- Email: [email protected]
Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses
Osteoarthritis and Cartilage xxx (2015) 1e10
Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses €fer y k, R. Bauer y z, Z. Jenei-Lanzl x, R.H. Springorum k, S. Gra €ssel y k * J. Lorenz y k, N. Scha y Centre for Medical Biotechnology, BioPark I, University of Regensburg, Regensburg, Germany z Department of Oral and Maxillofacial Surgery, University of Regensburg, Germany x Internal Medicine 1, Division of Rheumatology, University of Regensburg, Germany k Department of Orthopedic Surgery, University of Regensburg, Bad Abbach, Germany
a r t i c l e i n f o
s u m m a r y
Article history: Received 20 January 2015 Accepted 18 August 2015
Objective: Norepinephrine (NE) was measured in synovial fluid of trauma patients and sympathetic nerve fibers were detected in healthy and osteoarthritic (OA) joint tissues indicating that cartilage pathophysiology might be influenced by sympathetic neurotransmitters. The aim of this study was to elucidate the mostly unknown role of NE in OA chondrocyte metabolism and inflammatory responses. Methods: Articular cartilage was received after total knee replacement surgery from OA patients. Expression of adrenergic receptors (AR) and tyrosine hydroxylase (TH) was tested with end point polymerase chain reaction (PCR) and immunohistochemistry. 3-dimensional (3D) cell cultures were employed to analyze effects of NE on chondrocyte cell metabolism and the expression of interleukins (ILs), matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), glycosaminoglycan (GAG) and collagen II under non- and inflammatory conditions. Chondrocyte monolayer cultures were used to specify AR subtypes, to analyze cell cycle distribution and to determine catecholamines in cell culture supernatants. Results: AR subtypes and TH were detected in chondrocytes, whereas NE was not released in measurable amounts. 106 M NE reversed IL-1b induced changes in IL-8, MMP-13, GAG and collagen II expression/ production indicating for b-AR signaling. Additionally, NE caused cell cycle slow down and decreased proliferation via b-AR signaling. 108 M NE increased the number of proliferating cells and induced apoptosis via a1-AR signaling. Conclusions: NE affects chondrocytes from OA cartilage regarding inflammatory response and its cell metabolism in a dose dependent manner. The sympathetic nervous system (SNS) may have a dual function in OA pathology with preserving a stable chondrocyte phenotype via b-AR signaling and OA pathogenesis accelerating effects via a-AR signaling. © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Osteoarthritis Articular chondrocytes Norepinephrine IL-1b Sympathetic nervous system
Introduction OA is the most common form of arthritis and a leading cause of chronic pain. The social burden of OA is immense as it affects 9.6% of men and 18% of woman over 60 years of age worldwide1 and currently compromising the lives of 40 million Europeans2. The etiology of OA is multifactorial and still incompletely understood.
€ssel, Department of * Address correspondence and reprint requests to: S. Gra Orthopedic Surgery, Experimental Orthopedics, University of Regensburg, ZMB/ BioPark 1, Josef-Engert-Str. 9, 93053 Regensburg, Germany. Tel: 49-941-943-5065; Fax: 49-941-943-5066. E-mail address: [email protected] (S. Gr€ assel).
Hallmark features are structural changes including articular cartilage destruction as well as alterations in synovium and subchondral bone. Increased proteolytic activity leads to degradation of major cartilage extracellular matrix components as collagens and the large proteoglycans. In this process, MMPs and their inhibitors play a key role. Regulation of several highly expressed MMPs (MMP-2, -3 and -13) in end stage OA cartilage supports this concept3e5. Regarding metabolism of chondrocytes, early OA is characterized by proliferation accompanied with increased matrix production, whereas end stage OA chondrocytes express hypertrophy markers such as MMP-13 and collagen X and presumably undergo apoptosis6e8. Interestingly, intermittent inflammatory flares also occur in the synovial membrane in the course of OA suggesting
http://dx.doi.org/10.1016/j.joca.2015.08.007 1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
2
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
inflammation is associated with this disease. Although not a primary phenomenon, synovial inflammation contributes to OA progression. Among pro-inflammatory mediators involved in OA, IL-1b seems to play a key role9. It increases the synthesis of various MMPs and some short time pro-inflammatory mediators such as IL-6 and IL-88,10. NE belongs to the catecholamine family of tyrosine-derived sympathetic neurotransmitters. This subtype of sympathetic nerve fibres, characterized by the expression of TH, has been identified in synovium, bone marrow, periosteum, and in boneadherent ligaments11e13, indicating that growth and metabolic activity of synovial joint tissues are regulated by sympathetic neurotransmitters. Catecholaminergic effects are mediated by the adrenergic receptor (AR) family. At high concentrations (>107 M) NE acts via a- and b-AR, whereas low concentrations (107 M) are mediated mainly via a-AR14. Functional a1-, a2-and b2-AR are present on murine costal chondrocytes and chondrogenic ATDC5 cells15,16. Previously, we observed a decrease in apoptosis in murine costal chondrocytes after treatment with NE via b-AR signaling and an increase in focal adhesion contacts15. Additionally, it is described that NE may also induce apoptosis in several other cell types17,18 and to regulate the proliferation of osteoblasts, osteoblast-like cells, and mesenchymal stem cell lines through b- and a1-AR mediated signaling19e21. Furthermore, catecholamines play a decisive role in inflammatory processes characteristic for rheumatoid arthritis22. With respect to above disease entity, b-AR activation predominantly mediates anti-inflammatory effects, whereas activation of a-AR, if located on immune cells, mediates a priori pro-inflammatory effects (reviewed in23). Articular cartilage is not deeply innervated by nerve fibers, although there are hints in literature that catecholamines influence articular chondrocytes especially in OA. High levels of NE were measured in synovial fluid of patients with joint trauma24 along with a high incidence to develop post-traumatic OA25. Suri et al. have localized both sensory and sympathetic nerve fibers in similar distributions to the articular cartilage in human tibiofemoral OA. In the pathogenesis of OA, nerves grow into joint structures through vascular channels mainly from subchondral bone breaching through the tidemark26. In this study, we have addressed the response of human articular OA chondrocytes to NE with respect to inflammatory markers and chondrocyte metabolism.
Materials and methods Study design In this study we analyzed the localization of TH and AR subtypes in human OA cartilage, the response of human OA chondrocytes to NE under inflammatory conditions and their metabolic answer to NE stimulation. TH and AR subtype gene expression were measured with end point PCR after RNA isolation from monolayer- and 3D (fibrin gel) cultured chondrocytes under non- and inflammatory conditions (w/o 0.5 ng/ml IL-1b) TH, a1D-AR and b2-AR expression were approved by immunohistochemistry staining on cryo-embedded cartilage slides (N 3). Responses of human OA chondrocytes to NE under inflammatory conditions were tested with real time PCR, enzyme-linked immunosorbent assays (ELISA) and dimethyl methylene blue (DMMB) assays under non- and inflammatory conditions (w/o 0.5 ng/ml IL-1b). Chondrocytes of the same donor were cultured under following conditions: Treatment of fibrin gels with (1) vehicle (PBS with 0.5 mM acetic acid), (2) IL-1b and (3) IL-1b þ NE
(106 M and 108 M) respectively (4) vehicle (PBS with 0.5 mM acetic acid) and (5) NE (106 M and 108 M). (N 6) (Suppl. Fig. 1A). Metabolic (proliferation and apoptosis) response of OA chondrocytes to NE were analyzed in 3D culture under noninflammatory conditions after staining of proliferating cell nuclear antigen (PCNA, proliferation) and TdT-mediated dUTP-biotin nick end labeling (TUNEL, apoptosis) on paraffin-embedded micromass pellet sections. To specify AR subtypes involved in metabolic responses, chondrocytes were cultured in monolayer and AR antagonists were included in BrdU (proliferation) and caspase 3/7 (apoptosis) assays. To further analyze proliferation effects, cell cycle was analyzed via flow cytometer. (N 6) (Suppl. Fig. 1B).
Isolation and culture of chondrocytes Articular cartilage was obtained from OA patients after total endoprothesis surgery. This had been approved by the local Ethics €t Committee (Az:14-101-0189; Ethikkommission an der Universita Regensburg, email: [email protected]) and specimens were taken with patients’ written consent. For this study knee joints were obtained from 61 different donors (29 male and 32 female) within an age range of 48e84 (mean age of 66,4). Macros-copically normal looking cartilage slices were digested over night with collagenase II (PAA) in Dulbecco's modified Eagle medium (DMEM)/ F12 (Invitrogen) containing 1% penicillin/streptomycin (P/S) at 37 C. Isolated chondrocytes were passed through a 70 mm nylon mesh (Falcon) to remove residual cartilage matrix fragments, centrifuged at 200 g for 5 min, and resuspended in DMEM/F12 supplemented with 10% fetal calf serum and 1% P/S. Chondrocytes were seeded at a density of 20,000 cells/cm2 and initially cultured in monolayer for maximal 10 days in a humidified 37 C/5% CO2 incubator.
RNA isolation, end point PCR and real-time PCR RNA from chondrocyte monolayer culture was isolated with Aglient RNA Absolutely Miniprep Kit. 3D cultivated chondrocytes were first homogenized with a PowerGen 1000 Homogenizer (Fisher scientific) for 45 sec in 1 ml peqgold trifast (Peqlab) and afterwards RNA was isolated according to manufactures instructions. That was followed by a second RNA isolation step with RNA Absolutely Nanoprep Kit (Aglient). To translate RNA in cDNA, AffinityScript QPCR cDNA Synthesis Kit (Aglient) was applied. Gene expression of TH and subtypes of a- and b-ARs was determined using one-Step RT-PCR (Invitrogen), where each reaction consisted of 100 ng of cDNA. Gene expression of GAPDH was used as loading control. Primers were designed from universal probe library system assay design center (Roche) and listed in Suppl. Table 1. Commercial available RNA of human brain and arteria tissue served as positive controls and the identity of amplified PCR products was confirmed by sequencing. Pure RNA served as negative control because most AR subtypes contain only one exon, excluding intron-spanning primer design. Relative quantitative PCR for IL-6, IL-8, MMP-2, MMP-3, MMP13, TIMP-1, TIMP-2 and TIMP-3 was performed in Mx3005P qPCR system with Brilliant II SYBR Green QPCR Mstr Mx (Aglient), 30 ng cDNA and in triplicates. Primers are listed in Suppl. Table 2. Relative quantification was determined with MxPro QPCR software using GAPDH as endogenous loading control and IL-1b stimulated respectively untreated cells as calibrator. Log fold changes to calibrator (base 2) values were plotted using Graph Pad Prism 5 software.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
3
Localization of TH, a1D- and b2-adrenergic receptors in articular cartilage
combined either with vehicle or with NE (106 and 108 M, SigmaeAldrich).
To visualize TH, a1D- and b2-AR, articular cartilage slices were immersed in OCT embedding compound (Sakura Finetek Europe) and immediately frozen in liquid nitrogen. Transverse sections were cut with a cryostat at 10 mm, mounted onto clean glass microscopic slides and frozen at 70 C. Sections were fixed in cold acetone (20 C) and washed in phosphate buffered saline. For b2AR staining, sections were treated as described previously27 followed by blocking with 1% normal goat serum (SigmaeAldrich) for 1 h and overnight incubation with an anti-b2-AR antibody (Abcam) at 4 C. After washing, sections were incubated with appropriately diluted biotinylated secondary antibody as described previously27. For TH and a1D-AR staining, endogenous peroxidase was blocked for 5 min with 3% (volume/volume) H2O2 followed by blocking with 5% normal swine serum (Dako) and 1% (weight/volume) bovine serum albumin (SigmaeAldrich) in PBS for 1 h. TH staining was performed with rabbit anti human TH antibody (1:250 in blocking solution, Abcam) and a1D-AR staining with rabbit anti human a1DAR antibody (1:100 in blocking solution, Alomone Labs) overnight at 4 C. After washing, sections were incubated with a biotinylated swine anti rabbit antibody (1:500 in blocking solution, Dako) and further procedure was performed as described previously27. Sections incubated without the primary antibody or with pre-immune serum served as negative control.
ELISA for IL-8 and MMP-13
Determination of catecholamines To measure catecholamines, passage 0 chondrocytes were incubated for 10 days in monolayer culture. Afterwards cells were treated for 24 h with TH specific cofactors BH4 (6R-5,6,7,8tetrahydrobiopterin dihydrochloride) (105 M; SigmaeAldrich), respectively bivalent iron (Fe2þ sulfate heptahydrate) (105 M; SigmaeAldrich) and with inhibitors for NE degradation monoaminooxidase inhibitor bifemelane hydrochlorid acid (105 M; Tocris) respectively catechol-O-methyltransferase inhibitor OR-486 (105 M; Bristol). Catecholamines in cell culture supernatants were assayed by high performance liquid chromatography (HPLC) as previously described28. The detection limit for catecholamines in HPLC is about 1010 M.
Release of IL-8 and MMP-13 was analyzed with appropriate ELISA Kits (RayBiotec). Culture supernatants of chondrocytes cultured in fibringels were harvested after 24 h stimulation and different dilutions were subjected to ELISA according to manufacturers’ instructions. Biochemical analyses To analyze the amount of sulfated GAG, collagen II and dsDNA of chondrocyte-containing fibringels, gels were digested in 0.05 M acetic acid plus 0.5 M NaCl (pH 2.9e3.0) supplemented with 10 mg/ ml pepsin, and dissolved in 0.05 M acetic acid on the rotator for 48 h at 4 C. Next steps of digestion and DMMB assays were performed as described previously29. Collagen II Detection Kit 6019 (Chondrex) was used according to manufactures instructions and DNA content was analyzed with Quant-iT dsDNA Assay Kit (Invitrogen). The amount of GAG and collagen II of each pellet were related to the corresponding amount of DNA. Proliferation and apoptosis in micromass pellet culture In micromass pellets, number of apoptotic cells was monitored by TUNEL on paraffin-embedded pellet sections. Apoptotic cells were identified by TUNEL according to instructions of the fluorescent In Situ Cell Death detection kit (Roche Diagnostics). Proliferation was monitored by detection of PCNA on paraffinembedded pellet sections. PCNA Staining Kit (Invitrogen) and hematoxylin modified after Gil III (Merck) were used according to manufactures instructions. For both staining methods, 4e5 sections in approximately 30 mm intervals were stained and overview photographs were taken of 7e8 different patients with NE treatment and four apoptosis control patients (without ITS). Percentages of positive cells to total cell number were calculated from each section after standardized morphometric analysis with Adobe Photoshop CS6. Proliferation and apoptosis assays in monolayer cultures
3D culture of chondrocytes To address the impact of NE on proliferation and apoptosis rate, the 3D micromass pellet system was used. Therefore, 2 105 chondrocytes in 200 ml aliquots were placed into 96-well plates with conical bottom (Nunc/Fisher Scientific) and pelleted at 200 g for 10 min at RT. Pellets were maintained up to 21 days in DMEM/ F12 supplemented with 1% P/S, 1 mM cysteine, 1 mM pyruvate, 50 mg/ml ascorbate-2-phosphate (SigmaeAldrich) and ITSþpremix (BD). During the whole incubation time, cells were incubated every 2e3 days either with vehicle or with NE (106 and 108 M, SigmaeAldrich). For experiments under inflammatory conditions, chondrocytes were kept in a 3D fibringel matrix. Therefore, 2 106 chondrocytes were resuspended in 10 ml fibrinogen (100 mg/ml, SigmaeAldrich) and mixed with 17 ml thrombin (5 U/ml, Baxter). Cell fibrin suspension was seeded as a droplet into 48-well plates and incubated for 45 min at 37 C. To maintain a stable chondrocyte phenotype, chondrocytes were cultured in chondrogenic medium (DMEM high glucose [Gibco] supplemented with 1% P/S, 0.1 mM dexamethasone, 50 mg/ml ascorbate-2-phosphate, 40 mg/ml proline, 110 mg/ml pyruvate [SigmaeAldrich] and ITSþpremix [BD]) without TGF-b. After 7 days cells were treated for 24 h with IL-1b (0.5 ng/ml, Biomol)
Proliferation of chondrocytes cultured in monolayer was determined using bromodeoxyuridine (BrdU) cell proliferation kit (Roche). Cells in passage 1 were incubated with BrdU labeling medium in 96well plates for 24 h in presence of vehicle, IL-1b (0.5 ng/ml) and/or NE (106 and 108 M). BrdU incorporation was quantified according to manufactures instructions in a microplate reader (Tecan). Apoptosis of chondrocytes was determined using Apo-ONE caspase 3/7 assay (Promega). Cells in 96-well plates were treated with vehicle, IL-1b (0.5 ng/ml) and/or NE (106 and 108 M) for 24 h, following a 20 h incubation time with assay reagents. Fluorescence was measured using a microplate reader (Tecan). For receptor subtype specification, a1-antagonist doxazosin (105 M; SigmaeAldrich), a2-antagonist yohimbine (105 M; SigmaeAldrich), or b1e3-antagonist nadolol (105 M; SigmaeAldrich) were included into the incubation medium as indicated. Cell cycle analysis in monolayer culture Cell cycle of chondrocytes was monitored after propidium iodide (pI) staining with flow cytometry. Cells in passage 1 were placed in 75 cm2 culture flasks and incubated for 24 h in the presence of vehicle or NE (106 and 108 M). Chondrocytes were
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
4
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
fixed with methanol/aceton mixture (3:1) for 10 min on ice and RNA was digested with 50 mg RNase (Life Technologies) per 106 cells for 1 h at 37 C. After pI staining, cell cycle was monitored with at least 50,000 cells in a flow cytometer (BD, Calibur). Percentages of cells in different cell cycle phases were calculated using FlowJo software according the Dean-Jett-Fox model. Statistical analysis For statistical evaluation of different groups to a control group, column statistics with Wilcoxon signed rank test for hypothetical value (value ¼ 1 or 0) was used. To compare two groups, Wilcoxon matched-paired signed ranked test was performed. A two-tailed significance value of P < 0.05 was considered statistically significant. Data analysis was performed with GraphPad Prism 5 (San Diego). Each value represents an independent experiment and experiments were repeated with chondrocytes from different donors for at least six times (n > 6). Results AR profile in osteoarthritic chondrocytes OA chondrocytes expressed mRNA for various subtypes of aand b-AR [Fig. 1(A)]. Chondrocytes cultured in 2D- or 3D regimens under non- (IL-1b) and inflammatory conditions (þIL-1b) constitutively expressed a1D-, a2A-, a2B-, a2C- and b2-AR. A weak amplification of b1-AR mRNA was detected in 2D- and 3D cultured chondrocytes under inflammatory conditions, whereas a weak
expression of b3-AR was observed only in 3D cultured chondrocytes (/þIL-1b). Protein expression and localization of b2-and a1D-AR were determined in cryostat sections of OA articular cartilage [Fig. 1(B), (C)]. Immunohistochemistry for b2-AR revealed a positive signal in nearly all chondrocytes in the middle and deep cartilage zones [Fig. 1(B)]. Almost all chondrocytes located in the middle and deep zone were positive for a1D-AR while some chondrocytes of the superficial zone remained unstained [Fig. 1(C)]. TH and NE in OA chondrocytes OA chondrocytes cultured in 2D and 3D conditions expressed mRNA for TH, the rate-limiting enzyme for biosynthesis of catecholamines [Fig. 1(A)]. Immunohistochemistry for TH revealed that chondrocytes in all three cartilage zones were positive for TH with chondrocytes of the deep zone showing weakest staining intensity [Fig. 1(D)]. In order to determine if OA chondrocytes produce and secrete NE, we analyzed supernatants of 2D cultured chondrocytes. Neither NE nor the final product epinephrine (E) were detectable without or after addition of inhibitors for NE/E degradation as bifemelane hydrochloride acid and OR-486, and of TH specific cofactors BH4 and bivalent iron (data not shown). NE reversed IL-1b induced changes of IL-8, MMP-13, GAG and collagen II To analyze the impact of NE under inflammatory conditions, chondrocytes were cultured in 3D fibringels for 7 days and subsequently treated for 24 h with IL-1b w/o NE.
Fig. 1. Expression of TH and AR subtypes in OA chondrocytes. A) Gene expression of TH and AR subtypes was analyzed in monolayer (2D) and 3D cultured OA chondrocytes with and without IL-1b. GAPDH expression was used as a loading control. cDNA from human arteria and brain tissue served as positive controls (data not shown). BeD) Protein expression and localization of b2-AR (B), a1D-AR (C) and TH (D) in OA cartilage samples were analyzed on cryo-sections. Sections incubated with the appropriate isotype antibodies served as negative controls and showed no staining (data not shown). Bars: 100 mm.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
Stimulation with NE decreased IL-1b induced gene expression of IL-8 and MMP-13. Gene expression of MMP-3 and TIMP-2 was increased by trend after stimulation with IL-1b and 108 M NE whereas NE had no effect on gene expression levels of IL-6, MMP-2, TIMP-1 and TIMP-3 [Fig. 2(A)]. NE reversed IL-1b induced secretion of IL-8 and MMP-13 [Fig. 2(B)] and IL-1b induced decrease of GAG and collagen II production related to DNA content [Fig. 2(C)]. These NE effects were observed solely with 106 M NE strongly indicating for signaling via b-AR. To analyze the impact of NE under non-inflammatory conditions, OA chondrocytes were treated with NE (106 M and 108 M) in the absence of IL-1b (Suppl. Fig. 2). IL-8 and GAG were not altered compared to non-treated controls whereas MMP-13 and collagen II were significantly decreased after treatment with 106 M NE. NE modulates proliferation and apoptosis of 3D cultured chondrocytes To analyze the impact of NE on cell metabolism, chondrocytes were treated continuously with NE (106 M and 108 M) in a 3D micromass pellet model for 7 and 21 days. To visualize apoptotic cells, sections of paraffin-embedded pellets were stained with TUNEL, respectively with PCNA to indicate proliferating cells. TUNEL-positive cells were found after 7 and 21 days [Fig. 3(A)e(H)]. Numbers of TUNEL positive cells were increased significantly in sections treated with 108 M NE at both time points compared to vehicle treated controls [Fig. 3(I)]. PCNA-positive chondrocytes were found only after 21 [Fig. 4(A)e(C)] but not after 7 days in culture (data not shown).
5
Number of PCNA-positive chondrocytes was significantly decreased after treatment with 106 M NE, whereas increased numbers of PCNA-positive cells were observed after treatment with 108 M NE compared to vehicle controls [Fig. 4(G)]. NE induces apoptosis via a1-AR signaling To address AR subtypes involved in apoptosis induction, chondrocytes cultured in 2D were treated for 24 h with NE (106 M and 108 M) combined with a1-AR antagonist doxazosin, a2-AR antagonist yohimbine and b1-3-AR antagonist nadolol and analyzed for caspase-3/7 activity. In line with our results in 3D culture, 108 M NE without any antagonist increased significantly caspase-3/7 activity indicating apoptosis induction [Fig. 3(J)]. Increased caspase-3/7 activity was also observed after stimulation with 108 M NE in combination with yohimbine and nadolol. NE treatment combined with doxazosin prevented induction of apoptosis indicating for a1-AR signaling. Without concurrent NE treatment, adrenergic antagonists had no effect on apoptosis (data not shown). Notably, apoptosis induction with NE was not observed under inflammatory conditions (0.5 ng/ml IL-1b treatment). However, reduction of apoptosis was observed in the presence of IL-1b only (Suppl. Fig. 3A). NE causes cell cycle slow down via b-AR signaling To address AR subtypes involved in proliferation change, chondrocytes cultured in 2D were treated for 24 h with NE (106 M
Fig. 2. Impact of NE on 3D cultivated OA chondrocytes under inflammatory conditions. A) Expression of mRNA for IL-6, IL-8, MMP-2, MMP-3, MMP-13, TIMP-1, TIMP-2 and TIMP-3 in 3D cultivated chondrocytes was determined by real-time PCR. The relative expression level of each gene was calculated using GAPDH as endogenous loading control and IL-1b stimulated cells as calibrator (dotted line at 0). B) Concentrations of IL-8 and MMP-13 were determined in supernatants of 3D cultivated chondrocytes and plotted in relation to IL-1b stimulated controls (dotted line at 1). C) GAG and collagen II (Col II) content of each fibringel was related to DNA content and plotted in relation to IL-1b stimulated controls (dotted line at 1). Open box blots represent 3D cultured chondrocytes without any stimulation, grey box blots represent cells after 24 h stimulation with IL-1b and NE 106 M (light grey) or 108 M (dark grey). Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. n ¼ 6e8 independent experiments. (*) ¼ P 0.0625e0.05; * ¼ P < 0.05; ** ¼ P < 0.001 vs IL-1b stimulated controls.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
6
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
Fig. 3. Influence of NE on apoptosis of articular chondrocytes. AeH) Representative images of TUNEL stained micromass pellet sections were shown. 3D-cultured chondrocytes were treated for 7 days (AeD) and 21 days (EeH) with NE (106 M; B, F and 108 M; C, G). Vehicle treated pellets (A, E) served as negative controls, whereas eITS controls (D, H) served as positive apoptosis controls. I) Percentages of TUNEL-positive chondrocytes was determined and related to that of vehicle treated pellets (e solid black line). J) To analyze AR subtypes which mediate apoptosis induction, specific antagonists for AR (doxazosin for a1-AR; yohimbine for a2-AR; nadolol for b1e3-AR) were included in Caspase 3/7 assay. Unstimulated controls served as calibrator (e solid black line). IeJ) Data are shown as box plots. Light grey boxes indicate 106 M NE treatment; dark grey boxes indicate 108 M NE treatment and open boxes indicate minus ITS control. Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. (*) ¼ P 0.0625e0.05; * ¼ P < 0.05 vs unstimulated controls; Bars: 100 mm.
and 108 M) combined with appropriate AR inhibitors and analyzed for BrdU incorporation. In line with our results in 3D culture, 106 M NE decreased significantly BrdU incorporation compared to controls without NE indicating reduced proliferation. In contrast to our 3D results, treatment with 108 M NE had no effect on BrdU incorporation compared to vehicle treated control cells [Fig. 4(H)]. We observed a decrease in BrdU incorporation after stimulation with 106 M NE in combination with doxazosin and yohimbine. Treatment with 106 M NE and nadolol prevented decrease in proliferation, indicating for b-AR mediated signaling. Without concurrent NE treatment, AR antagonists had no effect on proliferation (data not shown). Notably, decrease in BrdU incorporation after NE treatment was not observed under inflammatory conditions (0.5 ng/ml IL-1b stimulation). However, reduction of proliferation was observed in the presence of IL-1b only (Suppl. Fig. 3B). To address changes in cell cycle phases induced by NE, cell cycle was monitored in monolayer cultured chondrocytes [Fig. 5(A)e(F)]. Stimulation with 106 M NE lead to a significant accumulation of chondrocytes in the G0/G1-phase [Fig. 5(D)] and a reduction in Sphase population compared to control [Fig. 5(E)]. We therefore suggest that NE caused a cell cycle slow down via b-AR mediated signaling. Discussion Whereas reports are available with respect to the effects of the SNS and catecholaminergic neurotransmitters on bone biology and
on chondrogenic differentiation of progenitor cells, not much is known about the impact of the SNS on adult articular cartilage physiology and pathophysiology (reviewed in30). In this study, expression of several AR subtypes in articular OA chondrocytes was observed indicating that articular chondrocytes are target cells of the SNS. Until now expression of AR subtypes was solely described for growth plate chondrocytes and for neonatal murine costal chondrocytes15,16,31. We further observed expression of TH in OA chondrocytes indicating for catecholamine synthesis. However, we did neither detect NE nor epinephrine (E) in chondrocyte culture supernatants suggesting that chondrocytes do not secrete and presumably also do not synthesize NE themselves. NE is a small molecule and as such presumably penetrates cartilage matrix easily and thus reaches cells via diffusion. As source of NE in joints serve synovium and subchondral bone which both are innervated by sympathetic nerve fibers. Our data indicate a different role of TH in OA chondrocytes apart from NE synthesis. The rare genetic disorder of tyrosine metabolism, alkaptonuria, might provide an indication as alkaptonuria leads to deposition of melanin-like pigments in cartilage of weight bearing joints resulting in severe OA32. In our in vitro inflammation model, NE modulated IL-1b induced effects in OA articular chondrocytes. An anti-inflammatory effect of NE in the osteoarticular system was reported earlier in a murine experimental arthritis model33. The authors described a significant reduction in clinical scores of collagen induced arthritis and cell infiltration in the paws after injection of TH expressing MSC which
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
7
Fig. 4. Influence of NE on proliferation of articular chondrocytes. AeF) Representative images of PCNA (AeC) and hematoxylin only (DeF) stained micromass pellet sections after vehicle (A, D) and NE treatment (106 M; B, E and 108 M; C, F) after 21 days. G) Percentage of PCNA-positive chondrocytes was determined and related to unstimulated pellets (e solid black line). H) To analyze AR subtypes which mediate proliferation changes, specific antagonists for AR (doxazosin for a1-AR, yohimbine for a2-AR, nadolol for b1e3-AR) were included in BrdU assay. Unstimulated controls served as calibrator (ee). GeH, Data are shown as box plots. Light grey boxes indicate 106M NE treatment and dark grey boxes 108 M NE treatment. Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. * ¼ P < 0.05 vs unstimulated controls; Bars ¼ 100 mm.
were differentiated towards neuronal cells in vitro producing and secreting NE. In our study, NE reversed IL-1b induced effects specifically at 106 M strongly indicating for signaling via b-AR which is associated with anti-inflammatory effects (reviewed in34) in inflammatory joint diseases22. Together with our data, we suggest that b-AR signaling has anti-inflammatory effects in OA chondrocytes. We observed a distinct concentration dependent impact of NE on metabolic activities of OA chondrocytes under 2D and 3D culture conditions. In the presence of 106 M NE, chondrocytes accumulated in the GO/G1-phase leaving a decreased cell number in the S-phase population. Proliferation decrease was prevented with nadolol indicating for b-AR signaling. This observation is in line with two studies on rat hepatocytes respectively rat oligodendrocyte progenitor cells which both observed a G1-phase arrest induced via b-AR signaling. Notably, 108 M NE induced proliferation in our 3D culture model but not in monolayer culture. This discrepancy might be due to different
timing of NE treatment (24 h vs 21 days) and/or different culture conditions (2D vs 3D). Several publications describe chondrocyte dedifferentiation during 2D culture35e37 latter presumably affecting proliferation activity. Differences of NE treatment solely depend on agonist concentration can be explained by the differential intracellular signaling pathways. High concentrations of NE mediate their effects by activating a- and b-AR signaling pathways and low concentrations mediate their effects mainly via a-AR signaling. b-AR signaling increases cAMP level38, whereas alpha AR signaling increases intracellular Ca2þ level39. Increased cAMP levels in chondrocytes are associated with a chondrocyte typical phenotype40, whereas increased Ca2þ level are described to induce apoptosis41. Also Lei et al.42 described a NE concentration dependent regulation of cell growth with a proliferation decrease after treatment with high doses and a proliferation increase with low doses NE. Taken together with our data we suggest a dual function of the SNS in OA chondrocytes with distinct impact of AR subtypes on metabolic
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
8
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
Fig. 5. Cell cycle analysis of articular chondrocytes in the presence of NE. AeC) Representative cell cycle histograms of vehicle (A) and NE treated (106 M; B and 108 M; C) chondrocytes are shown. Histograms were analyzed with Dean-Jett-Fox model to calculate percentages of cell populations in different cell cycle phases. DeF) Percentages of cell populations in G0/G1- (D), S- (E) and G2-phase (F) are shown as box plots. Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. * ¼ P < 0.05 vs unstimulated controls. n ¼ 6e7 independent experiments.
response. b-AR signaling rather promoted a stable articular chondrocyte phenotype with low proliferative activity. a-AR signaling might promote OA pathogenesis through induction of chondrocyte death and increased proliferation which is mostly associated with chondrocyte dedifferentiation6. Conclusion This study demonstrated that human OA chondrocytes are target cells of the SNS. NE treatment reversed IL-1b induced production of IL-8 and MMP-13 and IL-1b induced decrease of collagen II and GAG production presumptively via b-AR signaling. NE further induced apoptosis via a1-AR signaling whereas b-AR signaling lead to a cell cycle slow down resulting in decreased proliferation and ultimately in reduced cell growth. However, low doses of NE resulted in increased proliferation presumptively via a-AR signaling. We therefore suggest that b-AR signaling plays an antiinflammatory role in OA pathology and promotes a nonproliferative, metabolically stable articular chondrocyte phenotype which might counteract OA initiation or progression. Contrary, a-AR signaling might promote OA progression through induction of apoptosis and/or proliferation of articular chondrocytes.
RB made substantial contributions to the acquisition, analysis and interpretation of data. ZJL made substantial contributions to the acquisition and analysis of data. RHS provided study materials of patients, technical and logistic support and statistical expertise. SG made substantial contributions to conception and design of the study, to data interpretation, to drafting of the article, critical revision of the article for important intellectual content and final approval of the article. She also obtained the funding for this study. Competing interest statement The authors declare no competing interests. Funding source This work was funded with a grant from the German Research Society (DFG) to SG (GR 1301/9-1). Acknowledgements We want thank Anja Pasoldt for her expert and high quality technical assistance.
Author contributions
Abbreviations
JL made substantial contributions to the conception and design of the study, to the acquisition of data, analysis and interpretation. She also contributed critical to the drafting of the manuscript. NS made substantial contributions to the acquisition and analysis of data.
3D AR BH4 DMEM DMMB
3-dimensional adrenergic receptors 6R-5,6,7,8-tetrahydrobiopterin dihydrochloride Dulbecco's modified Eagle medium dimethyl methylene blue
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
ELISA GAG IL MMPs NE OA PCNA PCR pI P/S SNS TH TIMPs TUNEL
Enzyme-linked immunosorbent assay glycosaminoglycan interleukin matrix metalloproteases norepinephrine osteoarthritis/osteoarthritic proliferating cell nuclear antigen polymerase chain reaction propidium iodide penicillin/streptomycin sympathetic nervous system tyrosine hydroxylase tissue inhibitor of metalloproteases TdT-mediated dUTP-biotin nick end labeling
14.
15.
16.
Supplementary data
17.
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.joca.2015.08.007.
18.
References 1. Peat G, McCarney R, Croft P. Knee pain and osteoarthritis in older adults: a review of community burden and current use of primary health care. Ann Rheum Dis 2001;60:91e7. 2. Conaghan PG, Kloppenburg M, Schett G, Bijlsma JW, committee Eoah. Osteoarthritis research priorities: a report from a EULAR ad hoc expert committee. Ann Rheum Dis 2014;73: 1442e5. 3. Kevorkian L, Young DA, Darrah C, Donell ST, Shepstone L, Porter S, et al. Expression profiling of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum 2004;50:131e41. 4. Martel-Pelletier J, Pelletier JP. New insights into the major pathophysiological processes responsible for the development of osteoarthritis. Semin Arthritis Rheum 2005;34:6e8. 5. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 2000;1477:267e83. 6. Pitsillides AA, Beier F. Cartilage biology in osteoarthritiselessons from developmental biology. Nat Rev Rheumatol 2011;7:654e63. 7. Dreier R. Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther 2010;12:216. 8. Goldring MB, Goldring SR. Osteoarthritis J Cell Physiol 2007;213:626e34. 9. Melchiorri C, Meliconi R, Frizziero L, Silvestri T, Pulsatelli L, Mazzetti I, et al. Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chondrocytes from patients with osteoarthritis. Arthritis Rheum 1998;41:2165e74. 10. Goldring SR, Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res 2004:S27e36. 11. Imai S, Matsusue Y. Neuronal regulation of bone metabolism and anabolism: calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-containing nerves and the bone. Microsc Res Tech 2002;58:61e9. 12. Bjurholm A, Kreicbergs A, Terenius L, Goldstein M, Schultzberg M. Neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide-immunoreactive nerves in bone and surrounding tissues. J Auton Nerv Syst 1988;25: 119e25. 13. Pereira da Silva JA, Carmo-Fonseca M. Peptide containing nerves in human synovium: immunohistochemical evidence
19.
20.
21.
22. 23.
24.
25.
26.
27.
28.
29.
9
for decreased innervation in rheumatoid arthritis. J Rheumatol 1990;17:1592e9. Miller LE, Justen HP, Scholmerich J, Straub RH. The loss of sympathetic nerve fibers in the synovial tissue of patients with rheumatoid arthritis is accompanied by increased norepinephrine release from synovial macrophages. FASEB J 2000;14:2097e107. Opolka A, Straub RH, Pasoldt A, Grifka J, Grassel S. Substance P and norepinephrine modulate murine chondrocyte proliferation and apoptosis. Arthritis Rheum 2012;64:729e39. Takarada T, Hojo H, Iemata M, Sahara K, Kodama A, Nakamura N, et al. Interference by adrenaline with chondrogenic differentiation through suppression of gene transactivation mediated by Sox9 family members. Bone 2009;45: 568e78. Lai KB, Sanderson JE, Yu CM. High dose norepinephrineinduced apoptosis in cultured rat cardiac fibroblast. Int J Cardiol 2009;136:33e9. Communal C, Singh K, Pimentel DR, Colucci WS. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation 1998;98:1329e34. Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X, et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature 2005;434:514e20. Suzuki A, Palmer G, Bonjour JP, Caverzasio J. Catecholamines stimulate the proliferation and alkaline phosphatase activity of MC3T3-E1 osteoblast-like cells. Bone 1998;23:197e203. Takahata Y, Takarada T, Iemata M, Yamamoto T, Nakamura Y, Kodama A, et al. Functional expression of beta2 adrenergic receptors responsible for protection against oxidative stress through promotion of glutathione synthesis after Nrf2 upregulation in undifferentiated mesenchymal C3H10T1/2 stem cells. J Cell Physiol 2009;218:268e75. Straub RH, Harle P. Sympathetic neurotransmitters in joint inflammation. Rheum Dis Clin North Am 2005;31:43e59. viii. Lukewich MK, Rogers RC, Lomax AE. Divergent neuroendocrine responses to localized and systemic inflammation. Semin Immunol 2014 Oct;26(5):402e8, http://dx.doi.org/10.1016/ j.smim.2014.01.004. Jenei-Lanzl Z, Grassel S, Pongratz G, Kees F, Miosge N, Angele P, et al. Norepinephrine inhibition of mesenchymal stem cell and chondrogenic progenitor cell chondrogenesis and acceleration of chondrogenic hypertrophy. Arthritis Rheumatol 2014 Sep;66(9): 2472e81, http://dx.doi.org/10.1016/10.1002/art.38695. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum 2012;64:1697e707. Suri S, Gill SE, Massena de Camin S, Wilson D, McWilliams DF, Walsh DA. Neurovascular invasion at the osteochondral junction and in osteophytes in osteoarthritis. Ann Rheum Dis 2007;66:1423e8. Opolka A, Ratzinger S, Schubert T, Spiegel HU, Grifka J, Bruckner P, et al. Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice. Matrix Biol 2007;26:85e95. Kees MG, Pongratz G, Kees F, Scholmerich J, Straub RH. Via beta-adrenoceptors, stimulation of extrasplenic sympathetic nerve fibers inhibits lipopolysaccharide-induced TNF secretion in perfused rat spleen. J Neuroimmunol 2003;145: 77e85. Jenei-Lanzl Z, Straub RH, Dienstknecht T, Huber M, Hager M, Grassel S, et al. Estradiol inhibits chondrogenic differentiation of mesenchymal stem cells via nonclassic signaling. Arthritis Rheum 2010;62:1088e96.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
10
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
€ssel S. The role of peripheral nerve fibers and their neu30. Gra rotransmitters in cartilage and bone physiology and pathophysiology. Arthritis Res Ther 2014;16(6):485, http:// dx.doi.org/10.1186/s13075-014-0485-1. 31. Lai LP, Mitchell J. Beta2-adrenergic receptors expressed on murine chondrocytes stimulate cellular growth and inhibit the expression of Indian hedgehog and collagen type X. J Cell Biochem 2008;104:545e53. 32. Taylor AM, Boyde A, Wilson PJ, Jarvis JC, Davidson JS, Hunt JA, et al. The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria. Arthritis Rheum 2011;63:3887e96. 33. Jenei-Lanzl Z, Capellino S, Kees F, Fleck M, Lowin T, Straub RH. Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis. Ann Rheum Dis 2015 Feb;74(2):444e51, http:// dx.doi.org/10.1136/annrheumdis-2013-203925. 34. Ader R. Psychoneuroimmunology. San Diego, CA: ElsevierAcademic Press; 2007. 35. Cheung HS, Harvey W, Benya PD, Nimni ME. New collagen markers of 'derepression' synthesized by rabbit articular chondrocytes in culture. Biochem Biophys Res Commun 1976;68:1371e8.
36. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215e24. 37. Bonaventure J, Kadhom N, Cohen-Solal L, Ng KH, Bourguignon J, Lasselin C, et al. Reexpression of cartilagespecific genes by dedifferentiated human articular chondrocytes cultured in alginate beads. Exp Cell Res 1994;212: 97e104. 38. Levitzki A. From epinephrine to cyclic AMP. Science 1988;241: 800e6. 39. Piascik MT, Perez DM. Alpha1-adrenergic receptors: new insights and directions. J Pharmacol Exp Ther 2001;298:403e10. 40. Chosa E, Hamada H, Kitamura K, Kuwasako K, Yanagita T, Eto T, et al. Expression of adrenomedullin and its receptor by chondrocyte phenotype cells. Biochem Biophys Res Commun 2003;303:379e86. 41. Wang W, Xu J, Kirsch T. Annexin-mediated Ca2þ influx regulates growth plate chondrocyte maturation and apoptosis. J Biol Chem 2003;278:3762e9. 42. Lei B, Schwinn DA, Morris DP. Stimulation of alpha1a adrenergic receptors induces cellular proliferation or antiproliferative hypertrophy dependent solely on agonist concentration. PLoS One 2013;8:e72430.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses €fer y k, R. Bauer y z, Z. Jenei-Lanzl x, R.H. Springorum k, S. Gra €ssel y k * J. Lorenz y k, N. Scha y Centre for Medical Biotechnology, BioPark I, University of Regensburg, Regensburg, Germany z Department of Oral and Maxillofacial Surgery, University of Regensburg, Germany x Internal Medicine 1, Division of Rheumatology, University of Regensburg, Germany k Department of Orthopedic Surgery, University of Regensburg, Bad Abbach, Germany
a r t i c l e i n f o
s u m m a r y
Article history: Received 20 January 2015 Accepted 18 August 2015
Objective: Norepinephrine (NE) was measured in synovial fluid of trauma patients and sympathetic nerve fibers were detected in healthy and osteoarthritic (OA) joint tissues indicating that cartilage pathophysiology might be influenced by sympathetic neurotransmitters. The aim of this study was to elucidate the mostly unknown role of NE in OA chondrocyte metabolism and inflammatory responses. Methods: Articular cartilage was received after total knee replacement surgery from OA patients. Expression of adrenergic receptors (AR) and tyrosine hydroxylase (TH) was tested with end point polymerase chain reaction (PCR) and immunohistochemistry. 3-dimensional (3D) cell cultures were employed to analyze effects of NE on chondrocyte cell metabolism and the expression of interleukins (ILs), matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), glycosaminoglycan (GAG) and collagen II under non- and inflammatory conditions. Chondrocyte monolayer cultures were used to specify AR subtypes, to analyze cell cycle distribution and to determine catecholamines in cell culture supernatants. Results: AR subtypes and TH were detected in chondrocytes, whereas NE was not released in measurable amounts. 106 M NE reversed IL-1b induced changes in IL-8, MMP-13, GAG and collagen II expression/ production indicating for b-AR signaling. Additionally, NE caused cell cycle slow down and decreased proliferation via b-AR signaling. 108 M NE increased the number of proliferating cells and induced apoptosis via a1-AR signaling. Conclusions: NE affects chondrocytes from OA cartilage regarding inflammatory response and its cell metabolism in a dose dependent manner. The sympathetic nervous system (SNS) may have a dual function in OA pathology with preserving a stable chondrocyte phenotype via b-AR signaling and OA pathogenesis accelerating effects via a-AR signaling. © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Osteoarthritis Articular chondrocytes Norepinephrine IL-1b Sympathetic nervous system
Introduction OA is the most common form of arthritis and a leading cause of chronic pain. The social burden of OA is immense as it affects 9.6% of men and 18% of woman over 60 years of age worldwide1 and currently compromising the lives of 40 million Europeans2. The etiology of OA is multifactorial and still incompletely understood.
€ssel, Department of * Address correspondence and reprint requests to: S. Gra Orthopedic Surgery, Experimental Orthopedics, University of Regensburg, ZMB/ BioPark 1, Josef-Engert-Str. 9, 93053 Regensburg, Germany. Tel: 49-941-943-5065; Fax: 49-941-943-5066. E-mail address: [email protected] (S. Gr€ assel).
Hallmark features are structural changes including articular cartilage destruction as well as alterations in synovium and subchondral bone. Increased proteolytic activity leads to degradation of major cartilage extracellular matrix components as collagens and the large proteoglycans. In this process, MMPs and their inhibitors play a key role. Regulation of several highly expressed MMPs (MMP-2, -3 and -13) in end stage OA cartilage supports this concept3e5. Regarding metabolism of chondrocytes, early OA is characterized by proliferation accompanied with increased matrix production, whereas end stage OA chondrocytes express hypertrophy markers such as MMP-13 and collagen X and presumably undergo apoptosis6e8. Interestingly, intermittent inflammatory flares also occur in the synovial membrane in the course of OA suggesting
http://dx.doi.org/10.1016/j.joca.2015.08.007 1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
2
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
inflammation is associated with this disease. Although not a primary phenomenon, synovial inflammation contributes to OA progression. Among pro-inflammatory mediators involved in OA, IL-1b seems to play a key role9. It increases the synthesis of various MMPs and some short time pro-inflammatory mediators such as IL-6 and IL-88,10. NE belongs to the catecholamine family of tyrosine-derived sympathetic neurotransmitters. This subtype of sympathetic nerve fibres, characterized by the expression of TH, has been identified in synovium, bone marrow, periosteum, and in boneadherent ligaments11e13, indicating that growth and metabolic activity of synovial joint tissues are regulated by sympathetic neurotransmitters. Catecholaminergic effects are mediated by the adrenergic receptor (AR) family. At high concentrations (>107 M) NE acts via a- and b-AR, whereas low concentrations (107 M) are mediated mainly via a-AR14. Functional a1-, a2-and b2-AR are present on murine costal chondrocytes and chondrogenic ATDC5 cells15,16. Previously, we observed a decrease in apoptosis in murine costal chondrocytes after treatment with NE via b-AR signaling and an increase in focal adhesion contacts15. Additionally, it is described that NE may also induce apoptosis in several other cell types17,18 and to regulate the proliferation of osteoblasts, osteoblast-like cells, and mesenchymal stem cell lines through b- and a1-AR mediated signaling19e21. Furthermore, catecholamines play a decisive role in inflammatory processes characteristic for rheumatoid arthritis22. With respect to above disease entity, b-AR activation predominantly mediates anti-inflammatory effects, whereas activation of a-AR, if located on immune cells, mediates a priori pro-inflammatory effects (reviewed in23). Articular cartilage is not deeply innervated by nerve fibers, although there are hints in literature that catecholamines influence articular chondrocytes especially in OA. High levels of NE were measured in synovial fluid of patients with joint trauma24 along with a high incidence to develop post-traumatic OA25. Suri et al. have localized both sensory and sympathetic nerve fibers in similar distributions to the articular cartilage in human tibiofemoral OA. In the pathogenesis of OA, nerves grow into joint structures through vascular channels mainly from subchondral bone breaching through the tidemark26. In this study, we have addressed the response of human articular OA chondrocytes to NE with respect to inflammatory markers and chondrocyte metabolism.
Materials and methods Study design In this study we analyzed the localization of TH and AR subtypes in human OA cartilage, the response of human OA chondrocytes to NE under inflammatory conditions and their metabolic answer to NE stimulation. TH and AR subtype gene expression were measured with end point PCR after RNA isolation from monolayer- and 3D (fibrin gel) cultured chondrocytes under non- and inflammatory conditions (w/o 0.5 ng/ml IL-1b) TH, a1D-AR and b2-AR expression were approved by immunohistochemistry staining on cryo-embedded cartilage slides (N 3). Responses of human OA chondrocytes to NE under inflammatory conditions were tested with real time PCR, enzyme-linked immunosorbent assays (ELISA) and dimethyl methylene blue (DMMB) assays under non- and inflammatory conditions (w/o 0.5 ng/ml IL-1b). Chondrocytes of the same donor were cultured under following conditions: Treatment of fibrin gels with (1) vehicle (PBS with 0.5 mM acetic acid), (2) IL-1b and (3) IL-1b þ NE
(106 M and 108 M) respectively (4) vehicle (PBS with 0.5 mM acetic acid) and (5) NE (106 M and 108 M). (N 6) (Suppl. Fig. 1A). Metabolic (proliferation and apoptosis) response of OA chondrocytes to NE were analyzed in 3D culture under noninflammatory conditions after staining of proliferating cell nuclear antigen (PCNA, proliferation) and TdT-mediated dUTP-biotin nick end labeling (TUNEL, apoptosis) on paraffin-embedded micromass pellet sections. To specify AR subtypes involved in metabolic responses, chondrocytes were cultured in monolayer and AR antagonists were included in BrdU (proliferation) and caspase 3/7 (apoptosis) assays. To further analyze proliferation effects, cell cycle was analyzed via flow cytometer. (N 6) (Suppl. Fig. 1B).
Isolation and culture of chondrocytes Articular cartilage was obtained from OA patients after total endoprothesis surgery. This had been approved by the local Ethics €t Committee (Az:14-101-0189; Ethikkommission an der Universita Regensburg, email: [email protected]) and specimens were taken with patients’ written consent. For this study knee joints were obtained from 61 different donors (29 male and 32 female) within an age range of 48e84 (mean age of 66,4). Macros-copically normal looking cartilage slices were digested over night with collagenase II (PAA) in Dulbecco's modified Eagle medium (DMEM)/ F12 (Invitrogen) containing 1% penicillin/streptomycin (P/S) at 37 C. Isolated chondrocytes were passed through a 70 mm nylon mesh (Falcon) to remove residual cartilage matrix fragments, centrifuged at 200 g for 5 min, and resuspended in DMEM/F12 supplemented with 10% fetal calf serum and 1% P/S. Chondrocytes were seeded at a density of 20,000 cells/cm2 and initially cultured in monolayer for maximal 10 days in a humidified 37 C/5% CO2 incubator.
RNA isolation, end point PCR and real-time PCR RNA from chondrocyte monolayer culture was isolated with Aglient RNA Absolutely Miniprep Kit. 3D cultivated chondrocytes were first homogenized with a PowerGen 1000 Homogenizer (Fisher scientific) for 45 sec in 1 ml peqgold trifast (Peqlab) and afterwards RNA was isolated according to manufactures instructions. That was followed by a second RNA isolation step with RNA Absolutely Nanoprep Kit (Aglient). To translate RNA in cDNA, AffinityScript QPCR cDNA Synthesis Kit (Aglient) was applied. Gene expression of TH and subtypes of a- and b-ARs was determined using one-Step RT-PCR (Invitrogen), where each reaction consisted of 100 ng of cDNA. Gene expression of GAPDH was used as loading control. Primers were designed from universal probe library system assay design center (Roche) and listed in Suppl. Table 1. Commercial available RNA of human brain and arteria tissue served as positive controls and the identity of amplified PCR products was confirmed by sequencing. Pure RNA served as negative control because most AR subtypes contain only one exon, excluding intron-spanning primer design. Relative quantitative PCR for IL-6, IL-8, MMP-2, MMP-3, MMP13, TIMP-1, TIMP-2 and TIMP-3 was performed in Mx3005P qPCR system with Brilliant II SYBR Green QPCR Mstr Mx (Aglient), 30 ng cDNA and in triplicates. Primers are listed in Suppl. Table 2. Relative quantification was determined with MxPro QPCR software using GAPDH as endogenous loading control and IL-1b stimulated respectively untreated cells as calibrator. Log fold changes to calibrator (base 2) values were plotted using Graph Pad Prism 5 software.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
3
Localization of TH, a1D- and b2-adrenergic receptors in articular cartilage
combined either with vehicle or with NE (106 and 108 M, SigmaeAldrich).
To visualize TH, a1D- and b2-AR, articular cartilage slices were immersed in OCT embedding compound (Sakura Finetek Europe) and immediately frozen in liquid nitrogen. Transverse sections were cut with a cryostat at 10 mm, mounted onto clean glass microscopic slides and frozen at 70 C. Sections were fixed in cold acetone (20 C) and washed in phosphate buffered saline. For b2AR staining, sections were treated as described previously27 followed by blocking with 1% normal goat serum (SigmaeAldrich) for 1 h and overnight incubation with an anti-b2-AR antibody (Abcam) at 4 C. After washing, sections were incubated with appropriately diluted biotinylated secondary antibody as described previously27. For TH and a1D-AR staining, endogenous peroxidase was blocked for 5 min with 3% (volume/volume) H2O2 followed by blocking with 5% normal swine serum (Dako) and 1% (weight/volume) bovine serum albumin (SigmaeAldrich) in PBS for 1 h. TH staining was performed with rabbit anti human TH antibody (1:250 in blocking solution, Abcam) and a1D-AR staining with rabbit anti human a1DAR antibody (1:100 in blocking solution, Alomone Labs) overnight at 4 C. After washing, sections were incubated with a biotinylated swine anti rabbit antibody (1:500 in blocking solution, Dako) and further procedure was performed as described previously27. Sections incubated without the primary antibody or with pre-immune serum served as negative control.
ELISA for IL-8 and MMP-13
Determination of catecholamines To measure catecholamines, passage 0 chondrocytes were incubated for 10 days in monolayer culture. Afterwards cells were treated for 24 h with TH specific cofactors BH4 (6R-5,6,7,8tetrahydrobiopterin dihydrochloride) (105 M; SigmaeAldrich), respectively bivalent iron (Fe2þ sulfate heptahydrate) (105 M; SigmaeAldrich) and with inhibitors for NE degradation monoaminooxidase inhibitor bifemelane hydrochlorid acid (105 M; Tocris) respectively catechol-O-methyltransferase inhibitor OR-486 (105 M; Bristol). Catecholamines in cell culture supernatants were assayed by high performance liquid chromatography (HPLC) as previously described28. The detection limit for catecholamines in HPLC is about 1010 M.
Release of IL-8 and MMP-13 was analyzed with appropriate ELISA Kits (RayBiotec). Culture supernatants of chondrocytes cultured in fibringels were harvested after 24 h stimulation and different dilutions were subjected to ELISA according to manufacturers’ instructions. Biochemical analyses To analyze the amount of sulfated GAG, collagen II and dsDNA of chondrocyte-containing fibringels, gels were digested in 0.05 M acetic acid plus 0.5 M NaCl (pH 2.9e3.0) supplemented with 10 mg/ ml pepsin, and dissolved in 0.05 M acetic acid on the rotator for 48 h at 4 C. Next steps of digestion and DMMB assays were performed as described previously29. Collagen II Detection Kit 6019 (Chondrex) was used according to manufactures instructions and DNA content was analyzed with Quant-iT dsDNA Assay Kit (Invitrogen). The amount of GAG and collagen II of each pellet were related to the corresponding amount of DNA. Proliferation and apoptosis in micromass pellet culture In micromass pellets, number of apoptotic cells was monitored by TUNEL on paraffin-embedded pellet sections. Apoptotic cells were identified by TUNEL according to instructions of the fluorescent In Situ Cell Death detection kit (Roche Diagnostics). Proliferation was monitored by detection of PCNA on paraffinembedded pellet sections. PCNA Staining Kit (Invitrogen) and hematoxylin modified after Gil III (Merck) were used according to manufactures instructions. For both staining methods, 4e5 sections in approximately 30 mm intervals were stained and overview photographs were taken of 7e8 different patients with NE treatment and four apoptosis control patients (without ITS). Percentages of positive cells to total cell number were calculated from each section after standardized morphometric analysis with Adobe Photoshop CS6. Proliferation and apoptosis assays in monolayer cultures
3D culture of chondrocytes To address the impact of NE on proliferation and apoptosis rate, the 3D micromass pellet system was used. Therefore, 2 105 chondrocytes in 200 ml aliquots were placed into 96-well plates with conical bottom (Nunc/Fisher Scientific) and pelleted at 200 g for 10 min at RT. Pellets were maintained up to 21 days in DMEM/ F12 supplemented with 1% P/S, 1 mM cysteine, 1 mM pyruvate, 50 mg/ml ascorbate-2-phosphate (SigmaeAldrich) and ITSþpremix (BD). During the whole incubation time, cells were incubated every 2e3 days either with vehicle or with NE (106 and 108 M, SigmaeAldrich). For experiments under inflammatory conditions, chondrocytes were kept in a 3D fibringel matrix. Therefore, 2 106 chondrocytes were resuspended in 10 ml fibrinogen (100 mg/ml, SigmaeAldrich) and mixed with 17 ml thrombin (5 U/ml, Baxter). Cell fibrin suspension was seeded as a droplet into 48-well plates and incubated for 45 min at 37 C. To maintain a stable chondrocyte phenotype, chondrocytes were cultured in chondrogenic medium (DMEM high glucose [Gibco] supplemented with 1% P/S, 0.1 mM dexamethasone, 50 mg/ml ascorbate-2-phosphate, 40 mg/ml proline, 110 mg/ml pyruvate [SigmaeAldrich] and ITSþpremix [BD]) without TGF-b. After 7 days cells were treated for 24 h with IL-1b (0.5 ng/ml, Biomol)
Proliferation of chondrocytes cultured in monolayer was determined using bromodeoxyuridine (BrdU) cell proliferation kit (Roche). Cells in passage 1 were incubated with BrdU labeling medium in 96well plates for 24 h in presence of vehicle, IL-1b (0.5 ng/ml) and/or NE (106 and 108 M). BrdU incorporation was quantified according to manufactures instructions in a microplate reader (Tecan). Apoptosis of chondrocytes was determined using Apo-ONE caspase 3/7 assay (Promega). Cells in 96-well plates were treated with vehicle, IL-1b (0.5 ng/ml) and/or NE (106 and 108 M) for 24 h, following a 20 h incubation time with assay reagents. Fluorescence was measured using a microplate reader (Tecan). For receptor subtype specification, a1-antagonist doxazosin (105 M; SigmaeAldrich), a2-antagonist yohimbine (105 M; SigmaeAldrich), or b1e3-antagonist nadolol (105 M; SigmaeAldrich) were included into the incubation medium as indicated. Cell cycle analysis in monolayer culture Cell cycle of chondrocytes was monitored after propidium iodide (pI) staining with flow cytometry. Cells in passage 1 were placed in 75 cm2 culture flasks and incubated for 24 h in the presence of vehicle or NE (106 and 108 M). Chondrocytes were
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
4
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
fixed with methanol/aceton mixture (3:1) for 10 min on ice and RNA was digested with 50 mg RNase (Life Technologies) per 106 cells for 1 h at 37 C. After pI staining, cell cycle was monitored with at least 50,000 cells in a flow cytometer (BD, Calibur). Percentages of cells in different cell cycle phases were calculated using FlowJo software according the Dean-Jett-Fox model. Statistical analysis For statistical evaluation of different groups to a control group, column statistics with Wilcoxon signed rank test for hypothetical value (value ¼ 1 or 0) was used. To compare two groups, Wilcoxon matched-paired signed ranked test was performed. A two-tailed significance value of P < 0.05 was considered statistically significant. Data analysis was performed with GraphPad Prism 5 (San Diego). Each value represents an independent experiment and experiments were repeated with chondrocytes from different donors for at least six times (n > 6). Results AR profile in osteoarthritic chondrocytes OA chondrocytes expressed mRNA for various subtypes of aand b-AR [Fig. 1(A)]. Chondrocytes cultured in 2D- or 3D regimens under non- (IL-1b) and inflammatory conditions (þIL-1b) constitutively expressed a1D-, a2A-, a2B-, a2C- and b2-AR. A weak amplification of b1-AR mRNA was detected in 2D- and 3D cultured chondrocytes under inflammatory conditions, whereas a weak
expression of b3-AR was observed only in 3D cultured chondrocytes (/þIL-1b). Protein expression and localization of b2-and a1D-AR were determined in cryostat sections of OA articular cartilage [Fig. 1(B), (C)]. Immunohistochemistry for b2-AR revealed a positive signal in nearly all chondrocytes in the middle and deep cartilage zones [Fig. 1(B)]. Almost all chondrocytes located in the middle and deep zone were positive for a1D-AR while some chondrocytes of the superficial zone remained unstained [Fig. 1(C)]. TH and NE in OA chondrocytes OA chondrocytes cultured in 2D and 3D conditions expressed mRNA for TH, the rate-limiting enzyme for biosynthesis of catecholamines [Fig. 1(A)]. Immunohistochemistry for TH revealed that chondrocytes in all three cartilage zones were positive for TH with chondrocytes of the deep zone showing weakest staining intensity [Fig. 1(D)]. In order to determine if OA chondrocytes produce and secrete NE, we analyzed supernatants of 2D cultured chondrocytes. Neither NE nor the final product epinephrine (E) were detectable without or after addition of inhibitors for NE/E degradation as bifemelane hydrochloride acid and OR-486, and of TH specific cofactors BH4 and bivalent iron (data not shown). NE reversed IL-1b induced changes of IL-8, MMP-13, GAG and collagen II To analyze the impact of NE under inflammatory conditions, chondrocytes were cultured in 3D fibringels for 7 days and subsequently treated for 24 h with IL-1b w/o NE.
Fig. 1. Expression of TH and AR subtypes in OA chondrocytes. A) Gene expression of TH and AR subtypes was analyzed in monolayer (2D) and 3D cultured OA chondrocytes with and without IL-1b. GAPDH expression was used as a loading control. cDNA from human arteria and brain tissue served as positive controls (data not shown). BeD) Protein expression and localization of b2-AR (B), a1D-AR (C) and TH (D) in OA cartilage samples were analyzed on cryo-sections. Sections incubated with the appropriate isotype antibodies served as negative controls and showed no staining (data not shown). Bars: 100 mm.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
Stimulation with NE decreased IL-1b induced gene expression of IL-8 and MMP-13. Gene expression of MMP-3 and TIMP-2 was increased by trend after stimulation with IL-1b and 108 M NE whereas NE had no effect on gene expression levels of IL-6, MMP-2, TIMP-1 and TIMP-3 [Fig. 2(A)]. NE reversed IL-1b induced secretion of IL-8 and MMP-13 [Fig. 2(B)] and IL-1b induced decrease of GAG and collagen II production related to DNA content [Fig. 2(C)]. These NE effects were observed solely with 106 M NE strongly indicating for signaling via b-AR. To analyze the impact of NE under non-inflammatory conditions, OA chondrocytes were treated with NE (106 M and 108 M) in the absence of IL-1b (Suppl. Fig. 2). IL-8 and GAG were not altered compared to non-treated controls whereas MMP-13 and collagen II were significantly decreased after treatment with 106 M NE. NE modulates proliferation and apoptosis of 3D cultured chondrocytes To analyze the impact of NE on cell metabolism, chondrocytes were treated continuously with NE (106 M and 108 M) in a 3D micromass pellet model for 7 and 21 days. To visualize apoptotic cells, sections of paraffin-embedded pellets were stained with TUNEL, respectively with PCNA to indicate proliferating cells. TUNEL-positive cells were found after 7 and 21 days [Fig. 3(A)e(H)]. Numbers of TUNEL positive cells were increased significantly in sections treated with 108 M NE at both time points compared to vehicle treated controls [Fig. 3(I)]. PCNA-positive chondrocytes were found only after 21 [Fig. 4(A)e(C)] but not after 7 days in culture (data not shown).
5
Number of PCNA-positive chondrocytes was significantly decreased after treatment with 106 M NE, whereas increased numbers of PCNA-positive cells were observed after treatment with 108 M NE compared to vehicle controls [Fig. 4(G)]. NE induces apoptosis via a1-AR signaling To address AR subtypes involved in apoptosis induction, chondrocytes cultured in 2D were treated for 24 h with NE (106 M and 108 M) combined with a1-AR antagonist doxazosin, a2-AR antagonist yohimbine and b1-3-AR antagonist nadolol and analyzed for caspase-3/7 activity. In line with our results in 3D culture, 108 M NE without any antagonist increased significantly caspase-3/7 activity indicating apoptosis induction [Fig. 3(J)]. Increased caspase-3/7 activity was also observed after stimulation with 108 M NE in combination with yohimbine and nadolol. NE treatment combined with doxazosin prevented induction of apoptosis indicating for a1-AR signaling. Without concurrent NE treatment, adrenergic antagonists had no effect on apoptosis (data not shown). Notably, apoptosis induction with NE was not observed under inflammatory conditions (0.5 ng/ml IL-1b treatment). However, reduction of apoptosis was observed in the presence of IL-1b only (Suppl. Fig. 3A). NE causes cell cycle slow down via b-AR signaling To address AR subtypes involved in proliferation change, chondrocytes cultured in 2D were treated for 24 h with NE (106 M
Fig. 2. Impact of NE on 3D cultivated OA chondrocytes under inflammatory conditions. A) Expression of mRNA for IL-6, IL-8, MMP-2, MMP-3, MMP-13, TIMP-1, TIMP-2 and TIMP-3 in 3D cultivated chondrocytes was determined by real-time PCR. The relative expression level of each gene was calculated using GAPDH as endogenous loading control and IL-1b stimulated cells as calibrator (dotted line at 0). B) Concentrations of IL-8 and MMP-13 were determined in supernatants of 3D cultivated chondrocytes and plotted in relation to IL-1b stimulated controls (dotted line at 1). C) GAG and collagen II (Col II) content of each fibringel was related to DNA content and plotted in relation to IL-1b stimulated controls (dotted line at 1). Open box blots represent 3D cultured chondrocytes without any stimulation, grey box blots represent cells after 24 h stimulation with IL-1b and NE 106 M (light grey) or 108 M (dark grey). Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. n ¼ 6e8 independent experiments. (*) ¼ P 0.0625e0.05; * ¼ P < 0.05; ** ¼ P < 0.001 vs IL-1b stimulated controls.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
6
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
Fig. 3. Influence of NE on apoptosis of articular chondrocytes. AeH) Representative images of TUNEL stained micromass pellet sections were shown. 3D-cultured chondrocytes were treated for 7 days (AeD) and 21 days (EeH) with NE (106 M; B, F and 108 M; C, G). Vehicle treated pellets (A, E) served as negative controls, whereas eITS controls (D, H) served as positive apoptosis controls. I) Percentages of TUNEL-positive chondrocytes was determined and related to that of vehicle treated pellets (e solid black line). J) To analyze AR subtypes which mediate apoptosis induction, specific antagonists for AR (doxazosin for a1-AR; yohimbine for a2-AR; nadolol for b1e3-AR) were included in Caspase 3/7 assay. Unstimulated controls served as calibrator (e solid black line). IeJ) Data are shown as box plots. Light grey boxes indicate 106 M NE treatment; dark grey boxes indicate 108 M NE treatment and open boxes indicate minus ITS control. Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. (*) ¼ P 0.0625e0.05; * ¼ P < 0.05 vs unstimulated controls; Bars: 100 mm.
and 108 M) combined with appropriate AR inhibitors and analyzed for BrdU incorporation. In line with our results in 3D culture, 106 M NE decreased significantly BrdU incorporation compared to controls without NE indicating reduced proliferation. In contrast to our 3D results, treatment with 108 M NE had no effect on BrdU incorporation compared to vehicle treated control cells [Fig. 4(H)]. We observed a decrease in BrdU incorporation after stimulation with 106 M NE in combination with doxazosin and yohimbine. Treatment with 106 M NE and nadolol prevented decrease in proliferation, indicating for b-AR mediated signaling. Without concurrent NE treatment, AR antagonists had no effect on proliferation (data not shown). Notably, decrease in BrdU incorporation after NE treatment was not observed under inflammatory conditions (0.5 ng/ml IL-1b stimulation). However, reduction of proliferation was observed in the presence of IL-1b only (Suppl. Fig. 3B). To address changes in cell cycle phases induced by NE, cell cycle was monitored in monolayer cultured chondrocytes [Fig. 5(A)e(F)]. Stimulation with 106 M NE lead to a significant accumulation of chondrocytes in the G0/G1-phase [Fig. 5(D)] and a reduction in Sphase population compared to control [Fig. 5(E)]. We therefore suggest that NE caused a cell cycle slow down via b-AR mediated signaling. Discussion Whereas reports are available with respect to the effects of the SNS and catecholaminergic neurotransmitters on bone biology and
on chondrogenic differentiation of progenitor cells, not much is known about the impact of the SNS on adult articular cartilage physiology and pathophysiology (reviewed in30). In this study, expression of several AR subtypes in articular OA chondrocytes was observed indicating that articular chondrocytes are target cells of the SNS. Until now expression of AR subtypes was solely described for growth plate chondrocytes and for neonatal murine costal chondrocytes15,16,31. We further observed expression of TH in OA chondrocytes indicating for catecholamine synthesis. However, we did neither detect NE nor epinephrine (E) in chondrocyte culture supernatants suggesting that chondrocytes do not secrete and presumably also do not synthesize NE themselves. NE is a small molecule and as such presumably penetrates cartilage matrix easily and thus reaches cells via diffusion. As source of NE in joints serve synovium and subchondral bone which both are innervated by sympathetic nerve fibers. Our data indicate a different role of TH in OA chondrocytes apart from NE synthesis. The rare genetic disorder of tyrosine metabolism, alkaptonuria, might provide an indication as alkaptonuria leads to deposition of melanin-like pigments in cartilage of weight bearing joints resulting in severe OA32. In our in vitro inflammation model, NE modulated IL-1b induced effects in OA articular chondrocytes. An anti-inflammatory effect of NE in the osteoarticular system was reported earlier in a murine experimental arthritis model33. The authors described a significant reduction in clinical scores of collagen induced arthritis and cell infiltration in the paws after injection of TH expressing MSC which
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
7
Fig. 4. Influence of NE on proliferation of articular chondrocytes. AeF) Representative images of PCNA (AeC) and hematoxylin only (DeF) stained micromass pellet sections after vehicle (A, D) and NE treatment (106 M; B, E and 108 M; C, F) after 21 days. G) Percentage of PCNA-positive chondrocytes was determined and related to unstimulated pellets (e solid black line). H) To analyze AR subtypes which mediate proliferation changes, specific antagonists for AR (doxazosin for a1-AR, yohimbine for a2-AR, nadolol for b1e3-AR) were included in BrdU assay. Unstimulated controls served as calibrator (ee). GeH, Data are shown as box plots. Light grey boxes indicate 106M NE treatment and dark grey boxes 108 M NE treatment. Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. * ¼ P < 0.05 vs unstimulated controls; Bars ¼ 100 mm.
were differentiated towards neuronal cells in vitro producing and secreting NE. In our study, NE reversed IL-1b induced effects specifically at 106 M strongly indicating for signaling via b-AR which is associated with anti-inflammatory effects (reviewed in34) in inflammatory joint diseases22. Together with our data, we suggest that b-AR signaling has anti-inflammatory effects in OA chondrocytes. We observed a distinct concentration dependent impact of NE on metabolic activities of OA chondrocytes under 2D and 3D culture conditions. In the presence of 106 M NE, chondrocytes accumulated in the GO/G1-phase leaving a decreased cell number in the S-phase population. Proliferation decrease was prevented with nadolol indicating for b-AR signaling. This observation is in line with two studies on rat hepatocytes respectively rat oligodendrocyte progenitor cells which both observed a G1-phase arrest induced via b-AR signaling. Notably, 108 M NE induced proliferation in our 3D culture model but not in monolayer culture. This discrepancy might be due to different
timing of NE treatment (24 h vs 21 days) and/or different culture conditions (2D vs 3D). Several publications describe chondrocyte dedifferentiation during 2D culture35e37 latter presumably affecting proliferation activity. Differences of NE treatment solely depend on agonist concentration can be explained by the differential intracellular signaling pathways. High concentrations of NE mediate their effects by activating a- and b-AR signaling pathways and low concentrations mediate their effects mainly via a-AR signaling. b-AR signaling increases cAMP level38, whereas alpha AR signaling increases intracellular Ca2þ level39. Increased cAMP levels in chondrocytes are associated with a chondrocyte typical phenotype40, whereas increased Ca2þ level are described to induce apoptosis41. Also Lei et al.42 described a NE concentration dependent regulation of cell growth with a proliferation decrease after treatment with high doses and a proliferation increase with low doses NE. Taken together with our data we suggest a dual function of the SNS in OA chondrocytes with distinct impact of AR subtypes on metabolic
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
8
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
Fig. 5. Cell cycle analysis of articular chondrocytes in the presence of NE. AeC) Representative cell cycle histograms of vehicle (A) and NE treated (106 M; B and 108 M; C) chondrocytes are shown. Histograms were analyzed with Dean-Jett-Fox model to calculate percentages of cell populations in different cell cycle phases. DeF) Percentages of cell populations in G0/G1- (D), S- (E) and G2-phase (F) are shown as box plots. Each box represents 5e95% percentile and whiskers min to max. Lines inside the boxes represent the median. * ¼ P < 0.05 vs unstimulated controls. n ¼ 6e7 independent experiments.
response. b-AR signaling rather promoted a stable articular chondrocyte phenotype with low proliferative activity. a-AR signaling might promote OA pathogenesis through induction of chondrocyte death and increased proliferation which is mostly associated with chondrocyte dedifferentiation6. Conclusion This study demonstrated that human OA chondrocytes are target cells of the SNS. NE treatment reversed IL-1b induced production of IL-8 and MMP-13 and IL-1b induced decrease of collagen II and GAG production presumptively via b-AR signaling. NE further induced apoptosis via a1-AR signaling whereas b-AR signaling lead to a cell cycle slow down resulting in decreased proliferation and ultimately in reduced cell growth. However, low doses of NE resulted in increased proliferation presumptively via a-AR signaling. We therefore suggest that b-AR signaling plays an antiinflammatory role in OA pathology and promotes a nonproliferative, metabolically stable articular chondrocyte phenotype which might counteract OA initiation or progression. Contrary, a-AR signaling might promote OA progression through induction of apoptosis and/or proliferation of articular chondrocytes.
RB made substantial contributions to the acquisition, analysis and interpretation of data. ZJL made substantial contributions to the acquisition and analysis of data. RHS provided study materials of patients, technical and logistic support and statistical expertise. SG made substantial contributions to conception and design of the study, to data interpretation, to drafting of the article, critical revision of the article for important intellectual content and final approval of the article. She also obtained the funding for this study. Competing interest statement The authors declare no competing interests. Funding source This work was funded with a grant from the German Research Society (DFG) to SG (GR 1301/9-1). Acknowledgements We want thank Anja Pasoldt for her expert and high quality technical assistance.
Author contributions
Abbreviations
JL made substantial contributions to the conception and design of the study, to the acquisition of data, analysis and interpretation. She also contributed critical to the drafting of the manuscript. NS made substantial contributions to the acquisition and analysis of data.
3D AR BH4 DMEM DMMB
3-dimensional adrenergic receptors 6R-5,6,7,8-tetrahydrobiopterin dihydrochloride Dulbecco's modified Eagle medium dimethyl methylene blue
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
ELISA GAG IL MMPs NE OA PCNA PCR pI P/S SNS TH TIMPs TUNEL
Enzyme-linked immunosorbent assay glycosaminoglycan interleukin matrix metalloproteases norepinephrine osteoarthritis/osteoarthritic proliferating cell nuclear antigen polymerase chain reaction propidium iodide penicillin/streptomycin sympathetic nervous system tyrosine hydroxylase tissue inhibitor of metalloproteases TdT-mediated dUTP-biotin nick end labeling
14.
15.
16.
Supplementary data
17.
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.joca.2015.08.007.
18.
References 1. Peat G, McCarney R, Croft P. Knee pain and osteoarthritis in older adults: a review of community burden and current use of primary health care. Ann Rheum Dis 2001;60:91e7. 2. Conaghan PG, Kloppenburg M, Schett G, Bijlsma JW, committee Eoah. Osteoarthritis research priorities: a report from a EULAR ad hoc expert committee. Ann Rheum Dis 2014;73: 1442e5. 3. Kevorkian L, Young DA, Darrah C, Donell ST, Shepstone L, Porter S, et al. Expression profiling of metalloproteinases and their inhibitors in cartilage. Arthritis Rheum 2004;50:131e41. 4. Martel-Pelletier J, Pelletier JP. New insights into the major pathophysiological processes responsible for the development of osteoarthritis. Semin Arthritis Rheum 2005;34:6e8. 5. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 2000;1477:267e83. 6. Pitsillides AA, Beier F. Cartilage biology in osteoarthritiselessons from developmental biology. Nat Rev Rheumatol 2011;7:654e63. 7. Dreier R. Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther 2010;12:216. 8. Goldring MB, Goldring SR. Osteoarthritis J Cell Physiol 2007;213:626e34. 9. Melchiorri C, Meliconi R, Frizziero L, Silvestri T, Pulsatelli L, Mazzetti I, et al. Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chondrocytes from patients with osteoarthritis. Arthritis Rheum 1998;41:2165e74. 10. Goldring SR, Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res 2004:S27e36. 11. Imai S, Matsusue Y. Neuronal regulation of bone metabolism and anabolism: calcitonin gene-related peptide-, substance P-, and tyrosine hydroxylase-containing nerves and the bone. Microsc Res Tech 2002;58:61e9. 12. Bjurholm A, Kreicbergs A, Terenius L, Goldstein M, Schultzberg M. Neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide-immunoreactive nerves in bone and surrounding tissues. J Auton Nerv Syst 1988;25: 119e25. 13. Pereira da Silva JA, Carmo-Fonseca M. Peptide containing nerves in human synovium: immunohistochemical evidence
19.
20.
21.
22. 23.
24.
25.
26.
27.
28.
29.
9
for decreased innervation in rheumatoid arthritis. J Rheumatol 1990;17:1592e9. Miller LE, Justen HP, Scholmerich J, Straub RH. The loss of sympathetic nerve fibers in the synovial tissue of patients with rheumatoid arthritis is accompanied by increased norepinephrine release from synovial macrophages. FASEB J 2000;14:2097e107. Opolka A, Straub RH, Pasoldt A, Grifka J, Grassel S. Substance P and norepinephrine modulate murine chondrocyte proliferation and apoptosis. Arthritis Rheum 2012;64:729e39. Takarada T, Hojo H, Iemata M, Sahara K, Kodama A, Nakamura N, et al. Interference by adrenaline with chondrogenic differentiation through suppression of gene transactivation mediated by Sox9 family members. Bone 2009;45: 568e78. Lai KB, Sanderson JE, Yu CM. High dose norepinephrineinduced apoptosis in cultured rat cardiac fibroblast. Int J Cardiol 2009;136:33e9. Communal C, Singh K, Pimentel DR, Colucci WS. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway. Circulation 1998;98:1329e34. Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X, et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature 2005;434:514e20. Suzuki A, Palmer G, Bonjour JP, Caverzasio J. Catecholamines stimulate the proliferation and alkaline phosphatase activity of MC3T3-E1 osteoblast-like cells. Bone 1998;23:197e203. Takahata Y, Takarada T, Iemata M, Yamamoto T, Nakamura Y, Kodama A, et al. Functional expression of beta2 adrenergic receptors responsible for protection against oxidative stress through promotion of glutathione synthesis after Nrf2 upregulation in undifferentiated mesenchymal C3H10T1/2 stem cells. J Cell Physiol 2009;218:268e75. Straub RH, Harle P. Sympathetic neurotransmitters in joint inflammation. Rheum Dis Clin North Am 2005;31:43e59. viii. Lukewich MK, Rogers RC, Lomax AE. Divergent neuroendocrine responses to localized and systemic inflammation. Semin Immunol 2014 Oct;26(5):402e8, http://dx.doi.org/10.1016/ j.smim.2014.01.004. Jenei-Lanzl Z, Grassel S, Pongratz G, Kees F, Miosge N, Angele P, et al. Norepinephrine inhibition of mesenchymal stem cell and chondrogenic progenitor cell chondrogenesis and acceleration of chondrogenic hypertrophy. Arthritis Rheumatol 2014 Sep;66(9): 2472e81, http://dx.doi.org/10.1016/10.1002/art.38695. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum 2012;64:1697e707. Suri S, Gill SE, Massena de Camin S, Wilson D, McWilliams DF, Walsh DA. Neurovascular invasion at the osteochondral junction and in osteophytes in osteoarthritis. Ann Rheum Dis 2007;66:1423e8. Opolka A, Ratzinger S, Schubert T, Spiegel HU, Grifka J, Bruckner P, et al. Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice. Matrix Biol 2007;26:85e95. Kees MG, Pongratz G, Kees F, Scholmerich J, Straub RH. Via beta-adrenoceptors, stimulation of extrasplenic sympathetic nerve fibers inhibits lipopolysaccharide-induced TNF secretion in perfused rat spleen. J Neuroimmunol 2003;145: 77e85. Jenei-Lanzl Z, Straub RH, Dienstknecht T, Huber M, Hager M, Grassel S, et al. Estradiol inhibits chondrogenic differentiation of mesenchymal stem cells via nonclassic signaling. Arthritis Rheum 2010;62:1088e96.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007
10
J. Lorenz et al. / Osteoarthritis and Cartilage xxx (2015) 1e10
€ssel S. The role of peripheral nerve fibers and their neu30. Gra rotransmitters in cartilage and bone physiology and pathophysiology. Arthritis Res Ther 2014;16(6):485, http:// dx.doi.org/10.1186/s13075-014-0485-1. 31. Lai LP, Mitchell J. Beta2-adrenergic receptors expressed on murine chondrocytes stimulate cellular growth and inhibit the expression of Indian hedgehog and collagen type X. J Cell Biochem 2008;104:545e53. 32. Taylor AM, Boyde A, Wilson PJ, Jarvis JC, Davidson JS, Hunt JA, et al. The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria. Arthritis Rheum 2011;63:3887e96. 33. Jenei-Lanzl Z, Capellino S, Kees F, Fleck M, Lowin T, Straub RH. Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis. Ann Rheum Dis 2015 Feb;74(2):444e51, http:// dx.doi.org/10.1136/annrheumdis-2013-203925. 34. Ader R. Psychoneuroimmunology. San Diego, CA: ElsevierAcademic Press; 2007. 35. Cheung HS, Harvey W, Benya PD, Nimni ME. New collagen markers of 'derepression' synthesized by rabbit articular chondrocytes in culture. Biochem Biophys Res Commun 1976;68:1371e8.
36. Benya PD, Shaffer JD. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982;30:215e24. 37. Bonaventure J, Kadhom N, Cohen-Solal L, Ng KH, Bourguignon J, Lasselin C, et al. Reexpression of cartilagespecific genes by dedifferentiated human articular chondrocytes cultured in alginate beads. Exp Cell Res 1994;212: 97e104. 38. Levitzki A. From epinephrine to cyclic AMP. Science 1988;241: 800e6. 39. Piascik MT, Perez DM. Alpha1-adrenergic receptors: new insights and directions. J Pharmacol Exp Ther 2001;298:403e10. 40. Chosa E, Hamada H, Kitamura K, Kuwasako K, Yanagita T, Eto T, et al. Expression of adrenomedullin and its receptor by chondrocyte phenotype cells. Biochem Biophys Res Commun 2003;303:379e86. 41. Wang W, Xu J, Kirsch T. Annexin-mediated Ca2þ influx regulates growth plate chondrocyte maturation and apoptosis. J Biol Chem 2003;278:3762e9. 42. Lei B, Schwinn DA, Morris DP. Stimulation of alpha1a adrenergic receptors induces cellular proliferation or antiproliferative hypertrophy dependent solely on agonist concentration. PLoS One 2013;8:e72430.
Please cite this article in press as: Lorenz J, et al., Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses, Osteoarthritis and Cartilage (2015), http://dx.doi.org/10.1016/j.joca.2015.08.007