, Li LS. The expression of B7 molecules in the transplanted renal tissue. Chin J Nephrol 1998; 14: 143-7.
• 6 Wuthrich RP, Jevnikar AM, Takei F, Glimcher LH, Kelley VE. Intercellular adhesion molecule-1 (ICAM-1) is upregulated in autoimmune lupus nephritis. Am J Pathol 1990; 136: 441-50.
• 7 Jia L. Chemistry and pharmacology and clinical application of the plants of Tripterygium family. Chin Pharm Bull 1985; 20: 101-5.
• 8 Tao XL, Davis LS, Lipsky PE. Effect of the Chinese herbal remedy Tripterygium wilfordii Hook F on human immune responsiveness. Arthritis Rheum 1991; 34: 1274-81.
• 9 Gu WZ, Brandwein SR, Banerjee S. Inhibition of type II collagen induced arthritis in mice by an immunosuppressive extract of Tripterygium wilfordii Hook f. J Rheumatol 1992; 19: 682-8.
• 10 Gu WZ, Banerjee S, Rauch J, Brandwein SR. Suppression of renal disease and arthritis, and prolongation of survival in MRL-1pr mice treated with an extract of Tripterygium wilfordii Hook f. Arthritis Rheum 1992; 35: 1381-6.
• 11 Yang JW, Fan ZM, Dai CS, Liu ZH, Li LS. Effects of triptolide in treating of acute allograft rejection. Chin J Nephrol Dialysis Transplant 1997; 6: 214-23.
• 12 Gu WZ, Chen R, Brandwein S, McAlpine J, Burres N. Isolation, purification, and characterization of immunosuppressive compounds from Tripterygium: triptolide and tripdiolide. Int J Immunopharmacol 1995; 17: 241-51.
• 13 Yang Y, Liu ZH, Tolosa E, Yang JW, Li LS. Triptolide induces apoptotic death of T lymphocyte. Immunophar-macology 1998; 40: 139-49.
• 14 Wen XY, Zheng FL, Gao RT, Li Y, Sun Y, Zhang XM. Transdifferentiation of cultured human renal tubular epithelial cells induced by aristolochic acid I. Chin J Nephrol Dialysis Transplant 2000; 9: 206-9.
• 15 Kutney JP, Hewitt GM, Lee G, Piotrowska K, Roberts M, Rettig SJ. Studies with tissue cultures of the Chinese herbal plant, Tripterygium wilfordii. Isolation of metabolites of interest in rheumatoid arthritis, immunosuppression, and male contraceptive activity. Can J Chem 1992; 70: 1455-80.
• 16 Kupchan SM, Court WA, Dailey RG Jr, Gilmore CJ, Bryan RF. Triptolide and tripdiolide, novel antileukemic diterpenoid triepoxides from Tripterygium wilfordii. J Am Chem Soc 1972; 94: 7194-95.
• 17 Li LS, Liu ZH. The prospect of Tripterygium wilfordii application in the field of nephrology. Chin J Nephrol Dial Tranplant 1997; 6: 2-4.
• 18 Chan MA, Kohlmeier JE, Branden M, Jung M, Benedict SH. Triptolide is more effective in preventing T cell proliferation and interferon-¦Ãamma production than is FK506. Phytother Res 1999; 13: 464-7.
• 19 Pu LX, Zhang TM. Effects of triptolide on T lymphocyte function in mice. Acta Pharmacol Sin 1990; 11: 76-9.
• 20 Yang SX, Xie SS, Gao HL, Ma DL, Long ZZ. Triptolide suppresses T lymphocyte proliferation by inhibiting interleukin-2 receptor expression, but spares interleukin-2 production and mRNA expression. Int J Immunopharmacol 1994; 16: 895-904.
• 21 Yang JW, Dai CS, Chen ZH, Liu ZH, Li LS. Effects of triptolide on T cell functions and the relationship with cAMP/PKA system. Chin J Nephrol Dialysis Transplant 1997; 7:106-17.
• 22 Liu H, Liu ZH, Chen ZH, Yang JW, Li LS. Triptolide: a potent inhibitor of NF-kB in T-lymphocytes. Acta Pharmacol Sin 2000; 21: 782-6.
• 23 Collins TL, Kassner PD, Bierer BE, Burakoff SJ. Adhesion receptors in lymphocyte activation. Curr Opin Immunol 1994; 6: 385-93.
• 24 June CH, Bluestone JA, Nadler LM, Thompson CB. The B7 and CD28 receptor families. Immunol Today 1994; 17: 321-31.
• 25 Hagerty DT, Allen PM. Processing and presentation of self and foreign antigens by the renal proximal tubule. J Immunol 1992; 148: 2324-30.