Production of neuropeptide substance P by synovial fibroblasts from patients with rheumatoid arthritis and osteoarthritis

Production of neuropeptide substance P by synovial fibroblasts from patients with rheumatoid arthritis and osteoarthritis

Neuroscience Letters 303 (2001) 149±152 www.elsevier.com/locate/neulet Production of neuropeptide substance P by synovial ®broblasts from patients w...

296KB Sizes 0 Downloads 55 Views

Neuroscience Letters 303 (2001) 149±152

www.elsevier.com/locate/neulet

Production of neuropeptide substance P by synovial ®broblasts from patients with rheumatoid arthritis and osteoarthritis Hideo Inoue a,*, Yoshihito Shimoyama a, Kazuhiro Hirabayashi a, Hiroshi Kajigaya b, Seizou Yamamoto c, Hiromi Oda d, Yasuko Koshihara e a

Research Laboratory, Minophagen Pharmaceutical Co., 2-2-3, Komatsubara, Zama-shi, Kanagawa 228-0002, Japan b Nipon Professional School of Medicinal Technology, 1-7-1, Kyonan-cho, Musashino, Tokyo 180-8602, Japan c Department of Orthopedic Surgery, Tokyo Metropolitan Geriatric Hospital, Itabashi-ku, Tokyo 173-0015, Japan d Department of Orthopedic Surgery, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan e Department of Nutrition, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 173-0015, Japan Received 16 January 2001; received in revised form 1 March 2001; accepted 2 March 2001

Abstract We examined the production of substance P (SP) in synovial ®broblasts derived from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunoreactive SP was observed in non-stimulated RA ®broblasts. The expression of b-preprotachykinin-A (b-PPT-A) mRNA was con®rmed by reverse transcription-polymerase chain reaction analysis. SP contents in culture medium were increased by treatment of RA ®broblasts with transforming growth factor-b (TGFb) (10 ng/ml). Levels of SP release were elevated at 12 h after TGFb stimulation whereas the expression of b-PPT-A mRNA was enhanced at 3 h. Furthermore, SP production in response to TGFb was dose-dependently enhanced by basic ®broblast growth factor (bFGF). OA ®broblasts also signi®cantly released SP in the presence of TGFb (10 ng/ml) plus bFGF (50 ng/ml). These results suggest that SP produced by synovial ®broblasts may participate in joint diseases. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Substance P; Synovial ®broblasts; Rheumatoid arthritis; Osteoarthritis; Transforming growth factor-b; Basic ®broblast growth factor; Preprotachykinin mRNA; Human

The peripheral nervous system is involved in the ethiology and pathogenesis of joint diseases [10]. Neuropeptide substance P (SP) is synthesized in the dorsal root ganglion and then transported along the axons to the terminal nerve endings [18]. SP released after stimulation of articular C nerve ®bers appears to mediate extravasation of plasma proteins into the synovial cavity [5] and to be involved in the mechanism of joint destruction in experimental arthritis [12]. In fact, SP induces secretion of IL-1 and TNF by monocytes [11,14], and stimulates synovial cells to produce prostaglandin E2 [13], collagenase [13], gelatinase A [6], and oxygen free radicals [20]. Furthermore, high levels of SP have been detected in synovial ¯uid from patients with rheumatoid arthritis (RA) [2,16]. In the present study, we examined whether human synovial ®broblasts could produce SP. The synovial tissues were obtained from patients with RA * Corresponding author. Tel.: 181-462-51-1965; fax: 181-46251-5871. E-mail address: [email protected] (H. Inoue).

and osteoarthritis (OA), and synovial ®broblasts were isolated according to the method previously described [19]. Brie¯y, the minced tissue was incubated in RPMI 1640 medium containing 1 mg/ml collagenase and 0.15 mg/ml DNase I with shaking for 90 min at 378C. Synovial ®broblasts were isolated by density-gradient centrifugation through Ficoll/Paque and suspended in a-minimum essential medium (MEM) containing 10% heat-inactivated fetal calf serum (FCS) and 60 mg/ml kanamycin sulfate. Isolated ®broblasts were cultured at 378C in a humidi®ed atmosphere of 5% CO2/95% O2 air. When the cells reached con¯uence, they were passaged by brief trypsinization and used at more than ®ve passages at which they were homogeneous populations of ®broblasts. To assess SP release, synovial ®broblasts were seeded at 1:5 £ 104 cells/250 ml in 48-well plates and cultured up to con¯uence in the standard condition. Con¯uent cells (7 £ 104 cells) were replaced in a-MEM containing 0.5% FCS, kept for 24 h, and stimulated with either human recombinant transforming growth factor-b (TGFb) (Becton Dick-

0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 71 3- X

150

H. Inoue et al. / Neuroscience Letters 303 (2001) 149±152

Fig. 1. The presence of SP in synovial ®broblasts derived from RA patients. (A) SP immunoreactivity in RA ®broblasts. Positive staining was indicated by the presence of brown pigment. Original magni®cation, £ 400. (B) RT-PCR analysis of b-PPT-A mRNA expression in total RNA isolated from RA ®broblasts.

Fig. 2. Time course of SP production by RA synovial ®broblasts treated with TGFb. (A) Amounts of SP released from ®broblasts at the indicated time after TGFb (10 ng/ml) stimulation (n ˆ 6, points show mean ^ SEM). *P , 0:05 or **P , 0:01 vs control (ttest). (B) Changes of b-PPT-A mRNA expression in ®broblasts after TGFb (10 ng/ml) stimulation.

inson Labware, Bedford, MA), interleukin 1b (IL-1b) (Becton Dickinson Labware), tumor necrosis factor-a (TNFa) (Becton Dickinson Labware), nerve growth factor (NGF) (Pepro Tech EC, St James' Square, London), endothelial growth factor (VEGF) (Genzyme, Cambridge, MA), epidermal growth factor (EGF) (R&D systems, Mineapolis, MN) or basic ®broblast growth factor (bFGF) (Becton Dickinson Labware) for 48 h. Concentration of SP (detection limit roughly 5 pg/ml) in the culture supernatant was measured with an ELISA kit (Peninsula Laboratories, Belmont, CA). Data are expressed as mean ^ SEM. Signi®cance was evaluated by the Turkey±Kramer multiple comparison test after one-way analysis of variance or by the paired Student's t-test, and all positive results mentioned were signi®cant (P , 0:05 or P , 0:01). The expression of preprotachykinin (PPT) mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from con¯uent cells (2 £ 106 cells) with ISOGEN w (Nippon Gene, Toyama, Japan) and cDNA was synthesized from 1 mg RNA by using AMV reverse transcritpase XL (Life Sciences, St. Petersburg, FL) with an oligo dT-adaptor primer. cDNA was prepared for PCR using a TaKaRa Taq e kit (Takara, Osaka, Japan) and ampli®ed using the following conditions: 948C for 5 min, followed by 40 cycles of 948C for 30 s, 638C for 30 s, 728C for 1 min, followed by a ®nal of 728C for 5 min, on a Gene Amp PCR system 9700 (PE Applied Biosystems, Forest City, CA). The primer used for PPT was sense 5 0 - TTGCAGAAGAAATAGGAGCC-3 0 and antisense 5 0 -GCTATCACAACACATCATAC-3 0 . Human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) primers correspond to neucleotides 586±615 (forward) and 1017±1036 (reverse). PCR products were analyzed by a 2% agarose gel visualized using ethidium bromide. No PCR products were ampli®ed without RT reactions, and the ampli®ed product was con®rmed to be b-PPT-A by the sequence analysis. For SP immunohistochemistry, RA synovial ®broblasts were cultured in chamber slide until con¯uence and ®xed 4% paraformaldehyde. SP immunostaining was obtained using rabbit anti-serum (Chemicon international, Temecular, CA) (1:1000), secondary biotinylated antibody (1:200) (Histo®ne SAB-POR w, Nichirei Co., Tokyo, Japan) to rabbit IgG, and avidin-biotin-horse radish peroxidase (Nichirei). Negative controls were incubated in the absence of primary antibodies (data not shown). Immunoreactivity was visualized using diaminobenzidine as chromogen. First, we examined immunohistochemically whether SP is located in synovial ®broblast cell lines established from RA donors. Speci®c cytoplasmic staining of SP was observed in non-stimulated RA synovial ®broblasts (Fig. 1A). Furthermore, the expression of b-PPT-A mRNA, which encodes SP and neurokinin A [17], was con®rmed in total RNA isolated from ®broblasts by RT-PCR (Fig. 1B). However, RA synovial ®broblasts themselves released a little amount of SP (19 ^ 2 pg/ml, n ˆ 6) when kept in

H. Inoue et al. / Neuroscience Letters 303 (2001) 149±152

Fig. 3. Enhancement by bFGF of TGFb-induced SP production in RA synovial ®broblasts. Fibroblasts were incubated with bFGF in the presence of TGFb for 48 h (n ˆ 6, bars show mean ^ SEM). **P , 0:01 vs control (t-test). ²²P , 0:01 vs TGFb (Turkey± Kramer multiple test).

a-MEM containing 0.5% FCS for 48 h. Average contents of SP released after exposure to 1 ng/ml IL-1b, 20 ng/ml TNFa, 100 ng/ml NGF, 50 ng/ml bFGF, 100 ng/ml VEGF, and 100 ng/ml EGF were, 14 ^ 2, 11 ^ 1, 13 ^ 2, 26 ^ 5, 11 ^ 2, and 16 ^ 1 pg/ml (n ˆ 6), respectively, which did not differ signi®cantly from that (19 ^ 2 pg/ml, n ˆ 6) of untreated ®broblasts. In contrast, TGFb at a dose of 10 ng/ml signi®cantly (P , 0:01, Turkey±Kramer multiple test) induced SP release (69 ^ 6 pg/ml, n ˆ 6) from RA synovial ®broblasts. We further examined time-course of SP release by RA ®broblasts stimulated with TGFb (Fig. 2A). There was a signi®cant (P , 0:01) difference in amounts of SP at 12 h post-stimulation between control and TGFb-stimulated ®broblasts. The peak of SP release reached at 48 h after the stimulation. However, the expression of b-PPT-A mRNA was increased at 3 h and decreased at up to 24 h after TGFb stimulation (Fig. 2B). bFGF dose-dependently enhanced the release of SP in the presence of TGFb (Fig. 3), although bFGF alone failed to signi®cantly induce SP release. Average contents of SP released after treatment with NGF, VEGF, and EGF in the presence of TGFb for 48 h were, 44 ^ 6, 38 ^ 3, and 42 ^ 4 pg/ml (n ˆ 6), respectively, which were not different from that (48 ^ 2 pg/ml, n ˆ 6) of TGFb alone. These results indicate that bFGF plays as a promoter in the SP production of RA synovial ®broblasts. Moreover, levels of SP released from RA ®broblasts (276 ^ 56 pg/ml, three strains, n ˆ 6) in response to TGFb plus bFGF were significantly (P , 0:05, t-test) higher than those of OA ®broblasts (80 ^ 18 pg/ml. three strains, n ˆ 6).

151

SP released from sensory nerve terminals in response to electrical and chemical stimulation induces neurogenic in¯ammatory responses comprising vasodilation, plasma extravasation, and mast cell activation [7,18]. Therefore, it has been suggested that SP participates in the pathogenesis of chronic in¯ammatory conditions such as asthma, rheumatoid arthritis or atopic dermatitis. In this study, we demonstrated for the ®rst time that human synovial ®broblasts are able to produce SP. Cytokines and growth factors play an important role for developing synovial in¯ammatory responses in joint diseases [4,9]. The present results showed that TGFb induces SP production in RA synovial ®broblasts whereas IL-1b, TNFa, NGF, VEGF, EGF, and bFGF have little effect on the release of SP. TGFb is a cytokine capable of inhibiting or stimulating many aspects of cell proliferation and immune responses [8,15]. Others have reported that both latent and active TGFb contained in synovial ¯uid of patients with arthritis and its levels are higher in RA than in non-RA [1,3]. This fact supports that TGFb contained in synovial ¯uid contributes to the production of SP in synovial ®broblasts of RA patients. We also found that there is a time-lag between increased mRNA encoding PPT and peptide release in response to TGFb. SP is ®rstly synthesized as biologically inactive glycine-extended precursors and liberated by the sequential action of two enzymes. However, there was no change of SP immunoreactivity in ®broblasts treated with TGFb during 48 h (data not shown), indicating that the time-lag may be due to the post-transcriptional process of SP synthesis rather than the process of peptide release. As RA synovial ®broblasts induce SP production in response to TGFb alone and TGFb plus bFGF, we extended our study to OA synovial ®broblasts. OA cell strains also produced SP in the presence of TGFb plus bFGF, although the SP release was lower in OA ®broblast cell lines than in RA cell lines. It is, therefore, conceivable that the ability to produce SP in RA ®broblasts is greater than that of OA ®broblasts. In fact, SP levels detected in synovial ¯uid are higher in RA than in OA [16]. However, it seems that the production of SP by synovial ®broblasts is basically regulated by environmental conditions but not intrinsic in RA. In conclusion, we have demonstrated that synovial ®broblasts derived from patients with RA and OA are able to produce the neuropeptide SP. We also showed that TGFb and bFGF play an important role as an inductor or a promoter for the production of SP in synovial ®broblasts. The present data suggest that SP released from synovial ®broblasts, as well as peripheral nerve endings, participates in the development of joint diseases and pain. We are grateful to the late Mr N. Nagata and Dr M. Otsuka for their helpful advice, support, and discussion throughout this study. We also thank Dr T. Nishikawa and Mr T. Yoshida for their help in isolating synovial ®broblasts.

152

H. Inoue et al. / Neuroscience Letters 303 (2001) 149±152

[1] Brennan, F.M., Chantry, D., Turner, M., Foxwell, B., Maini, R. and Feldmann, M., Detection of transforming growth factor-beta in rheumatoid arthritis synovial tissue: lack of effect on spontaneous cytokine production in joint cell cultures, Clin. Exp. Immunol., 81 (1990) 278±285. [2] Devillier, P., Weill, B., Renoux, M., Menkes, C. and Pradelles, P., Elevated levels of tachykinin-like immunoreactivity in joint ¯uids from patients with rheumatic in¯ammatory diseases, N. Engl. J. Med., 314 (1986) 1323. [3] Fava, R., Olsen, N., Keski-Oja, J., Moses, H. and Pincus, T., Active and latent forms of transforming growth factor b activity in synovial effusions, J. Exp. Med., 169 (1989) 291±296. [4] Feldmann, M., Brennan, F.M. and Maini, R.N., Role of cytokines in rheumatoid arthritis, Annu. Rev. Immunol., 14 (1996) 397±440. [5] Ferrell, W.R. and Russell, N.J., Extravasation in the knee induced by antidromic stimulation of articular C ®bre afferents of anaesthetized cat, J. Physiol. (Lond.), 397 (1986) 407±416. [6] Hecker-Kia, A., Kolkenbrock, H., Orgel, D., Zimmermann, B., Sparmann, M. and Ulbrich, N., Substance P induces the secretion of gelatinase A from synovial ®broblasts, Eur. J. Clin. Chem. Clin. Biochem., 35 (1997) 655±660. [7] Holzer, P., Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, CGRP and other neuropeptides, Neuroscience, 24 (1988) 739±768. [8] Hoses, H.L., Yang, E.Y. and Pietenpol, J.A., TGF-b stimulation and inhibition of cell proliferation. New mechanistic insights, Cell, 63 (1990) 245±247. [9] Ivashkiv, L.B., Cytokine expression and cell activation in in¯ammatory arthritis, Adv. Immunol., 63 (1996) 337±376. [10] Kidd, B.L., Mapp, P.I., Gibson, S.J., Polak, J.M., O'Higgins, F., Buckland-Wright, J.C. and Blake, D.R., A neurogenic mechanism for symmetrical arthritis, Lancet, 11 (1989) 1128±1130.

[11] Kimball, E.S., Persico, F.J. and Vaught, J.L., Substance P, neurokinin A and neurokinin B induce generation of interleukin-1-like activity by P388D1 cells. Possible relevance to arthritic disease, J. Immunol., 141 (1988) 3564±3569. [12] Levine, J.D., Clark, R., Devor, M., Helms, C., Moskowitz, M.A. and Basbaum, A.I., Intraneuronal substance P contributes to the severity of experimental arthritis, Science, 226 (1984) 547±549. [13] Lotz, M., Carson, D.A. and Vaughan, J.H., Substance P activation of rheumatoid synoviocytes: neural pathway in pathogenesis of arthritis, Science, 235 (1987) 893±895. [14] Lotz, M., Vaughan, J.H. and Carson, D.A., Effect of neuropeptides on production of in¯ammatory cytokines by human monocytes, Science, 241 (1988) 1218±1221. [15] Massagne, J., The transforming growth factor-beta family, Annu. Rev. Cell Biol., 6 (1990) 597±641. [16] Menkes, C.J., Renoux, M., Laoussadi, S., Mauborgne, A., Bruxelle, J. and Cesselin, F., Substance P levels in the synovium and synovial ¯uid from patients with rheumatoid arthritis and osteoarthritis, J. Rheumatol., 20 (1993) 714± 717. [17] Nawa, H., Kotani, H. and Nakanishi, S., Tissue-speci®c generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing, Nature, 312 (1984) 729± 734. [18] Otsuka, M. and Yoshioka, K., Neurotransmitter functions of mammalian tachykinins, Physiol. Rev., 73 (1993) 229±308. [19] Takayanagi, H., Oda, H., Yamamoto, S., Kawaguchi, H., Tanaka, S., Nishikawa, T. and Koshihara, Y., A new mechanism of bone destruction in rheumatoid arthritis: synovial ®broblasts induce osteoclastogenesis, Biochem. Biophys. Res. Commun., 240 (1997) 279±286. [20] Tanabe, T., Otani, H., Mishima, K., Ogawa, R. and Inagaki, C., Mechanisms of oxyradical production in substance P stimulated rheumatoid synovial cells, Rheumatol. Int., 16 (1996) 159±167.