598 Annals ofthe Rheumatic Diseases 1990; 49: 598-602 Ann Rheum Dis: first published as 10.1136/ard.49.8.598 on 1 August 1990. Downloaded from Effects of capsaicin on the metabolism of rheumatoid arthritis synoviocytes in vitro

M Matucci-Cerinic, S Marabini, S Jantsch, M Cagnoni, G Partsch

Abstract to capsaicin is the damage of mitochondria and The effects of capsaicin, the ingredient of hot the change of the nuclear shape. These modifi- pepper, on rheumatoid arthritis synoviocytes cations and the dilatation of endoplasmic have been studied. Capsaicin was shown to reticulum and Golgi apparatus have been shown have a direct action on the metabolism of in a major subpopulation ofB type neurones. " l synovial cells. Thus at 10-6 moVl cell pro- The toxic effects of capsaicin have also been liferation was noted while at 10-8 mol/l and at described in endothelial cells.'2 higher doses DNA synthesis was restored to In rats with adjuvant arthritis subcutaneous the control level. Capsaicin at both doses pretreatment with capsaicin has been shown to induced an increase in the synthesis of col- moderate synovitis'3 and attenuate the increase lagenase and at the lower concentration (10-8 in substance P content, due to arthritis, in mol/V) only of prostaglandins. various nervous tissues.'3 14 Inman and These results indicate that the different coworkers showed that intra-articular capsaicin effects of capsaicin on cellular proliferation can moderate the severity of synovitis in cats.'5 and on metabolic activities are dependent on On the other hand, acute administration of dose. The responses seen in rheumatoid capsaicin to adult rats results in a substantial arthritis synoviocytes in vitro might not be depletion, from primary afferent fibres, of mediated by tachykinins if the synovial tissue substance P and other tachykinins'6 17 that are is still able to produce neuropeptides in the considered the putative mediators of neuro- absence of neuronal afferents. These results genic inflammation.'8 19 suggest that capsaicin, in addition to its direct Yaksh showed that when capsaicin was given action on the afferent nervous fibres and the intra-articularly in vivo in the cat substance consequent release of tachykinins, may also P like immunoreactivity was evoked in the have a direct action on the cells. The mech- synovial fluid.20 anisms by which capsaicin stimulates DNA It thus seems that (a) tachykinins are present synthesis and production of collagenase and in the synovial fluid, (b) they can be released by http://ard.bmj.com/ prostaglandin E2, in a manner dependent on capsaicin, and (c) capsaicin treatment has bene- dose, remain to be determined. ficial effects in adjuvant arthritis. But is there a correlation between depletion of tachykinins and the beneficial effect of capsaicin? Might Rheumatoid arthritis (RA) is a disease charac- capsaicin have a direct action on the synovial terised by chronic synovial inflammation lead- tissue independent of its effect on afferent ing to cartilage destruction, which is believed to fibres? At present there are no published on September 25, 2021 by guest. Protected copyright. result from the production of metabolites of reports on the effects of capsaicin on RA arachidonic acid and proteases.' 2 synovial cell cultures. Explants of RA synovial tissue are now in In our study we investigated the effect of widespread use for in vitro culture of synovio- capsaicin on various metabolic functions of RA Institute for Clinical cytes. These cultures, deprived of nervous synoviocytes, studying both DNA and RNA Medicine IV and Medical system control, provide a useful experimental synthesis and also the production of collagenase Pathology I, University of Florence, tool to study the cellular metabolism and the and prostaglandin E2 (PGE2). Italy response to substances that may interfere with M Matucci-Cerinic the inflammatory process. S Marabini M Cagnoni Capsaicin, a homovanillic acid derivative (8- Materials and methods has been RADIOLABELLED SUBSTANCES Ludwig Boltzmann- methyl-N-vanillyl-6-nonenamide), Institute for used as an experimental tool to investigate the Methyl [3H]thymidine (specific activity 3-1 Rheumatology and functional and neurochemical characteristics of TBq/mmol, 5-[3H]uridine (specific activity Balneology, A-1107 peptide containing primary sensory neurones.3 4 11 TBq/mmol), and 1-['4C]acetic anhydride Vienna-Oberlaa, Austria G Partsch Recently, it has been used on humans for (specific activity 370 MBq/mmol), all from of cluster NEN Chemicals, USA, were used. Orthopaedic topical treatment headache,5 herpes Department/ zoster,6 and vasomotor rhinitis.7 Pulmological Center of Systemic administration of capsaicin to the City of Vienna, rats results in an selective PREPARATION OF SYNOVIAL CELLS Austria newborn irreversible, S Jantsch degeneration of a distinct population of small Synovial tissue was obtained from patients with afferent neurones located in defmite RA undergoing reconstructive joint Correspondence to: size B type primary Dr Marco Matucci-Cerinic, spinal and cranial sensory ganglia.8 Likewise, surgery. via P Thouar 18, 50122, to adult rats Cultures of cells were grown in Florence, Italy. capsaicin given systematically synovial in the same fibres.9 Parker 199 medium (supplemented with 2 mM Accepted for publication induces reversible damage 8 December 1989 One of the most important neurotoxic responses L-glutamine, 100 U/ml penicillin, 100 ,Ig/ml Effects ofcapsaicin on rheumatoid arthritis synoviocytes 599

streptomycin, and 15% fetal calf serum) (PAA, was studied according to the method of

Austria), hereafter referred to as the medium. Johnson-Wint.23 Ann Rheum Dis: first published as 10.1136/ard.49.8.598 on 1 August 1990. Downloaded from Cells were used between the fifth and the 10th Digestion of a '4C labelled collagen film was passages. During experiments cells were kept in used in this assay. The collagen type III 24 or % well culture plates (Nunclon Delta; (calfskin; Sigma Co, USA) was labelled with the Nunc, Denmark). Six synoviocyte cultures 1-['4C]acetic anhydride for two hours at 6°C were plated in quadruplicate except for the (instead of one hour as indicated by Johnson- control samples (six sets). All cultures and Wint). The specific activity of the radiolabelled incubations were obtained in a CO2 incubator at collagen was about 11 000 dpm/mg collagen. A 37°C in an atmosphere of 5% CO2. The same 3 ml aliquot of this '4C labelled collagen cultures were used for each experiment. solution was dialysed (three times against 800 The effect of capsaicin (Sigma Co, USA) on ml 0-15 M sodium phosphate puffer, pH 7-4). synoviocytes was tested at doses between 108 14C labelled collagen solution (25 VI) was added and I0-3 mol/l. to each well of a 96 well, flat bottom Nunclon tissue culture plate (in an angle of 450) and treated further as described by Johnson-Wint. DNA SYNTHESIS The mitogenic effect of capsaicin was measured according to Partsch et a12' with [3H]thymidine Collagenase assay as a precursor for DNA. Briefly: 2x I 04 cells in Release of collagenase was measured in the 100 ,ul medium were seeded into microwell medium of six RA synoviocyte cultures. Cells plates. After 24 hours the Parker 199 medium (104) were plated in 24 well tissue culture plates, was changed and replaced by new medium. as described by Lotz et al.24 After six days of Capsaicin (10 pA) was added to obtain the final culture the cells were washed three times with concentrations shown in the figures. After 24 Hanks's solution and replaced by 0'5 ml hours, synoviocytes were labelled with 37 kBq medium (Parker 199) free from fetal calf serum. [3H]thymidine and incubated for a further 18 After the addition of 50 VIl of capsaicin (final hours. The medium was discharged, the cells concentration as given in 'Methods') cells were were washed several times with Hanks's solution incubated for a further 48 hours. The super- and then covered with 100 [L of 10% trichloro- natants were centrifuged at 12 000 rpm and acetic acid for 20 minutes. Methanol (100 [L) frozen at -70°C. The supernatant (500 RI) was was added, removed after 10 minutes, and the treated with 5 ,ul trypsin (100 jig/ml medium, cells were dried by a stream of air. The cells type III; Sigma, USA) for 10 minutes at 250C to were lysed in 100 pi 0-3 N NaOH for three activate latent collagenase. Then 10 pA soybean hours at 37°C, the supernatant was well mixed, trypsin inhibitor (type I-S; Sigma, USA) was and the radioactivity in 50 pi of supernatant was added and the solution left for 20 minutes at a Packard scintillation counter room temperature. Portions of the solution (200 measured by http://ard.bmj.com/ using Instagel (Packard, USA). ,ul) were placed in the wells coated with 14C labelled collagen. After an incubation of three hours at 370C the radioactivity in 150 [xi was RNA SYNTHESIS counted by liquid scintillation spectrometry RNA synthesis was evaluated by increasing [3H]- using Instagel (Packard, USA). Fresh culture uridine incorporation into confluent synovial medium that had been treated in the same way cells to the methods of Partsch et al' was used as control. The disintegrations per

according on September 25, 2021 by guest. Protected copyright. and Schmid et a122 with some modifications. minute of the control were substracted from the Cells (2-5x 104) were seeded in 24 well culture mean values obtained from the synoviocyte plates (0'5 ml medium) and kept for six days supernatant. until they were confluent. The medium was changed at day 3. Confluent cells were held for one hour in 0 5 ml of a protein free medium PROSTAGLANDIN (Hanks's solution), which was replaced by 0-5 The influence ofcapsaicin on the PGE2 synthesis ml of medium (Parker 199 plus 2 g/l human ofsynoviocyteswas studied bythemethodofLotz albumin and 1 FM uridine: Sigma Co, USA). et al,24 with some modifications. The substances under study (50 ,lI) were added Cells for culture were kept in 24 well Nunclon and the cells incubated for four hours. Cells plates with 0 5 ml medium. Cells (2 x 104) were were labelled with 37 kBq [3H]uridine and inoculated in medium plus 15% fetal calf serum incubation continued for 18 hours. Trichloro- and HEPES (N-2-hydroxyethylpiperazine-N'- acetic acid treatment and washing were the 2-ethanesulphonic acid). The next day the same as for the DNA experiments. The cells medium was replaced by fresh medium and the were lysed in 0'3 ml N NaOH for two hours at cells cultivated for one week up to confluency. 37°C, the solution was diluted with 0'7 ml The medium was then changed and replaced by distilled water, and 0-9 ml solution used for the fresh medium containing capsaicin (50 pA). The measurement ofradioactivity (Instagel; Packard, cells were incubated for a further 48 hours and USA). then the medium was collected and centrifuged. Cell free supernatant (450 pi) was put into polypropylene tubes and stored at -70°C for COLLAGENASE further treatment. Extraction of PGE2 was as Preparation of radiolabelled collagen follows: 450 IL of the medium were brought to Collagenase release from RA synoviocytes pH 3-5 with 0-5 N HC1. Ethyl acetate (2 ml) was cultured in medium, in the presence ofcapsaicin, added, the samples shaken for 30 seconds, and 600 Matucci-Gerinic, Marabini,jantsch, Cagnoni, Partsch

centrifuged at 1500 g. From the organic phase

200 til were taken and evaporated to dryness. Ann Rheum Dis: first published as 10.1136/ard.49.8.598 on 1 August 1990. Downloaded from 600[ The residue was dissolved in 600 ,ul distilled water and from this solution 20 1d used in a radioimmunoassay kit (NEN Products, West I Germany). The radioactivity was measured in a E 4000 gammacounter. Standard PGE2 was also added E. to the medium and extracted in the same way as the samples. Recovery of PGE2 was about 96%. IV :2 The counts were calculated by a standard curve 0 and the results expressed as a percentage of the control PGE2. oI 0 1tO 10t 104 -3 ANALYSIS OF THE RESULTS NS p<005 NS NS The statistical significance of the results for the Capsaicin (mol/l) six cultures was examined with a parameter free Figure 1: Thymidine incorporation into synovoyes was stimulated by capsaicin at I1o6 Wilcoxon signed rank test. The course of the molll. Capsaicin at le, 10', and lt molll had no significant influence on DNA.synthesis individual cultures and their mean values are in comparison with the control. The experiments were performed with six different cultures in both shown in the figures. quadruplicate.

Results NUCLEIC ACID SYNTHESIS 500r DNA synthesis Cell proliferation was studied by measuring -. thymidine incorporation into synoviocytes in la 400 2EJ° the presence of increasing concentrations of capsaicin (10-8 to I0-3 mol/l) (fig 1). In com- - - 3 parison with baseline controls the maximal 0 significant (p<005) increase of thymidine C) **44 incorporation was obtained with capsaicin at = 200 107' mol/l. Concentrations of 0 *. capsaicin 10-8 *0 molIl and higher did not induce a statistically S 2 significant thymidine incorporation. -2 100 C.) at http://ard.bmj.com/

0 RNA synthesis -8 -6 -4 -1 The results indicate that capsaicin did not cause 0 10 10 10 p<0-O5 p<0 05 p<005 p<05 any statistically significant change in uridine incorporation at any of the concentrations Capsaicin (mol/l) tested. Figure 2: Collagenase synthesis induced by capsaicin at different concentrations. The production ofcollagenase was increased by all capsaicin doses tested in this study. Thde six cell cultures were each performed in quadruplicate. on September 25, 2021 by guest. Protected copyright. COLLAGENASE Each concentration of capsaicin from 10-8 to 10-3 mol/l significantly increased the release of collagenase from synoviocytes into the medium 3 (fig 2). The highest stimulation occurred at a capsaicin concentration of 10-8 mol/l (p<005). Stimulation of collagenase synthesis was less but 2~~~ still significant at higher doses of capsaicin also (p

PROSTAGLANDIN E2 .CI Figure 3 shows the results for PGE2 pro- {S t/o~~~~"> duction. Clearly, rising concentrations of W * capsaicin increased the synthesis of PGE2 '*i by RA synoviocytes. In comparison with controls maximal stimulation of PGE2 production occurred at a capsaicin concen-

0 tration of 10-8 mol/l (p<0 05). Higher concentrations caused a statis- O 106 10 104 _ot3 p

Discussion directly on synovial cells. An increase of sodium This study shows that capsaicin exerts a accumulation of and influx, intracellular NaCl, Ann Rheum Dis: first published as 10.1136/ard.49.8.598 on 1 August 1990. Downloaded from direct action on the metabolism of synovial the consequent osmotic changes have been cells obtained from RA. The data suggest implicated as the mechanisms responsible for that capsaicin can induce cell proliferation the neurotoxic action of capsaicin in cultured at fairly low concentrations (106 mol/1) while sensory neurones.39 4 The mechanism by higher concentrations restore the cellular DNA which capsaicin stimulates DNA synthesis and synthesis to the level of controls. Capsaicin at production of collagenase and PGE2, however, low concentrations (108 mol/1) has also been in a manner dependent on dose, remains to be shown to increase the synthesis of collagenases determined. and prostaglandins. At higher concentrations the release of collagenases and prostaglandins into the culture medium by rheumatoid The authors thank Professor D Regoli, department of pharma- synoviocytes was reduced. These results suggest cology of the University of Sherbrooke, for his helpful advice and for text corrections. that capsaicin may have different actions on This work was supported by CNR, Rome, Italy (Progetto cellular proliferation and on metabolic activities. Finalizzato Chimica Fine Secondaria, grant No 85.00559.56) and Inflamed synovial tissue is known to proliferate in part by the Austrian National Bank (funds No. 2951). rapidly25 and to produce high concentrations of collagenase and prostaglandins.' 1 Dayer J M, Krane S M, Russel R G G, Robinson D R. Collagenase is a major product of fibro- Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. Proc Nad Acad Sci blasts,2 26 and it is apparent that fibroblasts USA 1976; 73: 945-9. have multiple mechanisms which regulate the 2 Harris E D Jr. Pathogenesis of rheumatoid arthritis. In: Kelley W N, Harris E D Jr, Ruddy S, Sledge C B, eds. activity and synthesis of the enzyme.2 27 28 Textbook of rheumatology. Philadelphia: Saunders, 1985: Cultures of RA synovial tissue produce 896-927. large amounts of collagenase, which can initiate 3 Holzer P. Local effector functions of capsaicin sensitive sensory nerve endings: involvement of tachykinins, CGRP breakdown of types I, II, and III interstitial and other peptides. Neuroscience 1988; 24: 739-68. thus giving this enzyme a major role 4 Maggi C A, Meli A. The sensory efferent function of collagens, capsaicin-sensitive sensory neurons. Gen Pharmacol 1988; in the degradation of connective tissue in both 19: 1-43. normal and pathological conditions.2 5 Sicuteri F, Fusco B, Marabini S, Fanciullacci M. Capsaicin as a potential medication for cluster headache. Medical Science Therefore production of collagenase and Research 1988; 16: 1079-80. 6 Watson C P, Evans R J, Watt V R. Post-herpetic neuralgia PGE2 by RA synoviocytes is of potential and topical capsaicin. Pain 1988; 33: 333-40. importance in the degradation of articular 7 Marabini S, Ciabatti G, Pofli G, et al. Effect of topical nasal treatment with capsaicin in vasomotor rhinitis [Abstract]. cartilage and adjacent joint structures. Indeed, Regul Pept 1988; 22: 1-2. PGE2, an inflammatory lipid mediator, may 8 Jancso G, Kiraly E, Jancso-Gabor A. Pharmacologically- role in provoking several induced selective degeneration of chemoreceptive primary have an additional sensory neurons. Nature 1977; 270: 741-3. effects, one of which might be inducing bone 9 Jancs6 G, Kiraly E, Joo F, Such G, Nagy A. Selective degeneration by capsaicin of a subpopulation of primary resorption. sensory neurons in the adult rat. Neurosci Leu 1985; 59: Other authors have shown that capsaicin can 209-14. http://ard.bmj.com/ 10 Solczany J, Jancso-Gabor A, Joo F. Functional and fine induce PGE2 release in homogenates of seminal structural characteristics of the sensory neurons blocking vesicles.29 In particular, it has been shown that effect of capsaicin. Naunyn Schmiedebergs Arch Pharnacol 1975; 287: 157-69. capsaicin induces PGE2 release after chronic 11 Chiba T, Masuko S, Kawano H. Correlation ofmitochondrial denervation of the rabbit ear, suggesting that swelling after capsaicin treatment and substance P and may exert other effects besides those somatostatin immunoreactivity in small neurons of dorsal capsaicin root ganglion in the rat. Neurosci Leu 1986;- 64: 311-6. mediated through sensory nerve stimulation.30 12 Kenins P, Hurley J V, Bell C. The role of substance P in the

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