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Keywords:

  • triptolide;
  • collagen-induced arthritis;
  • T-cell receptor;
  • interleukin-10

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Triptolide (TP) has been used in the treatment of rheumatoid arthritis (RA), but its mechanism of action is not understood. T-cell activation and associated release of cytokines appear to be major factors in the pathogenesis of RA. The overexpression of T-cell receptor (TCR) variable gene (V gene) fragments can cause the activation and infiltration of autoreactive T cells. This study examines the effects of TP on rats with collagen-induced arthritis (CIA). The levels of interleukin-10 (IL-10) in the serum were examined with ELISA. Compared to the CIA group, the levels of IL-10 were greater in the TP treatment group. Real-time quantitative polymerase chain reaction confirmed that the expression of TCR V beta (BV) 15 and TCR BV19 was increased in the CIA group, whereas in the TP treatment group, the expression was decreased. In this study, TP was found to enhance IL-10 levels and decrease the expression levels of TCR BV15 and TCR BV19. These changes might help explain the effectiveness of TP in the treatment of RA. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Rheumatoid arthritis (RA) is chronic autoimmune disease characterized by multiple joint inflammations, hyperplasia of synovial cells, and destruction of articular cartilage. About 0.37%–1% of the world's population is afflicted by RA (Brenol et al., 2007). Without appropriate early treatment, the disability rate is serious. The 2-year disability rate is 50% and the 3-year disability rate is 70%, meanwhile, RA decreases the mean expected life by 3–10 years (Rasker and Cosh, 1981). Indeed, as a common and highly disabling chronic condition, RA results in a considerable socioeconomic burden. However, the exact etiology and pathogenic mechanisms of RA are still unclear, and specific and effective therapies are lacking.

Currently available anti-RA drugs include nonsteroidal anti-inflammatory drugs, such as dichlorophen, loxoprofen, and nabumetone, cycloxygenase-2 inhibitors such as nabumetone (O'Dell, 2004), meloxicam, and nimesulide, disease-modifying antirheumatic drugs, such as methotrexate (JKR), leflunomide, and hydroxychloroquine (Jaimes-Hernandez et al., 2004), and glucocorticoids. The perennial vine Tripterygium wilfordii Hook. f. (Celastraceae) is an herb, also called “Lei Gong Teng” (Thunder God Vine or “three-wing nut”), with confirmed anti-inflammatory, immunosuppressive, and antiproliferative effects. It has been used in Chinese medicine for the treatment of autoimmune diseases, including RA, for more than four decades. Its effectiveness in treating RA is remarkable. In a 6-week randomized double-blind, placebo-controlled study of 61 patients with RA in China, T. wilfordii (TW) remarkably improved RA symptoms (Cibere et al., 2003). Tripterygium Hook. f. contains complex chemical constituents, and many monomers have been isolated from this plant (Brinker et al., 2007). One such monomer is triptolide (TP), which has shown considerable efficacy in the treatment of RA. Recent studies showed that the effect of TP on Peyer's patch immune cells might partially explain some of the immunosuppressive activities of TP (Xiao et al., 2009).

Many studies demonstrated that activated T cells played a central role in collagen-induced arthritis (CIA). It was believed that T-cell activation and cytokines produced during this process were the major preceding factors of CIA injuries (Benson et al., 2000). Specifically, activated antigen-reactive T-cell infiltration had been found in the synovium of the joints in RA patients (Catchpole et al., 2002). With the restricted expressions of some T-cell receptor (TCR) BV gene fragments, the profile of mature TCRs may be altered by exogenous presenting antigens and autoantigens. During specific antigen recognition, some genes in the TCR BV gene subfamily show dominant amplification; after its V region recognizes the antigen, the gene will rearrange, and mRNA expression level change (Chen et al., 1994). The pathologically relevant restricted V gene usage by such autoreactive T lymphocytes has become a topic of considerable research in autoimmune diseases (Bolon, 2011).

Therefore, research on TCR BV gene usage is important for identifying antigens that induce the pathogenesis of RA and for understanding the biological features of autoreactive T cells. In this study, using the rat model of CIA, the effectiveness of TP in treating CIA in rats was studied. Additionally, the effects on the TCR BV gene mRNA expression in peripheral blood were studied in an attempt to reveal the mechanism of action of TP in treating RA. The results from this study help explain the pathogenetic mechanisms of RA and identifying novel TCR-targeted therapies.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Animals

Five-week-old female Wistar rats, weighting 120 ± 15 g, were purchased from the Experimental Animal Center of the Second Affiliated Hospital, Harbin Medical University, Harbin, China. The studies were approved by the Ethics Committee for Animal Experiments at the College of Pharmacy, Harbin Medical University.

Reagents

Chicken Type II collagen, 5 mg (Sigma), Freund's adjuvant complete (Sigma), TP (Courtesy of Collage of Pharmacy, Heilongjiang University of Chinese Medicine), UltraPure™ diethypyrocarbonate (DEPC)-treated water (Invitrogen), rat interleukin-10 (IL-10) Enzyme linked immunosorbent assay (ELISA) reagent kid (Boster, Wuhan, China), TRIzol reagent (Invitrogen), RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Lithuania), and Maxima™ SYBR Green qPCR Master Mix (2×) kit (Fermentas) were used in this experiment. Primer synthesis was performed by Sangon Biotech (Shanghai), Shanghai, China.

Rat Model of CIA

An experimental rat model of CIA was established using methods previously described (Xu et al., 2011). The rats were randomly divided into three groups of 10 rats each: control nondisease group (normal group), CIA model control group (disease group), and CIA group treated with TP (therapy group). In the therapy group, 15 days after immunization, TP at a dose of 11.62 μg/(kg day) in a volume of 1 mL/100 g body weight was administered intragastrically daily for 6 consecutive weeks. Meanwhile, an equal volume of normal saline was administered intragastrically in the normal and disease control groups.

Assessment of Disease Activity in RA Using the Arthritis Index

The severity of arthritis manifested in each rat was determined by using the arthritis index (Wang et al., 2011). Based on the range and severity of joint swelling and redness, as well as the joint deformation, the score of each limb was graded from 0 to 4; 0, indicated no redness and swelling; 1, indicated swelling in toe joints; 2, indicated swelling in toe joints and vola pedis; 3, indicated swelling under the ankle; and 4, indicated swelling that involved the whole ankle. The highest possible score was 16. Average score in the arthritis index was the sum score divided by the sum number of rats. In the study, the average arthritis index score of CIA was 5.0 ± 0.4.

Imaging of Ankle Joints and Histopathological Examinations of the Ankles

Radiographs of ankle joints were taken after the rats were anesthetized by amobarbital. At necropsy, the ankle joints were harvested, then fixed in 4% paraformaldehyde, decalcified with 10% ethylene diamine tetraacetic acid (EDTA), and embedded in paraffin. Sections were prepared, stained with hematoxylin and eosin, and observed by light microscopy.

Spleen and Thymus Weights and Quantification of Peyer's Patches

Body weights and weights of the spleen and thymus were measured at necropsy. The spleen weights and thymus weights were normalized to body weights. Peyer's patches that exist in the small intestine, which can reflect the immune status of mucous membrane were counted by gross observation eye (Wang, 2000).

Determination of Serum IL-10 Levels by ELISA

Serum was prepared from rat peripheral blood and the IL-10 level was determined by ELISA (Wuhan Soster Bio-Engineering). The optical density (OD) value was read on an automatic ELISA reader. The concentration of IL-10 in the samples was then determined by comparing the OD of the samples to the standard curve.

Isolation of Rat Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque density gradient centrifugation from peripheral blood collected in EDTA-K2 tubes (Tao, 1983).

Total RNA Extraction and First-Strand cDNA Synthesis

RevertAid™ First Strand cDNA Synthesis Kits (MBI) were used in the study. Total RNA was extracted using the TRIzol reagent (Invitrogen) according to manufacturer's instructions. First-strand cDNA was synthesized according to the user's manual of the reverse transcriptase reagent (Xu et al., 1999).

Primer Design and Synthesis

Using established methods (Gold et al., 1992), Cβint5′-ATCTAAGCTTCTGATGGCTCAAACAAG-3′ was designed as the downstream primer. Rat β-actin was used as the endogenous reference gene, with an upstream primer sequence of 5′-GCCATGTACGTAGCCATCCA-3′ and downstream primer sequence of 5′-GAACCGCTCATT GCCGATAG-3′.

Real-Time Quantitative Polymerase Chain Reaction Determination of TCR BV Gene mRNA Expression

Using previously described methods (Hirokawa et al., 2000; Evans et al., 2001; Li et al., 2007), polymerase chain reaction (PCR) was performed using an ABI 7900 Sequence Detection System (Applied Biosystems). The expressions of individual BV genes, as reflected by the cycle threshold value, were calculated based on signal intensity of the PCRs. The expressions of TCR BV gene family members were measured in each group.

Statistical Analysis

SPSS 13.0 was used for all statistical analysis. Univariate multifactor analysis of variance and chi-squared tests were performed. All data were expressed as mean ± SD. P values less than 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Progression of CIA

The progression of the CIA in the disease group up to 3 weeks after immunization is presented in Table 1. In the disease group, the symptoms progressively increased reaching a peak on Week 5. In the therapy group treated with TP, however, the progression was significantly less compared to that in the corresponding disease group up to 6 weeks after immunization (P < 0.05).

Table 1. The progression of AI scores in therapy group and disease group
GroupTwo weeksThree weeksFour weeksFive weeksSix weeksSeven weeksEight weeksNine weeks
  1. Data are mean ± SD, n = 10.

Disease4.9 ± 2.696.2 ± 3.0110.4 ± 2.4111.9 ± 1.9712.7 ± 2.5812.8 ± 2.4912.9 ± 2.1313.0 ± 2.11
Therapy4.7 ± 2.835.9 ± 2.928.4 ± 2.177.3 ± 2.216.8 ± 2.576.5 ± 2.376.3 ± 2.586.5 ± 2.27

X-Ray

In the normal group (Fig. 1A), the ankles had normal joint spaces and the articular facets were smooth; no hyperostosis, joint stiffness, osteopenia, or swelling of soft tissue surrounding the joints was observed, as expected. In the disease group (Fig. 1C), the ankles had relatively normal joint spaces, but the articular facets were not smooth. The cortical bone was slightly thickened, but no overt osteopenia was observed. Obvious swelling of the soft tissue surrounding the joints was observed. In the therapy group treated with TP (Fig. 1B), the ankles had normal joint spaces and the articular facets were smooth; no hyperostosis, joint stiffness, or osteopenia was observed. The soft tissue surrounding the joints was, however, slightly swollen.

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Figure 1. X-ray imaging result of ankle joints in three groups. (A) X-ray imaging of the normal group. (B) X-ray imaging of the therapy group. (C) X-ray imaging of the disease group.

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Histopathological Findings

The histological sections of ankles of all mice were observed under the light microscope. In the normal group, clear articular cavity and obvious subsynovial cartilaginous tissue were observed. Additionally, no lymphocyte infiltrations were observed (Fig. 2A). In the disease group, a dense inflammatory infiltrate was found in the synovium tissue (Fig. 2B). In the therapy group treated with TP (Fig. 2C), inflammatory infiltrate was reduced when compared with the untreated disease group.

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Figure 2. Histopathology of the ankle joints. (A) Normal, nondisease control group. (B) The disease group. (C) Disease group treated with TP (×100). Compared to the disease group, the inflammatory infiltrate was reduced and the inflammatory response was less in the therapy group.

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Spleen and Thymus Weights

The spleen and thymus weights were normalized to body weights. The normalized spleen weights of disease group were significantly higher than the normal group (P < 0.01) (Fig. 3). The normalized spleen weights of the therapy group were significantly lower than the disease group (P < 0.05). No significant difference was found between the therapy group and the normal group. There were no significant differences in the normalized thymus weights among the three groups.

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Figure 3. The comparison of normalized spleen weight and thymus weight in the three experimental groups. The normalized spleen weight of the therapy group was significantly lower than the disease group. No significant difference was found between the therapy group and the normal group. No significant difference in normalized thymus weight was found among these three groups.

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Numbers of Peyer's Patches

The disease group had significantly more Peyer's patches than the normal group (P < 0.05). However, the number of Peyer's patches was much lower in the therapy group than in the disease group (Fig. 4).

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Figure 4. Numbers of Peyer's patches in the three experimental groups. The number of Peyer's patches was less in the therapy group than in the disease group, indicating the effectiveness of TP.

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Expression of Serum IL-10

The levels of IL-10 were remarkably decreased in the disease group compared with the nondisease controls. However, the levels of IL-10 increased in CIA group after treating with TP (Table 2).

Table 2. Expression of serum IL-10 in three groups (pg/mL)
GroupNOD450 nmIL-10P-value
  • Data are mean ± SD.

  • a

    P < 0.05, compared with normal group.

  • b

    P < 0.05, compared with disease group.

Normal100.223 ± 0.018133.10 ± 16.9 
Disease100.150 ± 0.01023.77 ± 4.97<0.05a
Therapy100.188 ± 0.01480.68 ± 10.92<0.05b

TCR BV Gene mRNA Expression in PBMC

The expression levels of TCR BV15 and TCR BV19 in PBMC were significantly greater in the disease group, suggesting the presence of biased usage of TCR BV genes under the stimulation of antigen. The expression profiles of TCR BV genes in PBMC in the therapy group indicating that TP could reduce or prevent the rise in the expression levels of both TCR BV15 and TCR BV19. (Fig. 5)

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Figure 5. mRNA expression of TCR BV15 and TCR BV19 in peripheral blood mononuclear cells (PBMCs). Compared to the disease group, the expression levels of TCR BV15 and TCR BV19 in PBMC was significantly less in the therapy group.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

RA is an autoimmune disorder characterized by the proliferation of autoantigen-reactive T cells and their invasion into peripheral joints (Fournier, 2005). The pathogenesis of RA is closely related to cell-mediated immunity, which is initially characterized by the sensitization and proliferation of T cells.

The TCR, a specific receptor of T cells that recognizes and binds to exogenous antigens, is an important molecule involved in specific immune recognition (Sundberg, 2009). As the characteristic surface marker of all T cells, the TCR is a heterodimer comprising alpha (α) and beta (β) chains, or gamma and delta (γ/δ) chains. Most T cells in the peripheral blood have α/β chains. In addition, α/β T cells are also the main cell population participate in the immune response (Geissinger et al., 2005). Some TCRs BV gene, which is one of subfamilies coding V region peptides of β chains of TCR, was found to be significantly upregulated in response to specific antigens. When incubated with peptide-specific Th1 cell clones in the absence of antigen-presenting cells or costimulatory molecules, only the cognate Toll-like receptor (TLR) induced partial activation through the TCR. This partial activation included rapid TCR δ-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. It provides a chance to identify the most appropriate site of pathogenic autoreactive T lymphocyte and the effective targets for specific treatment.

T. wilfordii Hook. f. (Celastraceae), also named “Lei Gong Teng” (Thunder God Vine), is a medicinal plant distributed widely in southern China. It shows dramatic efficacy in the treatment of RA and has been recommended to treat RA. T. wilfordii Hook. f. contains many molecular components including terpenoids, glucosides, alkaloids, and dulcitol, among which TP (Asano et al., 1997), a diterpenoid triepoxide, is believed to be the most important component because of its anti-inflammatory and immunosuppressive activities (Qiu and Kao, 2003).

Over the last decade, many studies have demonstrated that TP has multiple pharmacological effects including immunosuppression, anti-inflammatory activity, induction of apoptosis, and inhibition of blood vessel hyperplasia. TP can also directly inhibit the proliferation of T cells (Krakauer et al., 2005). Lai et al. (2001) found that TP-induced T-cells apoptosis and DNA damage require the activation of aspartate-specific cysteine proteinase (caspase-30), but is not related to Fas/Fas ligand (FasL) interactions. According to Hu et al. (2002), TP inhibits vascular endothelial growth factor expression and secretion in endothelial cells treated by 12-O-tetradecanoylphorbol-13-acetate and also inhibits the expression of proto-oncogene c-jun/c-fos mRNA in endotheliocytes, indicating that TP may influence the expression of vascular endothelial growth factor (VEGF) (and thus inhibit vascularization) by inhibiting the formation of activator protein-1.

In previous studies, two TCR BV gene segments (TCR BV3S1 and TCR BV18) in RA were analyzed. The research found that neither allelic frequencies nor genotypic frequencies differ among RA and healthy individuals, suggesting that there was no direct association among the TCR BV allelic variants studied and the development of RA (Dresch et al., 2003). We analyzed the TCR BV gene mRNA expression in the experimental rats. In the normal group, no skewed BV gene expression was found. In the disease group, higher expressions of TCR BV15 and TCR BV19 were observed. There are two conditions that may increase the expression of a certain BV gene subfamily. One way is the absolute increase of specific BV T-cell count. Another away is activated production of more mRNA.

In this study, using rat CIA models, we observed the therapeutic activity of TP in treating experimental RA. To elucidate the specific mechanism of action of TP in the treatment of RA, the perspective of molecular immunology was used in the study. The results showed that, in the rat CIA model, TP can relieve the swelling of foot joints, remarkably lower ankle AI, decrease the proliferation of synoviocytes, alleviate the inflammation of synovial tissues, decrease the number of Peyer's patches, and increase the serum IL-10 level. However, the exact cause of the increased BV gene subfamily could not be determined from this study. In consideration of the excessive overexpression of TCR BV gene fragments that can cause the activation and infiltration of autoreactive T cells, a possible reason for the treatment effect against RA is the ability of TP to reduce the expression levels of TCR BV15 and TCR BV19. In other words, TP may be an effective therapeutic against RA by influencing TCR BV gene mRNA expression.

IL-10 genes confer susceptibility to RA. Previous research certified that the methylation of the proximal CpGs in the IL-10 promoter may regulate gene transcription in RA (Fu et al., 2011). It is also reported that IL-10 GGC haplotype is associated with severe progression of radiographic damage, which was caused by arthritis (Ursum et al., 2010). In our study, IL-10 level in serum was much higher in the therapy group than in the normal group. This result suggested that a possible mechanism for treating RA by TP was the increase in serum IL-10 levels.

To further elucidate the therapeutic mechanism of TP in treating RA, in future experiments, it would be advantageous to study the effects of TP on the expressions of TCR BV genes and their mRNA and relevant proteins in synovial fluid and tissues. These studies could also be extended to clinical studies and trials.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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