Differential Effects of Chlorogenic Acid on Various

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Despite chlorogenic acid (CGA) being widely present in nature, particularly in the human diet, there is very little information .... homogenized suspension in distilled water and adminis- ..... and soluble CD14 in patients with rheumatoid arthritis.
PHYTOTHERAPY RESEARCH Phytother. Res. (2011) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.3684

Differential Effects of Chlorogenic Acid on Various Immunological Parameters Relevant to Rheumatoid Arthritis Prashant Singh Chauhan,1* Naresh Kumar Satti,2 Punita Sharma,2 Vijay Kumar Sharma,2 Krishan Avtar Suri2 and Sarang Bani1 1

Cell Biology Laboratory, Division of Pharmacology, Indian Institute of Integrative Medicine, Jammu Tawi, Jammu and Kashmir -180001, India Natural Product Chemistry Division, Indian Institute of Integrative Medicine, Jammu Tawi, Jammu and Kashmir -180001, India

2

Despite chlorogenic acid (CGA) being widely present in nature, particularly in the human diet, there is very little information regarding its pharmacological activities. The present investigation was carried out to investigate the antiarthritic activities of this compound in adjuvant induced-arthritis in male Wistar rats, and to explore the underlying mechanisms of actions in view of immunological responses. We observed that CGA effectively controlled the total (CD3) and differentiated (CD4 and CD8) T cells count at the dose of 40 mg/kg. We also assessed the effect on co-stimulatory molecules (CD28, CD80/86) and found that CGA efficiently suppressed CD80/86 but failed to bring any changes in the CD28 count, whereas ibuprofen (standard drug) resulted in highly significant inhibition of both. We next examined the effect on CD4+ T cells specific Th1/Th2 cytokines by flow cytometry and observed that CGA suppressed the Th1 cytokines in a highly significant manner but elevated Th2 cytokines with dose dependence. Results of the present investigation suggest that CGA is a potent antiarthritic agent. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: Chlorogenic acid; T cells; cytokines; co-stimulatory molecules.

INTRODUCTION Rheumatoid arthritis (RA) is one of the most common joint diseases and has been the root cause of physical disability in the elderly. As a result of its unknown aetiology, there is still no cure or effective measures for the prevention of this disease. The current model holds that T cells and macrophages are recruited into the synovium sublining layer where they induce a hyperplastic reaction of resident fibroblasts, eventually causing tissue destruction and cartilage and bone invasion (Feldmann, 2001; McInnes, 2001) by secretion of inflammatory mediators and proinflammatory cytokines. The most convincing evidence for the involvement of T cells in the pathogenesis of RA comes from the analysis of RA synovial biopsies (Duke et al., 1982), from studies demonstrating an association of the human leucocytes antigen (HLA)-DR4 with disease susceptibility and outcome (Zebcn et al., 1991), the presence of large numbers of activated T lymphocytes in affected joints (Haraoui et al., 1991), similarities in the cellular infiltrate in delayed type hypersensitivity (DTH) reactions of the skin and RA synovial tissue (Klareskog et al., 1982) and intervention studies directed against T cells that were effective in the treatment of RA (Paulus et al., 1977). T cells require at least two signals to become fully activated. Signal 1 is antigen-specific and signal 2 is * Correspondence to: Prashant Singh Chauhan, Cell Biology Laboratory, Division of Pharmacology, Indian Institute of Integrative Medicine, Jammu Tawi, Jammu and Kashmir-180001, India. E-mail: [email protected]

Copyright © 2011 John Wiley & Sons, Ltd.

delivered by binding of the co-stimulatory receptor on T cells to the ligand on antigen presenting cells. In the absence of the second signal, T cells may be rendered poorly responsive to otherwise optimal subsequent stimulation, or may undergo apoptosis (Mueller et al., 1989). Therefore inhibition of T cells activation by blocking receptor binding to their ligand represents an important tool in the prevention of RA. Cytokines regulate a broad range of inflammatory processes that are implicated in the pathogenesis of RA. In rheumatoid joints, it is well known that an imbalance between pro- and antiinflammatory cytokine activities favours the induction of autoimmunity, chronic inflammation and thereby joint damage (Iain and Georg, 2007); therefore, modulation of Th1/Th2 balance has become a new paradigm for immunomodulatory therapy (Liblau et al., 1995). Today there is an increasing demand and belief that traditionally used preparations of medicinal plants could provide a safe, effective and challenging opportunity for the prevention and/or treatment of RA, since most of the available over-the-counter antiarthritic drugs are very expensive and are associated with severe side effects. In our efforts to find out the effective antiarthritic agent, we focused on chlorogenic acid (CGA), a phenolic compound isolated from the aqueous fraction of Bidens pilosa, and carried out pharmacological studies. Chlorogenic acid is widely present in nature, particularly in the human diet, and it is known for its antioxidant, anticarcinogenic and antiinflammatory properties (Kono et al., 1995; Tsuchiya et al., 1996; Santos et al., 2006). However, despite the aforementioned biological activities there are very few reports Received 27 November 2010 Revised 03 September 2011 Accepted 18 September 2011

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concerning the effect of CGA on RA, and to the best of our knowledge the mechanisms of action are not clear. Therefore to evaluate the antiinflammatory activities of CGA, we studied its effects on the lipopolysaccharide (LPS)-induced rat knee joint inflammation model and in order to delineate the mode of action we produced chronic inflammation by the injection of heat killed Mycobacterium tuberculosus in the widely used adjuvantinduced arthritis model in male Wistar rats.

MATERIALS AND METHODS Chlorogenic acid. Chlorogenic acid was isolated from the aqueous fraction of B. pilosa by the method described elsewhere (Jassbi and Naturforsch, 2003), and its NMR and LC-MS spectral data were compared with those in the literature, which confirmed that the isolated compound was CGA. The chemical structure of CGA is given in Fig. 1. Animals. Experiments were performed on Wistar rats (weighing 130–150 g each) and Swiss albino mice (weighing 20–25 g each) of either sex after obtaining clearance from the Institutional Animals Ethics Committee. All animals were kept in standard cages and maintained under standard laboratory conditions (temperature 22  2  C with a 12 h light/12 h dark cycle) with free access to pellet food (Lipton India Ltd) and water ad libitum throughout the study. The room was well ventilated (> 10 air changes/h.) with 100% fresh air, according to the Indian Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. In accordance with ethical regulations on animal research, all animals used in the experimental work received humane care. Test material was prepared as a homogenized suspension in distilled water and administered orally by gavage to the experimental animals for the duration of experiment. Chemicals and drugs used. Concanavalin A (Con A), lipopolysaccharide (LPS), 3-(4, 5-diamethyl-2-thiazolyl) 2, 5 diphenyl-2H-tetrazolium) (MTT), RPMI-1460, L-glutamine, penicillin, streptomycin, HEPES, Mycobacterium tuberculosus, liquid paraffin, ibuprofen (Sigma Chemical Co., USA), fluorescein isothiocyanate (FITC) labelled anti-CD4, phycoerythrin (PE) labelled anti-CD3, CD8, CD28, CD80, CD86, IL-2, IFN-g, IL-12, IL-4 and IL-10 monoclonal antibodies,

Figure 1. Structure of chlorogenic acid. Copyright © 2011 John Wiley & Sons, Ltd.

fluorescence activated cell sorting (FACS) permeabilizing solution, FACS lysing solution and Golgi plug (B. D. Biosciences, Jammu, India). TNF-a and IL-1b enzyme linked immunosorbent assay (ELISA) kits were purchased from R & D Systems (Jammu, India). All other reagents used were of analytical grade. Experimental models Acute toxicity studies. The acute toxicity of CGA was evaluated in mice using the up and down procedure (Organization of Economic Co-operation and Development (OECD), Guideline No. 423). Mice of either sex (three females and three males; weight: 20–25 g; age: 4–6 weeks) received CGA (single dose) starting from 300 to 2000 mg/kg orally by gavage. The animals were observed for toxic symptoms continuously for the first 4 h after dosing. Finally, the number of survivors was noted after 24 h and these animals were then maintained for a further 13 days with observations made daily. The animals were also observed for any changes in general behaviour or other physiological activities. Rat knee joint inflammation. The following group configurations were maintained for the study, with 6 rats per group. Group I: LPS control – injected with LPS (100 mL of sterilized normal saline containing 10 mg LPS). Group II: LPS + CGA at 2.5, 5, 10, 20 and 40 mg/kg. Group III: LPS + reference drug – ibuprofen (100 mg/kg). Drugs were administered 1 h prior to LPS injection through gavage. For the induction of inflammation, the method of Singh et al. (1997) was followed with minor modifications, where the animals fasted overnight were injected intraarticular (i.a) with 100 mL of sterilized normal saline containing LPS with a 26 gauge needle into the left knee joint after 1 h of test drug administration. The injection site was previously shaved and treated with an alcohol solution. After 4 h of LPS injection, experimental animals were killed by ether overdose and synovial fluid collected after opening the knee joint. The synovial fluid so collected was then centrifuged and the supernatant stored for the estimation of TNF-a using ELISA kits according to the manufacturer’s instructions. Delayed type hypersensitivity reaction induced by SRBC. For the antigen, fresh sheep red blood cells (SRBC) collected aseptically were stored in sterile, cold Alsever’s solution, washed thrice with pyrogen-free sterile normal saline (0.9% NaCl) and adjusted to the final concentration of 5  109 SRBC/mL for immunization and challenge at the required time. Experimental animals (Swiss albino mice) were immunized by injecting 20 mL of SRBC subcutaneously into the right hind footpad and the test drug was administered orally and continued over consecutive days. Five days later the thickness of the left hind foot was measured and considered as control. These mice were then challenged by injecting the same amount of SRBC intradermally into the left hind foot pad. The foot thickness was measured again at 24 h after challenge with a volume differential meter and the increase in paw volume was determined by comparing it to the initial paw volume. Phytother. Res. (2011)

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Adjuvant-induced arthritis. The method of Newbold (1963) was employed for the induction of arthritis. Briefly, on day 0, rats were weighed and were marked directly above both ankle joints to provide an anatomical marker to minimize variability when measuring paw volume. The adjuvant was prepared by grinding M. tuberculosus in a mortar and mixing with liquid paraffin oil to a final concentration of 5 mg/mL. On day 0, arthritis was induced by a subplantar injection of adjuvant (0.1 mL) in the dorsum of the left hind paw between the third and fourth digits. The volume of both hind paws was measured on day 0 (before adjuvant injection) and then on alternate days using a digital plethysomometer (UGO Basile 7140, Italy). Any changes in paw volume were calculated as the difference between volumes on day 0 and volumes recorded on days 3, 5, 7, 9, 11 and 13. The study medications were initiated on day 1 (one day before the adjuvant injection) and daily doses were continued for 13 days. Lymphocyte proliferation assay. A RPMI-1640 medium was supplemented with 2 mM L-glutamine, 100 U/mL of penicillin, 100 mg/mL streptomycin, and 10% (v/v) heatinactivated fetal bovine serum (FBS) was used for the cell culture. On day 14 of arthritis induction, animals from all the groups (control groups as well as drug-treated groups) were sacrificed, and their spleens were excised aseptically and a single cell suspension was prepared by teasing the tissue between two glass slides. This suspension was centrifuged at 400  g for 10 min at 4  C. The viability of splenocytes was checked using Evan’s blue dye. Splenocytes (2  105 cells/well) were seeded in triplicate in a 96-well culture plate. Suboptimal concentrations of Con-A (0.5 mg/mL) were added to each well separately for priming T cells. Plates were incubated at 37  C in a humidified atmosphere of 5% CO2 for 72 h. After incubation, cell proliferation was determined by MTT assay [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (Mossmann, 1983). Absorbance was measured in an ELISA plate reader (Meglan, USA) at 570 nm. Cell surface markers and co-stimulatory molecules estimation by flow cytometry. For the flow cytometry estimation of T cell surface markers (CD3, CD4, CD8) and co-stimulatory molecules (CD28, CD80, CD86), whole blood was collected from the retro-orbital plexus of normal, arthritic and treated animals and monoclonal antibodies conjugated to a fluorochrome and directed against CD3, CD4, CD8, CD28, CD80 and CD86 were added directly to 100 mL of whole blood, which was then lysed using whole-blood lysing reagent (B.D. Biosciences). Following the final washing in phosphate buffer saline (PBS), samples were resuspended in PBS (pH 7.4) and analysed directly on the flow cytometer (BD Biosciences) using Cell Quest Pro Software (B.D. Biosciences). Quantification of CD4 specific Th1/Th2 cytokines. For the estimation of CD4+ T cells specific Th1/Th2 cytokines, 100 mL of blood was added in separate falcon tubes and red blood cells were lysed by adding FACS lysing solution. Following centrifugation and washing in PBS, samples were incubated with FACS permeabilizing solution and incubation was carried out. After incubation, samples were washed with PBS and once again incubated Copyright © 2011 John Wiley & Sons, Ltd.

with monoclonal antibodies directed against FITC labelled CD4 and PE labelled IL-2, IFN-g, IL-12, IL-4 and IL-10 in the dark for 30 min. After 30 min samples were centrifuged. A total of 10000 events were collected from each sample for flow cytometry analysis. Statistical analysis. The results were subjected to statistical analysis using ANOVA with a post-Bonferroni test and expressed as the mean  SEM. ***p < 0.001; **p < 0.01; *p < 0.05.

RESULTS Acute toxicity studies Groups of animals treated with CGA at the doses of 300–2000 mg/kg showed no abnormal behaviour and mortality, which is indicative of the safety of CGA, even at higher doses. Chlorogenic acid inhibited TNF-a and IL-1b production To investigate the effect of CGA on proinflammatory cytokines we analysed the expression of TNF-a and IL1b in the supernatant of knee joint fluid by ELISA. TNF-a and IL-1b are major proinflammatory cytokines, the blockade of which has a global effect on inflammation and today represents a major advancement in the treatment of RA. When we analysed LPS activated knee joint fluid samples, it was found that CGA-treated groups showed less cytokines production as compared with the LPS control, however, the most significant suppression was obtained at the dose of 40 mg/kg (Fig. 2). This cytokines inhibition at 40 mg/kg was similar to the effect exhibited by the standard drug ibuprofen at 100 mg/kg. Chlorogenic acid inhibited SRBC-induced DTH response Delayed type hypersensitivity is a reaction triggered by antigen-specific T cells, and is an important in vivo manifestation of cell-mediated immune response. The DTH response is characterized by the expansion of

Figure 2. Effect of CGA on TNF-a and IL-1b expression in knee joint fluid of Wistar rats. Results are represented as mean  SEM with n = 6 in each group. p value; ***p < 0.001; **p < 0.01; *p < 0.05. The statistical test employed is ANOVA followed by a postBonferroni test. CGA, chlorogenic acid. Phytother. Res. (2011)

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antigen-specific Th1 type CD4+ T cells during the initial phase, and an inflammation response by Th1 cytokines released from CD4+ T cells during the effector phase. Chlorogenic acid inhibited DTH response in a dosedependent manner, with the most significant effect being obtained at the dose of 40 mg/kg, which suggests that CGA is a potent T cells response modifier (Fig. 3).

Quantification of paw oedema induced by Freund’s complete adjuvant To determine the effect of CGA on paw oedema (injected and uninjected), arthritis-induced animals were treated prophylactically with CGA at doses from 2.5 to 40 mg/kg and oedema was measured on alternate days. The determination of paw oedema is an apparently simple, sensitive and quick procedure for evaluating the

degree of inflammation and assessing the therapeutic effects of drugs. Paw oedema in injected paw (adjuvant injected paw) was apparent from the day of injection, but the uninjected paw (adjuvant not injected, used for the determination of secondary lesions) oedema developed on day 9 onwards and was at its maximum on day 13. Paw volume measurement reveals that CGA caused highly significant inhibition of both injected (Fig. 4A) and uninjected paw oedema (Fig. 4B) as compared with control, and at higher doses the effect was almost comparable to ibuprofen. The determination of paw oedema has been used in evaluating the antiarthritic activity and effectiveness of the treatment and the significant inhibition of oedema observed in our studies suggests that CGA is a potent antiarthritic agent.

Lymphocyte proliferation As lymphocytes are known to be key regulatory cells in chronic inflammatory diseases, we next examined the inhibitory effects of CGA on lymphocyte proliferation from splenocytes in the presence of common T cell mitogen (con-A) by MTT assay. As can be seen in Fig. 3, CGA displayed broad inhibitory spectrum between the doses of 2.5–40 mg/kg in a dose-dependent manner, however, the maximum inhibition of 48% was observed at the dose of 40 mg/kg. Ibuprofen also suppressed con-A-induced T cells proliferation with inhibitory activity of 50.15% at the dose of 100 mg/kg as compared with control (Fig. 5).

Figure 3. Effect of graded doses (2.5–40 mg/kg/body weight) of CGA and ibuprofen (100 mg/kg/body weight) on SRBC induced DTH response. Results are represented as mean  SEM with n = 6 in each group. ***p < 0.001; **p < 0.01; *p < 0.05. The statistical test employed is ANOVA followed by a post-Bonferroni test. CGA, chlorogenic acid.

Chlorogenic acid reduced T cells count Many studies have demonstrated that T cells and their different patterns of differentiation result in the activation of different inflammatory effector pathways that

Figure 4. Effect of CGA on injected (Fig. 4A) and uninjected (Fig. 4B) paw oedema in adjuvant induced arthritic rats. Results are represented as mean  SEM with n = 6 in each group. ***p < 0.001; **p < 0.01; *p < 0.05. The statistical test employed is ANOVA followed by a postBonferroni test. ap < 0.05; bp < 0.01; cp < 0.001. CGA, chlorogenic acid. Copyright © 2011 John Wiley & Sons, Ltd.

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inhibition of CD4+ T cells caused by CGA was equivalent to ibuprofen at the higher dose of 40 mg/kg, which suggests that CGA is able to inhibit one of the most important immunological events responsible for the damage seen in arthritis.

Chlorogenic acid suppressed CD80/86 but could not alter CD28 count

Figure 5. Effect of graded doses (2.5–40 mg/kg/body weight) of CGA and ibuprofen (100 mg/kg/body weight) on con-A induced lymphocyte proliferation assay. Results are represented as mean  SEM with n = 6 in each group. ***p < 0.001; **p < 0.01; *p < 0.05. Statistical test employed is ANOVA followed by a post-Bonferroni test. CGA, chlorogenic acid.

correlate with the extent and type of damage observed in RA. As can be seen in Fig. 6, injection of adjuvant in rat footpad significantly increased the T cells count in the circulation as compared with normal control animals. In our studies, groups of animals treated with CGA at graded doses showed highly significant and dosedependent inhibition of T cells as compared with arthritis control. However, it was very interesting to see that

Keeping in view the crucial role of co-stimulatory molecules in T cells activation, we next examined the effect of CGA treatment on CD28 and CD80/86. As can be seen in Fig. 7, the group of animals treated with CGA at 40 mg/kg exhibited highly significant inhibition of CD80/86 but it did not alter the CD28 count, whereas ibuprofen was found to not only decrease CD28 but also CD80/86 at 100 mg/kg as compared with control, which suggests that the mode of action of CGA is different to that of ibuprofen.

Chlorogenic acid reduced the Th1 cytokines and elevated Th2 cytokines Experimental animals received 100 mL of M. tuberculosus in the left footpad and blood was collected from their retro-orbital plexus on day 14 for the estimation of

Figure 6. Flowcytometric evaluation of the effect of CGA (2.5–40 mg/kg/body weight) and ibuprofen (100 mg/kg/body weight) on CD3, CD4 and CD8+ T cells in adjuvant induced arthritis. Results are represented as mean  SEM with n = 6 in each group. ***p < 0.001; **p < 0.01; *p < 0.05. Statistical test employed is ANOVA followed by a post-Bonferroni test. CGA, chlorogenic acid; Ibu, ibuprofen. Copyright © 2011 John Wiley & Sons, Ltd.

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Figure 7. Flowcytometric evaluation of the effect of CGA (2.5–40 mg/kg/body weight) and ibuprofen (100 mg/kg/body weight) on CD28, CD80 and CD86 in adjuvant induced arthritis. Results are represented as mean  SEM with n = 6 in each group. ***p < 0.001; **p < 0.01; *p < 0.05. Statistical test employed is ANOVA followed by a post-Bonferroni test. CGA, chlorogenic acid; Ibu, ibuprofen.

cytokines by flow cytometery in CD4+ T cells. During flow cytometry analysis of cytokines, a gate was put around leucocytes and then PE labelled cytokines were detected in gated FITC labelled CD4+ T cells. The levels of both Th1 and Th2 cytokines were significantly enhanced by the injection of adjuvant as compared with normal control animals. We observed dose-dependent inhibition of IL-2, IFN-g and IL-12 by CGA (Fig. 8), whereas there was elevation in the levels of IL-4 and IL-10 (Fig. 9). These results indicate that CGA selectively inhibits Th1 cytokines, unlike ibuprofen, which resulted in highly significant inhibition of both Th1 and Th2 cytokines at the dose of 100 mg/kg. Copyright © 2011 John Wiley & Sons, Ltd.

DISCUSSION This study was carried out with the aim of developing a potent antiarthritic agent. We therefore carefully evaluated CGA for its immunopharmacological efficacy against adjuvant induced-arthritis and its relevant immune responses. First we tested the antiinflammatory potential of CGA using the rat knee joint inflammation model with LPS as an antigen. Lipopolysaccharide is known to trigger multiple intracellular proinflammatory signal transduction cascades accompanying the secretion of proinflammatory mediators such as TNF-a and IL-1b. Therefore it has been postulated that any kind Phytother. Res. (2011)

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Figure 8. Flowcytometric evaluation of the effect of CGA (2.5–40 mg/kg/body weight) and ibuprofen (100 mg/kg/body weight) on proinflammatory cytokines (IL-2, IFN-g and IL-12) in adjuvant induced arthritis. Results are represented as mean  SEM with n = 6 in each group. ***p < 0.001; **p < 0.01; *p < 0.05. Statistical test employed is ANOVA followed by a post-Bonferroni test. CGA, chlorogenic acid; Ibu, ibuprofen.

of agent that is able to block the LPS triggered TNF-a and IL-1b production, will act as a promising antiinflammatory agent in many inflammatory diseases (Lee and Lim, 2009), including RA. TNF-a and IL-1b have overlapping actions including local inflammation, enhancing adhesive properties of inflammatory cells, causing angiogenesis and bone resorption (Choy and Panayi, 2001). Furthermore, TNF-a and IL-1b are potent stimulators of mesenchymal cells, such as synovial fibroblasts, osteoclasts and chondrocytes that release tissuedestroying matrix metalloproteinases. They also inhibit the production of tissue inhibitors of metalloproteinases by synovial fibroblasts. These dual actions are thought to lead to joint damage. Perhaps by inducing the Copyright © 2011 John Wiley & Sons, Ltd.

production of IL-11, TNF-a stimulates the development of osteoclasts, which are responsible for bone degradation (Ernest and Gabriel, 2001). Therefore these proinflammatory cytokines have been major targets for therapeutic manipulation of RA and our current findings showed that the levels of these proinflammatory cytokines were significantly decreased in a dosedependent manner suggesting that CGA can block LPS triggered proinflammatory signal transduction cascades possibly through inhibition or blockade of p38 mitogen-activated protein (MAP) kinase, which have been considered to play a critical role in regulating the expression of proinflammatory mediators (Kyriakis and Avruch, 2001). Phytother. Res. (2011)

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Figure 9. Flowcytometric evaluation of the effect of CGA (2.5–40 mg/kg/body weight) and ibuprofen (100 mg/kg/body weight) on antiinflammatory cytokines (IL-4 and IL-10) in adjuvant induced arthritis. Results are represented as mean  SEM with n = 6 in each group. ***p < 0.001; **p < 0.01; *p < 0.05. Statistical test employed is ANOVA followed by a post-Bonferroni test. CGA, chlorogenic acid; Ibu, ibuprofen.

Adjuvant-induced arthritis is an autoimmune model of RA characterized by a systemic inflammation of peripheral joints. The model is similar to chronic human inflammatory arthritis with respect to clinical symptoms and histopathology and is considered to be an important experimental model of chronic inflammation related to RA (Karen et al., 2007). Arthritis-induced rats showed soft tissue swelling around the ankle joint, which was considered oedema. As the arthritis progressed, a more severe oedema developed in the injected paw and secondary lesions occurred after a delay of approximately 9 days and were characterized by inflammation of the uninjected paw. Reduction of primary and Copyright © 2011 John Wiley & Sons, Ltd.

secondary lesions by CGA may be due to immunological protection rendered by CGA. Several lines of evidences suggest that T cells play an important role in the pathogenesis of RA: (i) it is well established that a small number of antigen specific T cells are sufficient to drive inflammatory (immune and autoimmune) processes (Steinman, 1996); (ii) RA is a disease strongly associated with certain MHC class II alleles suggesting involvement of certain T cell receptors (Fox, 1997); (iii) treatment directed towards T cells, such as therapy with cyclosporin A, can lead to significant clinical effects in RA (Tugwell, 1992); and (iv) anti-CD4 monoclonal antibody therapy has been shown to lead to a decrease Phytother. Res. (2011)

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of monokines as well as to reduction of the inflammatory synovitis, including synovial lining cell hyperplasia (Horneff et al., 1993). In our studies we found that prophylactic treatment with CGA at graded doses significantly decreased the T cells count as compared with the control, however, only CD4+ T cells inhibition was comparable to ibuprofen at 40 mg/kg. The inhibitory effect of CGA on T cells function was confirmed by inhibition of con-A inducedlymphocyte proliferation assay and the SRBC-induced T cells mediated DTH model, where CGA showed highly significant inhibition of DTH response as assessed by paw volume measurement. To find out the possible mechanism behind T cells inhibition, we next examined the effect on co-stimulatory molecules, as optimal activation of T cells requires engagement of the T-cell receptor as well as a nonantigen-specific, co-stimulatory signal. The best characterized co-stimulatory molecules are CD80 and CD86. They are expressed by antigen-presenting cells, such as dendritic cells, macrophages and B cells. The interaction of CD80 or CD86 with the T-cell receptor, CD28, promotes T-cell responses by decreasing apoptosis and enhancing the expansion of antigen-specific T cells (Kobata et al., 2000). The CD80 and CD86 molecules have also been shown to regulate Th1/Th2 differentiation and are critical for antibody production and isotype switching. Taking into consideration the importance of these co-stimulatory molecules in T cells activation we analysed the effects of CGA on these parameters and found that CGA significantly suppressed the CD80/86 expression but showed no effect on CD28. On the other hand, ibuprofen suppressed the expression of both CD28 and CD80/86. It can be speculated here that inhibition of T cells by CGA might be due to inhibition of CD80/86, as in the absence of the required number of ligands CD28 might not have delivered the appropriate secondary signals required for T cells activation and differentiation, and this in turn would have induced T cells anergy or functional inactivation and finally apoptosis. Co-stimulation mediated by the CD28 molecule is also considered critical in the activation of CD4+ T cells (Pasquini et al., 1997), therefore, highly significant inhibition of CD4+ T cells obtained in our studies can be attributed indirectly to the inhibition of CD80/86. Absence of CD28–CD80/CD86 mediated co-stimulation is also reported to result in impaired T cells proliferation, reduced cytokine production and altered Th1/Th2 cytokines balance (Won et al., 1997), therefore we next examined the effect on Th1/Th2 cytokines balance. Cytokines play an important role in the inflammation cascade, and changes in these proteins can be monitored to determine how a given treatment is altering disease progression. The Th1 cytokines (IL-2, IFN-g and IL-12) are known to promote cell mediated immunity and they have been shown to have a major role in RA. Among the Th1 cytokines that are involved in the pathogenesis of several autoimmune diseases, IL-12 is the main stimulator of IFN-g production and of the development of Th1 autoimmune response. In patients

with RA, levels of IL-12 are elevated in serum and synovial fluids, with a direct correlation with disease activity (Kim et al., 2000). Besides activating the Jak/STAT pathway, IL-12 also activates the mitogen-activated protein kinase (MAPK) cascade. In particular, the p38 MAPK is enzymatically active upon IL-12 stimulation of T cells and activation of this MAPK is critical for IFN-g production in response to IL-12 (Zhang and Kaplan, 2000). The role of IFN-g in priming Th1 cell development has been demonstrated using transgenic T cells in vitro (Macatonia et al., 1993), and presumably reflects the capacity of IFN-g to promote IL-12Rb2 chain expression and the appearance of competent IL-12 receptors on activated CD4+ T cells (Szabo et al., 1995). IFN-g also induces IL-12 p40 production by macrophages (Ma et al., 1996), thus establishing an autocrine loop for the amplification of Th1 immunity. We found that after 13 days of CGA administration, there was highly significant inhibition of IL-12, which might be responsible for the significant suppression of adjuvant-induced arthritis. Th1/Th2 cytokines are mutually antagonist and each suppresses the activity of the other. While Th1 cytokines are responsible for disease initiation, Th2 cytokines activation correlates with disease regression. Th2 cytokines by virtue of antagonizing the effect of Th1 cytokines, suppressing the production of proinflammatory cytokines, blocking the inflammatory cells migration, stimulating the production of other antiinflammatory molecules and inhibiting the release of matrix metalloprotinases may prevent joint damage in RA (Agarwal and Malaviya, 2005). However, the very low levels of Th2 cytokines (IL-4 and IL-10) might account for disease perpetuation by a failure to suppress the production of Th1 cytokines. IL-4 inhibits the activation of Th1 cells, and this, in turn, decreases the production of TNF-a and IL-1b. It has also been shown to down-regulate IL-12Rb2 expression (Afkarian et al., 2002), and IL-10 is known to suppress the progression of arthritis in animal models, and clinical improvement has been reported in rheumatoid patients treated with recombinant human IL-10 (Katsikis et al., 1994). Keeping in view the significance of Th2 cytokines, we further studied the effect of CGA treatment on Th2 cytokines and found that Th2 cytokines were increased in a dose-dependent manner. Taken together, our study is of great significance, because CGA not only inhibited the T cells and Th1 cytokines but also elevated the natural capacity of the immune system to keep the Th1 cytokines under control by increasing IL-4 and IL-10. On the basis of the results obtained it can be concluded that CGA is a potent antiarthritic agent, the mode of action of which is associated with the significant inhibition of CD80/86 resulting in decreased T cells count, and elevation of Th2 cytokines resulting in inhibition of Th1 cytokines. Conflict of Interest The authors have declared that there is no conflict of interest.

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