Rheumatology 2001;40:1013–1021

The k- , asimadoline, alters cytokine gene expression in adjuvant arthritis K.A.Bush,B.W.Kirkham1 andJ.S.Walker School of Physiology and Pharmacology, University of New South Wales and 1Department of Rheumatology, St George Hospital, New South Wales, Australia

Abstract Objective. We have previously found that the k-opioid agonist, asimadoline, attenuates adjuvant arthritis in a dose-dependent, antagonist-reversible manner. To elucidate possible mechanisms, we investigated the effects of asimadoline (5 mgukguday i.p.) or vehicle on in vivo cytokine expression and T-cell recruitment in adjuvant arthritis. Methods. Arthritis severity was assessed every 3–4 days for 21 days. Rats were killed on days 0, 13 and 21 post-induction and synovial membrane and inguinal lymph nodes were removed for mRNA extraction. Changes in cytokine mRNA expression were measured using reverse transcription–polymerase chain reaction (RT–PCR) and densitometry. T cells in joints were quantified by immunohistochemistry. Results. Asimadoline significantly decreased arthritis severity at day 13, with a concomitant decrease in synovial membrane expression of cytokines interleukin-17 and transforming growth factor-b (TGF-b) mRNA at day 13, and no change in T cell numbers in the joints of arthritic rats. By contrast, in the inguinal lymph nodes, expression of tumour necrosis factor was increased at day 13 and TGF-b mRNA was increased throughout. Conclusion. An altered balance, therefore, in the pro- and anti-inflammatory effects of TGF-b by asimadoline might explain its striking anti-arthritic actions.

KEY WORDS: Cytokines,, Asimadoline, Adjuvant arthritis, k-Opioid agonist, Inflammation.

The immune system can be influenced by both Opioids have been shown to suppress immune cell endogenous and exogenous opioids w1x. Opioids have cytokine production in vitro. The prototype k-opioid been shown to regulate lymphocyte proliferation, anti- agonist, U-50,488H, inhibited gene transcription and body production and natural killer cell activity, as production of the cytokines tumour necrosis factor well as inhibiting the function of neutrophils, monocytes (TNF) and interleukin-1 (IL-1), by macrophages in vitro and macrophages w2x. They exert their effects on the w15x. Our laboratory has shown that asimadoline immune system via one of three families of opioid suppresses TNF production by lipopolysaccharide- receptors (m, k or d) located on cells of the immune stimulated peritoneal macrophages in vitro w16x.We system w1, 3–5x. Pharmacological doses of opioids have have also shown that k- significantly reduce been shown to have anti-inflammatory effects. For synovial macrophage and mast cell numbers in example, opioids can inhibit carrageenan-induced paw adjuvant arthritis w16x, as well as inhibit up-regulation swelling in the rat w6x and can attenuate the development of the adhesion molecule, ICAM-1, in vivo w17x. The of experimental arthritis w7–10x. Agonists acting at the effect of k-opioid agonists on the expression of TNF k-receptor are more potent immunosuppressive agents and other cytokines in in vivo models of inflammation, than those acting at the m-receptor w11, 12x. In rats, such as adjuvant arthritis, has not been studied. we have shown that opioids acting at the k-receptor Furthermore, the effect of k-agonists on T-cell can attenuate adjuvant arthritis, an immunologically recruitment has not been examined. Therefore, the aim mediated inflammatory arthritis w9, 10, 13x. For example, of this study was to investigate the effects of the the peripherally selective k-opioid agonist, asim- k-opioid agonist, asimadoline, on in vivo cytokine adoline, attenuated adjuvant arthritis by up to 80% in expression and T-cell trafficking in adjuvant arthritis. a dose-dependent, antagonist-reversible manner w13, 14x. However, the mechanism of this effect is not known. Materials and methods Animals Submitted 30 May 2000; revised version accepted 22 March 2001. Correspondence to: J. Walker, School of Physiology and Male Dark Agouti rats 4–8 weeks old weighing Pharmacology, Faculty of Medicine, University of New South Wales, 104–224 g were obtained from the University of Sydney, NSW, Australia, 2052. Queensland and housed in cages lined with cellulose

1013 ß 2001 British Society for Rheumatology 1014 K. A. Bush et al. bedding and shredded paper in a temperature controlled its absorbance at 260 nm on a QuantaGene spectro- room (22 " 18C) with a 12 h alternating light and dark photometer (The Australian Chromatography cycle. The animals were given food (rat chow, Gordon’s Company, Australia). Dithiothreitol (DTT, 0.1 M; Speciality Stockfeeds, Yandera, Australia) and water 0.8 ml; Gibco, Mulgrave, Australia) and 0.1 mlof ad libitum for 1–2 weeks prior to, and throughout, RNasin (per 20 ml RNA; Promega, Madison, WI, the experiments. All experiments were approved by the USA) were added to the final RNA preparation and Animal Care and Ethics Committee of the University of the RNA was stored at 2708C. New South Wales, Sydney, Australia. cDNA synthesis Induction of adjuvant arthritis To prepare cDNA, the RNA (0.13 mg) was added to 1 ml The animals were handled for 1–2 weeks prior to the of 53 first strand buffer (75 mM KCl; 50 mM Tris HCl, experiments and every 2–3 days throughout the 21-day pH 8.3; 3 mM MgCl; Gibco), 2 mM dNTP (10 mM each study. During this time, food and fluid intake and body dATP, dCTP, dTTP and dGTP; Promega), 0.025 ml weight were monitored. To induce adjuvant arthritis, of 0.0156 U (0.4 mg) random hexamers (Amersham the rats were anaesthetized with a (50 mgukg Pharmacia Biotech, Sydney, Australia), 20 U of i.p.; Parnell Laboratories, Alexandria, Australia) and Moloney murine leukaemia virus (M-MLV) reverse xylazine (5 mgukg i.p.; Troy Laboratories, Smithfield, transcriptase (Gibco), 0.001 U RNasin (Promega) and Australia) mixture and then injected with Freund’s RNase-free water to a final volume of 5 ml per complete adjuvant (0.1 ml of 10 mguml heat killed and cDNA. The reaction mix was then incubated at 428C dried Mycobacterium butyricum suspension in paraffin for 60 min to generate cDNA, and the final product oil and mannide monooleate; Difco Laboratories, was stored at 2208C. Detroit, MI, USA) intradermally into the tail base. Primers Severity of adjuvant arthritis Sense and anti-sense oligonucleotide primers used to amplify b-actin, IL-2, interferon-c (IFN-c), IL-4, TNF Disease progress was assessed every 3–4 days post- and transforming growth factor-b (TGF-b) have been adjuvant by measuring paw volume by plethysmometry previously published w19, 20x. IL-17 primers were (Ugo Basile, Comerio, Italy), body weight change, and designed using mRNA sequences from published clinical arthritis severity score (as judged by swelling, papers w21x and GenBank. The sequences for the necrosis and redness) over 21 days. The clinical arthritis IL-17 primers were as follows: sense = 59-TGGAC- severity score utilized a scoring system from 0 to 5; = = = = TCTGAGCCGCATTGA-39, anti-sense 59-GACG- 0 no arthritis, 1 mildly swollen, 2 red and moder- CATGGCGGACAATAGA-39. The primers were ately swollen, 3 = swollen with moderate necrosis, = = checked for specificity using GenBank and the poly- 4 severe necrosis and nodules, 5 very severe merase chain reaction (PCR) product was sequenced necrosis of entire foot. Gait was evaluated using a to confirm identity. An extensive set of preliminary scoring system from 0 to 3; 0 = normal, 1 = slight = experiments was undertaken to achieve the optimal lameness, 2 lameness with weight bearing on toes conditions for each cytokine (Table 1). only, 3 = non-weight bearing lameness w14, 17x. The same trained observer performed the measurements PCR throughout the study. The rats were euthanased according to institutional guidelines using pentobarbital We used semi-quantitative reverse transcription–PCR (60 mg i.p.; Virbac, Sydney, Australia). (RT–PCR) to measure the expression of relevant cytokines to evaluate the effect of asimadoline treat- ment. This method was found to show comparable Cytokine mRNA expression changes in cytokine expression to that using quantitative Tissue collection. Following death, the inguinal lymph nodes from both sides and synovium from the TABLE 1. Day 0 cytokine mRNA expression (as a percentage of right paw of each rat were removed, placed in cryo- positive control), and PCR cycle number in the synovium (SM) and tubes and immediately frozen in liquid nitrogen. The inguinal lymph nodes (ILN) of naı¨ve rats samples were stored at 2708C for subsequent RNA Cytokine expression extraction. (% positive control) RNA extraction. Synovial membrane and inguinal (mean " S.E.M.) Cycle number lymph node tissues were crushed using a custom-made metal tissue homogenizer and liquid nitrogen on ice. ILN SM ILN SM The resulting powder was dissolved in guanidinium TNF 34.2 " 5.3 6.2 " 1.2 28 35 isothiocyanate. Total RNA was extracted using the IFN-c 43.7 " 10.2 1.6 " 0.7 40 40 method of Chomczynski and Sacchi w18x. The RNA IL-17 9.7 " 3.5 22.0 " 6.4 30 36 pellet was dissolved in 25 ml of RNase-free water at IL-2 39.0 " 5.3 4.6 " 1.2 35 40 " " 428C for 15 min. RNA was quantified by diluting 5 ml IL-4 77.7 8.9 25.1 7.2 40 40 TGF-b 125 " 109 44.1 " 17.2 30 35 of RNA in 495 ml of RNase-free water and measuring Asimadoline and cytokine expression in arthritis 1015

PCR w22x. In previous human studies we have found that Densitometry if the different stages of the RT–PCR process are The density of the bands on the negative images of performed in the same experiment for the samples to be the gels was determined using densitometry (GS-700 compared, very reliable results are achieved with a densitometer, Biorad Laboratories, CA, USA) and com- w x variation of 10% 23 . A limited number of samples can puter analysis (Molecular Analyst Software, Biorad, be amplified in the same PCR reaction (i.e. 40 samples), CA, USA). Background readings of the gels were thus it is essential to use the same positive control in subtracted from the density of the samples. After each reaction so samples with and without treatment correction for background, the density of the samples can be directly compared. Like immunohistochemistry, was expressed as a percentage of the density of positive RT–PCR is only semi-quantitative, but it has the control samples amplified simultaneously in the PCR advantage that it can detect small quantities of cytokines reaction with the experimental samples. that are expressed weakly, for example in the synovium, cDNA for the synovial and inguinal lymph node w x where others have had difficulty detecting them 24 . samples for each rat was prepared simultaneously. RNase-free water (34.75 ml), 5 mlof103 reaction buffer Synovial samples were amplified in the same PCR reaction (Promega) and 0.25 ml (1.25 U) of Taq DNA polymer- at the optimal cycle number with appropriate positive ase (Amersham Pharmacia Biotech, Sydney, Australia) and negative controls. Inguinal lymph node samples were per PCR reaction were combined, vortexed and 40 ml amplified at the same time in a different PCR reaction. was aliquoted per tube. 59 sense primer (2.5 ml) and 39 Synovial and inguinal lymph node samples were often anti-sense primer (2.5 ml) were added to each tube and amplified at different cycle numbers, therefore the levels of vortexed. Each tube was then overlayed with 50 mlof cytokines in the two tissues cannot be compared directly, mineral oil (Sigma Chemicals, St Louis, MO, USA) and but changes in cytokine levels over time and between centrifuged for 1 min. treatments for each tissue are directly comparable. The tubes were placed in a thermal cycler (Hybaid Omn-E, Hybaid Ltd, Middlesex, UK) and heated at Experimental protocol 948C for 1 min prior to the addition of cDNA and u u cycling. Sample cDNA (5 ml) was added to each tube Drug-treated rats received asimadoline (5 mg kg day and cycled at various cycle numbers, depending on i.p.; EMD61753, kindly supplied by A. Barber and the cytokines being tested. Each cycle involved a G. Bartoszyk, Merck KGaA, Darmstadt, Germany). denaturation step at 948C for 1 min, an annealing step This regimen was chosen on the basis of our previous at 608C for 1 min, and an extension step at 728C for work with this drug that demonstrated the ED50 (dose which gives 50% maximal response) to be ;1mgukguday 2 min. b-actin primers amplified a 607 bp cDNA u u w x fragment and IFN-c, IL-2, IL-4, TNF, TGF-b and and a maximum effect was achieved at 5 mg kg day 13 . IL-17 primers produced PCR products of 405, 410, Control rats received the equivalent volume of vehicle 378, 292, 535 and 245 bp, respectively. (33% polyethylene glycol 400; Aldrich Chemical Co., Milwaukee, USA) in saline (0.9%). The rats were killed The primers were initially used at various cycle = numbers with cDNA from the target tissue, e.g. inguinal on days 0, 13 and 21 (n 6–8 at each time point) and lymph node and synovium, to determine which the inguinal lymph nodes and synovium were removed cycle numbers were on the linear portion of the and immediately snap frozen in liquid nitrogen for cycle number vs density curve. subsequent mRNA extraction. Controls Immunohistochemistry for T cells Inguinal lymph node cells stimulated for 4 h with For pragmatic reasons, immunohistochemistry was concanavalin A were used as a positive control which performed on rat ankle sections obtained in a separate expressed high levels of each of the cytokines. Negative experiment. Paraffin sections were cut from pre-fixed controls contained all components of the reaction mix ankle blocks at 4 mm on a rotary microtome and set except for cDNA. Each sample was tested for b-actin on slides coated with 0.3% gelatin and dried at 568C. expression to confirm that the sample RNA was not The slides were baked at 808C for 30 min in an oven degraded prior to testing with other cytokine primers. prior to dewaxing. The paraffin sections of rat ankle joints were dewaxed in xylene and ethanol and washed Agarose gel electrophoresis with Tris-buffered saline (TBS; Sigma Chemicals). The PCR product (4 ml of each) and 100 bp markers Endogenous peroxidase was blocked with 3% hydrogen (3 ml; Promega) were mixed with 5 ml of loading buffer peroxide in methanol for 5 min and the slides were then w28% Ficoll in 13 TAE (5 3 TAE contains 40 mM Tris washed in TBS. The antigen retrieval step consisted of base, 20 mM glacial acetic acid, 1 mM EDTA) plus heating the slides in an autoclave at 1218C for 20 min in bromophenol bluex and electrophoresed through a 2% Tris-EDTA citrate (TEC) retrieval buffer (0.001 M agarose gel (ICN, OH, USA) at 80 V for 60 min. The NaEDTA, 0.001 M Tris NaCitrate and 0.002 M Tris gel was stained with ethidium bromide (0.25 mguml) base). The slides were rinsed with distilled water and for 10 min and then photographed with Polaroid 665 then TBS. They were then incubated with skim milk (2% in film (Polaroid, Hertfordshire, UK) using ultraviolet water) for 15 min, dipped in distilled water and incubated illumination. with polyclonal rabbit anti-human CD3 (1 : 200; Dako, 1016 K. A. Bush et al.

Australia) for 1 h at 378C. Negative controls were (essentially non-arthritic rats). Differences between incubated with rabbit serum (Gibco) at the same pro- means were calculated using a one-way ANOVA. tein concentration. The slides were washed and then If a significant difference was found (P < 0.05) after incubated with goat anti-rabbit biotinylated secondary ANOVA, post-hoc t-tests utilizing Fisher’s multiple antibody (1 : 300; Dako, Australia) for 30 min. After comparison test were performed. All statistical computa- further washes with TBS, avidin biotin complex (ABC kit; tions were performed using the Number Crunching Vector Laboratories, CA, USA) was applied for 30 min. Statistical System (NCSS, UT, USA). The slides were again washed and sections developed in 0.01% DAB (3,39-diaminobenzidine tetrahydro- chloride; Sigma Chemicals) with 10 ml of hydrogen Results peroxide (30%) for 5 min and rinsed in TBS. The sec- Time course of adjuvant arthritis tions were counterstained in Harris’ haematoxylin for 10 s, washed in water and dipped in Scott’s blue for 5 s. The animals were judged to be arthritic if their paw The slides were then washed in water, dehydrated volume increased more than two standard deviations and cleared in alcohol and xylene, respectively, and from the mean of the non-arthritic control group at day mounted using DPX (BDH Laboratory Supply, UK). 21. Using this criterion, all but two rats treated with Freund’s complete adjuvant developed arthritis. The arthritic animals groomed themselves well and Quantification of T cells maintained their original body weight throughout the Stained cells were counted in high-power fields (3400) progress of the disease (day 0 vs day 21: 203 " 4 vs by means of a 0.02 mm2 graticule fitted in the eyepiece 209 " 10 g). Paw volume, arthritis severity and gait of the microscope. Up to 10 fields were counted for scores were significantly increased in the untreated all samples (coefficient of variation = 15%) by two rats at days 13 and 21 compared with day 0, P < 0.05 independent observers blind to the treatments (sections (Fig. 1). from some non-arthritic rats did not have enough tissue to count 10 fields). Negative controls for each individual Effect of asimadoline on adjuvant arthritis ankle joint were also included. The number of cells was Compared with untreated rats, asimadoline significantly expressed per mm2 of joint tissue. reduced paw volume at day 13 (P < 0.05), but not at day 21. Arthritis severity scores and gait followed a Statistical analysis similar pattern to paw volume (Fig. 1). Inflammation. Previous studies in our laboratory have shown that asimadoline significantly reduces measures of Effect of asimadoline on cytokine expression arthritis severity, assessed by oedema, radiology and w x The expression of cytokine mRNA at day 0 is sum- histology, to a similar extent 13 . For pragmatic reasons, marized in Table 1. The cycle numbers for the PCR radiology and histology could not be performed in the experiments for each tissue are also shown. In general, present study. Thus, the three indices of arthritic damage used were oedema, clinical arthritis severity and gait. Successive paw volumes (oedema) were averaged from bilateral measures. All measurements were made over the entire 21-day observation of the time course of the adjuvant arthritis and every measurement was normal- ized relative to the measurement of day 0. Maximum disease severity was at day 21. Thus, the averaged paw volumes, clinical arthritis severity and gait for each rat on days 13 and 21 were then adjusted to control (vehicle- treated animals) values on day 21 (100%). All graphical plots show mean values with standard error of the mean (S.E.M.). Multiple comparisons for each variable were made by performing a repeated measures ANOVA wthe factors wgroups and daysx being considered fixed and where the F-ratios indicated significant heterogeneity (P < 0.05)x. Cytokines. For each sample on a gel, densities were corrected for background. The density of the samples was then expressed as a percentage of the density of positive control samples amplified simultaneously in FIG. 1. Normalized paw volume, clinical arthritis scores and the PCR reaction with the experimental samples. To gait scores for rats with adjuvant arthritis treated with either express the data relative to non-arthritic control values, asimadoline 5 mgukguday or vehicle from day 0 (n = 6–16 at the individual values on days 13 and 21 were nor- each time point). *Significant difference from vehicle-treated malized relative to the averaged day 0 measurements rats (P < 0.05). Asimadoline and cytokine expression in arthritis 1017 cycle numbers to generate PCR product at the expo- (P < 0.05) at day 13 compared with untreated rats nential stage of product accumulation were higher in the (Fig. 2C, E). Asimadoline also significantly reduced synovium than inguinal lymph nodes, indicating lower mRNA expression of TGF-b (P < 0.05) in the syno- levels of cytokine mRNA in the synovial tissue. The vium at day 21 compared with untreated rats (Fig. 2E). patterns of cytokine mRNA expression in both the Although asimadoline reduced mRNA expression synovium and inguinal lymph nodes are presented in of TNF (P = 0.056; Fig. 2A) and IL-4 (P = 0.069; Figs 2 and 3, respectively. Fig. 2F), it did not quite reach the 5% level of In the synovium, asimadoline significantly reduced significance. Similarly, there was a trend for a reduction mRNA expression of IL-17 (P < 0.05) and TGF-b in IFN-c expression (P = 0.13) by asimadoline (Fig. 2B).

FIG. 2. mRNA expression (as a percentage of day 0 values) for (A) TNF, (B) IFN-c, (C) IL-17, (D) IL-2, (E) TGF-b and (F) IL-4 in the synovium of rats with adjuvant arthritis treated with either 5 mgukguday asimadoline or vehicle i.p. Filled panels represent vehicle-treated rats, open panels represent asimadoline-treated rats. *Significant difference from vehicle-treated rats (P < 0.05; n = 6–8). 1018 K. A. Bush et al.

FIG. 3. mRNA expression (as a percentage of day 0 values) for (A) TNF, (B) IFN-c, (C) IL-17, (D) IL-2, (E) TGF-b and (F) IL-4 in the inguinal lymph node of rats with adjuvant arthritis treated with either 5 mgukguday asimadoline or vehicle i.p. Filled panels represent vehicle-treated rats, open panels represent asimadoline-treated rats. *Significant difference from vehicle-treated rats (P < 0.05; n = 6–8).

By contrast, IL-2 expression remained unchanged with rats (P < 0.05) (Fig. 3C). Expression of IFN-c, IL-2 and asimadoline treatment (Fig. 2D). IL-4 remained unchanged with asimadoline treatment In the inguinal lymph nodes, an extra-articular site on both days 13 and 21 (Fig. 3B, D, F). thought to reflect the early stages of the disease, asimadoline increased mRNA expression of TNF Effect of asimadoline on T cell numbers (P < 0.05) at day 13 and TGF-b (P < 0.05) at days 13 In a separate group of rats, asimadoline treatment did and 21 (Fig. 3A, E). Asimadoline decreased mRNA not change the number of T cells at either day 13 or expression of IL-17 at day 21 compared with untreated day 21 in the synovium of rats with adjuvant arthritis Asimadoline and cytokine expression in arthritis 1019

(vehicle vs asimadoline: day 13: 565 " 259 vs 610 " 169 Asimadoline treatment did not prevent the increase in cellsumm2; day 21: 1012 " 214 vs 896 " 248 cellsumm2). synovial T cell numbers observed in adjuvant arthritis. Thus, the significantly reduced mRNA expression of IL-17 in the synovium at day 13, together with the Discussion trend for reduced synovial IFN-c transcription, in the asimadoline-treated rats probably reflects suppression There is increasing awareness of important interactions of synovial T-cell activation by opioids. Our results between endogenous and exogenous opioids with the support literature findings that opioids affect T-cell immune system w1x. Few studies have demonstrated function (see w1x for a review) and that b-endorphin that opioids have important effects on immune func- suppresses lymphocyte chemotactic factor release by tion in vivo. The present study confirms our previous peripheral blood mononuclear cells w28x. Alternatively, findings that the peripherally selective k-agonist, our results may also reflect a decreased number of asimadoline, attenuates adjuvant arthritis. In this monocytes and less co-stimulation, with no direct effect study we showed that asimadoline may exert its on T cells. The observed reduction in cytokine expres- anti-inflammatory actions via altered mRNA expres- sion with no change in T cell numbers is consistent with sion of pro- and anti-inflammatory cytokines in the studies in rheumatoid arthritis patients in which synovium and inguinal lymph nodes of rats with treatments such as leflunomide and methotrexate adjuvant arthritis. showed a reduction in cytokine expression with no The synovium is the site where pannus formation and change in T cell numbers w29x. joint destruction are observed in both adjuvant arthritis The inguinal lymph node is an important site of and rheumatoid arthritis. The reduced expression of immune activity in adjuvant arthritis, particularly in the IL-17, a pro-inflammatory cytokine, at day 13, but not first few days w30–33x. The overall lack of changes in day 21, in the synovial membrane is consistent with the cytokine expression at day 13 compared with day 0 in clinical signs of adjuvant arthritis (e.g. oedema) which the inguinal lymph node suggests that changes may begins to resolve after 21 days. have occurred before day 13 of adjuvant arthritis. The TNF is a pro-inflammatory cytokine thought to play increase in TNF mRNA expression in the inguinal a pivotal role in rheumatoid arthritis w25x. Our in vivo lymph nodes at day 13 with asimadoline treatment findings showing a trend for a reduction in synovial compared with vehicle-treated controls may reflect TNF mRNA expression with asimadoline treatment a delay in disease development, compatible with a support our previous studies in vitro showing reduced reduction in clinical arthritis score at day 13, but not TNF production by rat peritoneal macrophages w16x, at day 21. Alternatively, TNF may be promoting an and those of others demonstrating reduced TNF extra-articular pro-inflammatory effect. production and transcription by a macrophage cell line TGF-b is present in the synovium of rheumatoid by the selective k-opioid agonist U50,488H w15x. The arthritis patients and animal models of inflammatory present study is the first to show such effects in vivo arthritis. Its role, however, remains elusive. TGF-b and warrants further study. is a predominantly macrophage-derived cytokine, The changes in synovial TNF and TGF-b expression, which has been shown to have both pro- and both predominantly macrophage-derived cytokines, anti-inflammatory effects, depending on the site of may be due to suppression of macrophage activation action w34x. For example, TGF-b has been shown to or to reduced numbers of synovial macrophages. We have both pro- and anti-inflammatory effects in have previously shown that asimadoline reduces macro- collagen-induced arthritis and streptococcal cell wall phage trafficking to the joints of rats with adjuvant arthritis in mice. In one study, extra-articular injection arthritis w16x. In addition, the up-regulation of ICAM-1, of TGF-b protected against collagen-induced arthritis which is thought to be stimulated by pro-inflammatory and anti-TGF-b treatment exacerbated the disease cytokines such as TNF and IFN-c, has been shown to be w35, 36x. In contrast, intra-articular injection of TGF-b reduced by the k-agonist PD117302 w17x. The reduction has been shown to augment collagen-induced arthritis in TNF expression in the synovium observed in this w37x or induce synovitis in normal rats w38x. These study may explain the asimadoline-related suppression data suggest that intra-articular TGF-b may be of ICAM-1 up-regulation (a TNF-dependent function) pro-inflammatory, while extra-articular TGF-b may and macrophage trafficking in the synovium. be anti-inflammatory w39x. Our results show that IL-17 is a newly described, pro-inflammatory, T cell asimadoline treatment changed TGF-b expression in a cytokine with the potential to play an important role in way that would maximize the anti-inflammatory effects inflammatory arthritis. We have previously shown that of these opposing TGF-b functions. Asimadoline IL-17 mRNA is expressed over the time course of decreased synovial (intra-articular) TGF-b expression adjuvant arthritis w26x. We show here, for the first time, and increased inguinal lymph node (extra-articular) that asimadoline reduces the mRNA expression of TGF-b expression. An altered balance in the pro- and this cytokine in the synovium at day 13 by more than anti-inflammatory effects of TGF-b by asimadoline 50%, concordant with a decrease in paw swelling. Such might explain its striking anti-arthritic actions. This effects may contribute to the unique anti-inflammatory hypothesis requires further testing using TGF-b properties of k-opioids w27x. blocking strategies and in vitro experimentation. 1020 K. A. Bush et al.

One limitation of the present study is that changes on sensory nerves to inhibit nociception in inflammation. in mRNA expression may not reflect a proportional Proc Natl Acad Sci USA 1990;87:5935–9. change in protein in the synovium. In a preliminary 5. Stein C. Peripheral analgesic actions of opioids. J Pain study, we found that there is secreted TNF protein in Symptom Manage 1991;6:119–24. synovial cultures on day 17 post-adjuvant (K. A. Bush, 6. Joris J, Costello A, Dubner R, Hargreaves KM. suppress carrageenan-induced oedema and hyper- unpublished observations). Once rat antibodies to all the thermia at doses that inhibit hyperalgesia. Pain 1990; cytokines of interest are available, in particular IL-17 43:95–103. and TGF-b, studies are warranted to confirm that 7. Levine JD, Moskowitz MA, Basbaum AI. The contri- similar changes in cytokine proteins do occur. bution of neurogenic inflammation in experimental In summary, we have found that the peripherally arthritis. J Immunol 1985;155:843s–7s. selective k-opioid agonist, asimadoline, reduces synovial 8. Walker JS, Howlett CR, Nayanar V. Anti-inflammatory membrane cytokine production in rats with adjuvant effects of kappa-opioids in adjuvant arthritis. Life Sci arthritis, with no effect on T-cell recruitment to the 1995;57:371–8. joint. These results from an in vivo model, support our 9. Wilson JL, Nayanar V, Walker JS. The site of previous work and that of others which demonstrate anti-arthritic action of the k-opioid, U-50,488H, in adjuvant arthritis: importance of local administration. k-opioid suppression of inflammatory cell immune Br J Pharmacol 1996;118:1754–60. function. To date, k-opioids have only been used 10. Walker JS, Chandler AK, Wilson JL, Binder W, Day RO. clinically as analgesics and their anti-inflammatory and Effect of m-opioids and on anti-arthritic actions have largely gone unrecognized. the development of adjuvant arthritis in rats. Inflamm The majority of specific centrally acting k-agonists have Res 1996;45:557–63. shown little analgesic efficacy and the prevalence of 11. Taub DD, Eisenstein TK, Geller EB, Adler MW. central side-effects has ruled out many of these Immunomodulatory activity of m- and k-selective opioid compounds w40x. Asimadoline is a peripherally selective agonists. Proc Natl Acad Sci USA 1991;88:360–4. k-opioid agonist, which lacks these side-effects. Its 12. Radulovic J, Miljevic C, Djergovic D et al. Opioid analgesic actions have only been assessed in post- receptor-mediated suppression of humoral immune w x response in vivo and in vitro: involvement of k opioid operative pain in patients undergoing knee surgery 41 . receptors. J Neuroimmunol 1995;57:55–62. Our studies are the first to evaluate the anti-arthritic 13. Binder W, Walker JS. Effect of the peripherally selective actions of asimadoline w13x. k-opioid suppression k-opioid agonist, asimadoline, on adjuvant arthritis. of inflammatory immune cell function may therefore Br J Pharmacol 1998;124:647–54. be clinically advantageous in immune diseases such as 14. Binder W, Scott C, Walker JS. Involvement of sub- inflammatory arthritis. stance P in the anti-inflammatory effects of the peri- pherally selective k-opioid asimadoline and the NK1 antagonist GR205171. Eur J Neurosci 1999;11:2065–72. Acknowledgements 15. Belkowski SM, Alicea C, Eisenstein TK, Adler MW, Rogers TJ. Inhibition of interleukin-1 and tumor necrosis The authors thank Dr Waltraud Binder for her factor-a synthesis following treatment of macrophages assistance with the preparation of sections for the with the kappa opioid agonist U-50,488H. J Pharmacol T-cell immunohistochemisty, Dr Caroline Scott for Exp Ther 1995;273:1491–6. preparing RNA from each tissue and Angelina Enno 16. Walker J, Wilson J, Binder W et al. Immunopharmacology for her invaluable help with the histology and immuno- of opioids in inflammatory arthritis. 9th World Congress histochemistry. on Pain, 22–27 August, Vienna, Austria, 1999 (Abstract). This study was funded by grants from the National 17. Wilson JL, Walker JS, Antoon JS, Perry MA. Intercellular Health and Medical Research Council of Australia adhesion molecule-1 expression in adjuvant arthritis in (970851 to JSW), ARC and Glaxo Wellcome rats: inhibition by k-opioid agonist but not by NSAID. (Scholarship to KB). The authors thank Drs A. Barber J Rheumatol 1998;25:499–505. 18. Chomczynski P, Sacchi N. Single-step method of RNA and Gerd Bartoszyk from Merck (KGaA, Darmstadt, isolation by acid guanidinium thiocyanate–phenol– Germany) for the gift of asimadoline and for their chloroform extraction. Anal Biochem 1987;162:156–9. continuous support in this regard. 19. McKnight AJ, Barclay AN, Mason DW. 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