Daeganghwal-tang inhibits the stem cell factor-induced migration and inflammatory cytokines secretion in mast cells

Journal of Ethnopharmacology 85 (2003) 157–161

Daeganghwal-tang inhibits the stem cell factor-induced migration and inflammatory cytokines secretion in mast cells H.Y. Shin a , H.J. Jeong a , H.J. Na a , S.H. Hong a , S.K. Lee b , K.N. Lee b , Y.S. Song b , H.M. Kim a , T.Y. Shin c,∗ a

Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University, Iksan-city, Chonbuk 570-749, South Korea b Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan-city, Chonbuk 570-749, South Korea c College of Pharmacy, Woosuk University, Jeonju, Chonbuk 565-701, South Korea Received 3 June 2002; received in revised form 13 November 2002; accepted 28 November 2002

Abstract Traditional Oriental medicinal prescription, Daeganghwal-tang (DGHT) has been used for the treatment of rheumatoid arthritis (RA) in Korea. However, its effect in experimental models remains unknown. Recent reports suggest that in patients with RA, synovial mast cells increase in number and show signs of activation and inflammatory cytokines secretion. Our results show that stem cell factor (SCF) is a potent chemotactic factor for the mast cells in vitro. The chemotactic response to SCF was blocked by DGHT. When DGHT (1 mg/ml) was added, the secretion of tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤ and IL-6 was inhibited by 60.1, 81.8, 72.5%, respectively in phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187-stimulated HMC-1 cells. In addition, the expression of TNF-␣ mRNA in HMC-1 cells was inhibited by DGHT (1 mg/ml). These findings indicate that DGHT inhibits SCF-induced migration and PMA plus calcium ionophore-stimulated inflammatory cytokines secretion in mast cells. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Daeganghwal-tang; Stem cell factor; Rheumatoid arthritis; Inflammatory cytokines

1. Introduction Daeganghwal-tang (DGHT) has been used as a traditional Oriental medicine for the treatment of rheumatoid arthritis (RA) (Lee et al., 1994). Recently, DGHT has been successfully used for the regulation of immune response in South Korea (Hwang, 1986). RA is characterized by a relapsing remitting course of joint inflammation that leads to joint destruction and disability (Decker et al., 1984; Krane and Simon, 1986). During this process, profound hypertrophic changes of the synovium with infiltration of immune cells result in the formation of a synovial pannus that invades cartilage and bone (Feldmann et al., 1992). Although the pathogenesis of RA is still not well understood, a number of cell systems and humoral factors are thought to be involved in the initiation and propagation of synovitis (Decker et al., 1984; Krane and Simon, 1986; Feldmann et al., 1992). Numerous reports have described specific cellular components of the inflamed synovial tissue, such as macrophages, T lymphocytes, neutrophils, and fibroblasts, but only recently has the mast cells been ∗ Corresponding

author. Tel.: +82-63-290-1572; fax: +82-63-290-1567. E-mail address: [email protected] (T.Y. Shin).

recognized as an important component of the rheumatoid lesion. Several histological studies have reported increased mast cell numbers in rheumatoid synovia compared with normal synovium, including their presence at sites of cartilage erosion (Crisp et al., 1984; Godfrey et al., 1984; Malone et al., 1986; Gruber et al., 1986; Bridges et al., 1991; Kopicky-Burd et al., 1988; Castor, 1960). It was determined that two of the known mast cell chemotoxins, stem cell factor (SCF) and TGF-␤1, were expressed in the synovial membrane, and contributed to mast cell chemotactic activity in the synovial fluids (Nilsson et al., 1994; Gruber et al., 1994). These observations have suggested that SCF plays a role in the accumulation and activation of mast cells in the synovium of patients with RA. The mast cells store a number of mediator molecules in their granules and can secrete these mediators into the extra cellular space upon activation (Church and Caulfield, 1993; Galli, 1993; Schwartz, 1994). Activated mast cells also secrete inflammatory cytokines such as tumor necrosis factor (TNF)-␣, interleukin (IL)-1, and IL-6. (Nilsson et al., 1999; Gordon et al., 1990). Most of these cytokines can be detected in synovial fluid from patients with RA. These cytokines are likely to have primary roles in the pathogenesis of RA (Choy and Panayi, 2001).

0378-8741/02/$ – see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(02)00369-0

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H.Y. Shin et al. / Journal of Ethnopharmacology 85 (2003) 157–161

In the present study, we investigated the effect of DGHT to block SCF induced migration in rat peritoneal mast cells (RPMCs). We also investigated the effect of DGHT on phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187-induced secretion of TNF-␣, IL-1␤, IL-6 in HMC-1 cells. TNF-␣ mRNA expression was studied in HMC-1 cells by reverse transcription-polymerase chain reaction (RT-PCR).

2. Materials and method 2.1. Reagents Bovine serum albumin (BSA), ␣-minimum essential medium (␣-MEM) and cyclosporin A (CsA), PMA, calcium ionophore A23187, metrizamide and fetal bovine serum (FBS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant murine SCF (rmSCF), The rTNF-␣, rIL-1␤ and rIL-6 were purchased from R & D systems Inc, (Minneapolis, MN, USA). 2.2. Animals Wister rats were purchased from the Dae Han Experimental Animal Center (Taejon, Korea) and the animals were maintained at the College of Pharmacy, Wonkwang University. The animals were housed in a laminar air flow room maintained at a temperature of 22 ± 2 ◦ C and relative humidity of 55 ± 10% throughout the study. 2.3. Preparation of DGHT The plant materials were obtained from the Wonkwang Oriental Medicine Hospital (Chonju, Chonbuk) and authenticated by Professor Y. S. Lyu, College of Oriental Medicine, Wonkwang University. A voucher specimen (number 01-03-21) was deposited at the herbarium of the College of Pharmacy, Wonkwang University. An extract of DGHT was prepared by decocting the dried prescription of herbs with boiling distilled water. The duration of decoction was about 3 h. The decoction was filtered, lyophilized and kept at 4 ◦ C. The yield of extraction was about 14% (w/w). The DGHT water extract powder was dissolved in sterile saline (50 mg/ml). The ingredients of DGHT include 6 g of Angelicae koreanae radix, 6 g of Cimicifugae rhizoma, 4 g of Araliae cordatae radix, 2.8 g of Atractylodes rhizoma, 2.8 g of Sinomeni caulis et rhizoma, 2.8 g of Clematidis radix, 2.8 g of Atractylodis rhizoma alba, 2.8 g of Angelicae gigantis radix, 2.8 g of Poria, 2.8 g of Alismatis rhizoma, 2.8 g of Glycyrrhizae radix. 2.4. Isolation of RPMCs RPMCs were isolated as previously described (Kim et al., 1998). In brief, rats were anesthetized by ether

and infected with 20 ml of Tyrode buffer B (137 mM/l NaCl, 5.3 mM/glucose, 12 mM/l NaHCO3 , 2.7 mM/l KCl, 0.3 mM/l NaH2 PO4 ) containing 0.1% gelatin (Sigma Chemical Co.) into the peritoneal cavity and the abdomen was gently massaged for about 90 s. The peritoneal cavity was carefully opened and the fluid containing peritoneal cells was aspirated by a Pasteur pipette. Thereafter, the peritoneal cells were sedimented at 150 × g for 10 min at room temperature and resuspended in Tyrode buffer B. mast cells were separated from the major components of rat peritoneal cells, i.e. macrophages and small lymphocytes, according to the method described by Yurt et al. (Yurt et al., 1977). In brief, peritoneal cells suspended in 1 ml of Tyrode buffer B were layered on 2 ml of 22.5% w/v metrizamide (density, 1.120 g/ml, Sigma Chemical Co.) and centrifuged at room temperature for 15 min at 400×g. The cells remaining at the buffer-metrizamide interface were aspirated and discarded; the cells in the pellet were washed and resuspended in 1 ml of Tyrode buffer A (10 mM/l HEPES, 130 mM/l NaCl, 5 mM/l KCl, 1.4 mM/l CaC12 , 1 mM/l MgC12 , 5.6 mM/l glucose) containing 0.1% BSA (Sigma Chemical Co.). Mast cell preparations were about 95% pure as assessed by toluidine blue staining. More than 97% of the cells were viable as judged by trypan blue uptake. 2.5. Chemotaxis assay An amount of 10 ng/ml of SCF (500 ␮l) or the assay medium alone were applied into each well of 4-well culture plates (Nalge Nuck International, Roskilde, Denmark). After 10-mm tissue culture inserts (Nalge Nuck International) were placed into each well, 5 × 104 RPMCs (500 ␮l) were added into each insert. The lower compartment of the well was separated from the cell suspension in the upper compartment with an 8-␮m pore-size polycarbonate membrane of the culture inserts, RPMCs were incubated for 4 h at 37 ◦ C in a humidified atmosphere flushed with 5% carbon dioxide (CO2 ) in air. Following aspiration of nonadherent RPMCs in the upper compartment, cells adherent to the upper surface of the membrane were removed by scraping with a rubber blade. Migrated cells adherent to the lower surface of the membrane were fixed with methanol for 5 min and stained with 0.5% toluidine blue. The membranes were mounted on glass slides by routine histological methods. The total number of mast cells migrated across the membrane was counted under a light microscope. 2.6. Assay of TNF-α and IL-1β and IL-6 secretion TNF-␣, IL-1␤ and IL-6 secretion was measured by modification of an enzyme-linked immunosorbent assay (ELISA) as described previously (Scuderi et al., 1986). HMC-1 cells were cultured with ␣-MEM plus 10% FBS and resuspended in Tyrode buffer A. The cells were sensitized with PMA (20 nM) plus A23187 (1 ␮M) for 6–9 h in the absence or presence of DGHT. The ELISA was sensitive to TNF-␣

H.Y. Shin et al. / Journal of Ethnopharmacology 85 (2003) 157–161

and IL-1␤ concentrations in the medium above 0.01 ng/ml. The ELISA was performed by coating 96-well plates with 6.25 ng/well of murine monoclonal antibody with specificity for murine TNF-␣, IL-1␤ and IL-6 respectively. Before use and between subsequent steps in the assay, the coated plates were washed twice with PBS containing 0.05% Tween-20 and twice with PBS alone. All reagents used in this assay and the coated wells were incubated for 1 h at room temperature. For the standard curve, rTNF-␣, rIL-1␤ and rIL-6 were added to serum previously determined to be negative for endogenous TNF-␣, IL-1␤ and IL-6. After exposure to the medium, the assay plates were exposed sequentially to biotinylated anti-human TNF-␣, IL-1␤ and IL-6, 2,2 azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) tablets substrates. Optical density readings were made within 10 min of the addition of the substrate on a Titertek Multiscan (Flow Laboratories) with a 405 nm filter. Appropriate specificity controls were inclued. Inhibition (%) =

a−b × 100 a

where ‘a’ is cytokine secretion without DGHT and ‘b is cytokine secretion with DGHT. 2.7. RT-PCR analysis Total RNA was isolated from HMC-1 cells with Easy-blue according to the manufacturer’s instruction, PCR was performed with following primers for TNF-␣ (5 CGG GAC GTG GAG CTG GCC GAG GAG3 ; 5 CAC CAG CTG GTT ATC TCT CAG CTC 3 ). The actin (5 GTG GGG CGC CCC AGG CAC CA3 ; 5 GTC CTT AAT GTC ACG CAC GAT TTC3 ) was used to verify that equal amounts of RNA were used for reverse transcription and PCR amplification from different experimental conditions. The annealing temperature was 60 ◦ C for TNF-␣ and actin. Amplified fragment sizes for TNF-␣ and actin were 355 and 661 bp, respectively. Products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide.

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Fig. 1. DGHT inhibits rmSCF-induced migration in RPMCs. (A) Inhibitory effect of DGHT. The RPMCs were treated with DGHT (1 mg/ml) or rmSCF (10 ng/ml) or rmSCF (10 ng/ml) plus DGHT (1 mg/ml). After incubation for 4 h, the migrated cells were counted. Data were obtained from 5 separate experiments. ∗ P < 0.05, when compared with rmSCF alone. (B) The photograph of the migrated cells in the medium alone. (C) The photograph of the migrated cells treated with rmSCF.

were markedly migrated toward the lower surface of the polycarbonate membrane through an 8-␮m pores by rmSCF (Fig. 1C) compared to medium alone (Fig. 1B). DGHT inhibited significantly the rmSCF-induced migration of RPMCs. DGHT alone had no effect on the migration (Fig. 1A). 3.2. DGHT inhibits the inflammatory cytokines secretion from mast cells Next, we examined the effect of DGHT on the PMA plus A23187-stimulated secretion of TNF-␣, IL-1␤, and IL-6 from HMC-1 cells. Culture supernatants were assayed for each cytokines levels by ELISA method. As shown in Table 1, DGHT inhibited the secretion of TNF-␣, IL-1␤, and IL-6 in PMA plus A23187-stimulated HMC-1 cells. CsA was used as a reference drug at the same condition.

2.8. Statistical analysis Each datum represents the mean ± S.E.M. of the different experiments under the same conditions. The student’s t-test was used to make a statistical comparison between the groups. Results with P < 0.05 were considered statistically significant.

3. Results 3.1. DGHT inhibits the rmSCF-induced migration of RPMCs Initially, we investigated whether DGHT was able to block rmSCF-induced migration of RPMCs. The RPMCs

Table 1 Inhibitory effect of DGHT on PMA plus A23187-stimulated TNF-␣, IL-1␤, IL-6 secretion from HMC-1 cells Treatment

Saline CsA DGHT

Concentration (mg/ml)

Inhibition (%) TNF-␣

IL-1␤

IL-6

– 0.01 0.01 0.1 1

– 57.2∗ 28.7 47.5∗ 60.1∗

– 52.9∗ 30.9 76.4∗ 81.8∗

– 43.8∗ 25.6 35.7∗ 72.5∗

PMA plus A23187-stimulated HMC-1 cells (3 × 105 ) were incubated for 6 h in the absence or presence of DGHT. TNF-␣, IL-1␤, and IL-6 secreted into the medium are presented as the mean ± S.E.M. of three independent experiments. ∗ P < 0.05: significantly different from the saline value.

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Fig. 2. Inhibitory effect of DGHT on the expression of TNF-␣ mRNA in HMC-1 cells by RT-PCR. Total RNA was isolated from HMC-1 cells. HMC-1 cells were pretreated with DGHT (1 mg/ml) for 30 min prior to PMA plus A23187 stimulation. CsA used as a positive control. Lane 1, Control; Lane 2, PMA plus A23187; Lane 3, (0.01 mg/ml); Lane 4, PMA plus A23187 plus DGHT (1 mg/ml); Lane 5, PMA plus A23187 plus DGHT (0.1 mg/ml); Lane 6, PMA plus A23187 plus DGHT (0.01 mg/ml).

3.3. DGHT inhibits the expression of TNF-α mRNA in mast cells We examined the inhibitory effect of DGHT on the expression of TNF-␣ mRNA in HMC-1 cells stimulated with PMA plus A23187 through RT-PCR. RT-PCR products were generated from total RNA obtained from HMC-1 cells. PMA plus A23187-stimulated HMC-1 cells increased TNF-␣ mRNA expression while unstimulated HMC-1 cells expressed very little TNF-␣ mRNA. DGHT inhibited the increased TNF-␣ mRNA expression by PMA plus A23187 (Fig. 2). ␤-Actin was used as a control for RNA integrity and equal loading.

multifunctional cytokines, its potential contributions to processes of inflammation and matrix degradation have recently become evident (Tetlow and Woolley, 1995b; McNeil, 1996; Galli, 1993). Mast cell activation in rheumatoid synovial tissue has often been associated with TNF-␣ and IL-1␤ production by adjacent cell type (Woolley and Tetlow, 2000). The proinflammatory cytokines TNF-␣ and IL-1␤ are reported to play important roles in cartilage and bone degradation, TNF-␣ occupying cascade through its ability to upregulate production of other cytokines, including IL-1, IL-6, IL-8 and IL-10 (Feldmann et al., 1996; Brennan et al., 1998). We found that DGHT inhibited the secretion of TNF-␣, IL-1␤, IL-6 in PMA plus A23187-stimulated HMC-1 cells. DGHT also inhibited the main inflammatory cytokine TNF-␣ mRNA expression HMC-1 cells. These results suggest that DGHT may contribute to the down-regulation of initiation and progression of the destructive inflammatory process of RA. However, additional experiments that are critical to interpretation of these findings are required. Acknowledgements This work was supported by Wonkwang University in 2002. References

4. Discussion We have demonstrated that DGHT pretreatment profoundly inhibited SCF-induced migration and inflammatory cytokine secretion in mast cells. It was reported that mast cells in the synovial membrane of patients with arthritis do not proliferate (Ceponis et al., 1998). It is therefore likely that a direct migration of mast cells within tissues is an important mechanism for the increase in mast cell numbers evident in rheumatic diseases. Although mast cells have been considered to be stationary cells in the tissue, with low tendency to migrate, it is now recognized that they can migrate (Nilsson and Mercalfe, 1996). Ceponis et al. reported that SCF is expressed in the synovial membrane in arthritis (Ceponis et al., 1998). Furthermore, the same group has reported that synovial fibroblasts release SCF in vitro that can induce mast cell migration (Kiener et al., 2000). Thus the presence of SCF in the synovium may have important functions both for the activation and survival of the synovial mast cells and might thereby contribute to the increased number of mast cells in the synovium. We found that DGHT pretreatment blocked migration of RPMCs induced by SCF. This finding indicates that DGHT may block the SCF-induced migration of mast cells in the synovium. Mast cell activation has been reported for a significant proportion of rheumatoid specimens (Tetlow and Woolley, 1995a). Because the mast cells contain potent mediators, including histamine, heparin, proteinases, leukotrienes and

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