All mice were bred at the animal facilities of the Alexander Fleming Biomedical Sciences Research Center (BSRC) under specific pathogen–free conditions. Mice were housed at a temperature of 20–22°C, 55 ± 5% humidity, and a 12-h light–dark cycle; water and food were given ad libitum. Mice were bred and maintained in their respective genetic backgrounds for >10 generations. All experiments were approved by the Institutional Animal Ethic Committee of BSRC Fleming (#232), as well as by the Veterinary Service and Fishery Department of the local government agency. The generation and genotyping instructions of Tg197
; Keffer et al., 1991
(Kontoyiannis et al., 1999
(Armaka et al., 2008
(Koike et al., 2009
(Fotopoulou et al., 2010
), and Tg(a1t1
(Pamuklar et al., 2009
) mice have been previously described.
Animal models of arthritis.
Both inducible and spontaneous animal models of arthritis were used in this study (Kollias et al., 2011
). CIA was induced in C57BL/6 mice as previously described (Campbell et al., 2000
). In brief, mice were immunized with chicken collagen type II, followed by a boost immunization at day 21. Disease onset was observed at day 28; all experimental mice were sacrificed at day 60 after immunization. Tg197
) is a humanized TNF transgenic mouse with human TNF overexpression resulting in the spontaneous development of chronic, erosive, and symmetrical polyarthritis (Kollias et al., 2011
). Disease symptoms were observed 3 wk after birth; all experimental mice were sacrificed 5–6 wk after birth. TnfΔARE
is a mouse mutant carrying a deletion of the ARE (AU-rich elements), 3′ untranslated region regulatory element of TNF that results in loss of its posttranscriptional regulation. Deregulated TNF expression leads to the gradual development of spontaneous inflammatory polyarthritis and inflammatory bowel disease (Kontoyiannis et al., 1999
Human synovial tissues and SFs were obtained at the time of joint replacement surgery at the Schulthess Clinic (Zurich, Switzerland). Sera from patients were obtained from the Schulthess Clinic. All patients, classified according to the 1987 criteria of the American College of Rheumatology, signed an informed consent form where they agreed to the anonymous use of their samples for research purposes after approval of the Kantonale Ethikkommission, Spezialisierte Unterkommission für Spezialfächer (#475). All personal and clinical data of patients are included in Tables S1 and S2
1-oleoyl-LPA (18:1) and 1-oleoyl-LPC (18:1) were purchased from Avanti Polar Lipids, Inc. ERK inhibitor PD98059, p38 MAPK inhibitor SB202190, and JNK inhibitor SP600125 were obtained from EMD. Fatty acid–free BSA, choline oxidase, peroxidase, TMB (3,3′,5,5′-tetramethylbenzidine) substrate, phalloidin, and DAB (3,3′-diaminobenzidine) were obtained from Sigma-Aldrich. Recombinant ATX and mouse TNF were purchased from R&D Systems. Native ATX refers to the 10× concentrated supernatants of MDA-MB-43S cells (American Type Culture Collection), which are highly enriched in ATX. Fibronectin and Cytofix/Cytoperm solution were obtained from BD. Rabbit anti-ATX polyclonal antibodies (Abs) were purchased from Phoenix Pharmaceuticals and/or Cayman Chemical; rat monoclonal Abs (clone 4F1) against ATX were a gift from J. Aoki (Tohoku University, Aoba-ku, Sendai, Miyagi, Japan). Antivimentin Abs were purchased from Millipore, and collagen P4H Abs were purchased from Acris. Secondary Alexa Fluor 555–conjugated anti–rabbit Ab was obtained from Invitrogen; all secondary horseradish peroxidase (HRP)–conjugated Abs and isotype controls were obtained from and SouthernBiotech.
Histopathology and arthritic score.
Paraffin-embedded mouse joint tissue samples were sectioned and stained with hematoxylin and eosin (H&E) as previously described (Aidinis et al., 2005
; Armaka et al., 2008
). Arthritic histopathology in mice was assessed (in a blinded fashion from three independent reviewers) using a semiquantitative scoring system as previously described for CIA (0 = no detectable pathology; 1 = mild arthritis, minimal synovitis and cartilage loss; 2 = moderate arthritis, synovitis and bone erosions; and 3 = severe arthritis, synovitis, extensive erosions, and disrupted joint architecture; Campbell et al., 2000
). The scoring system for Tg197
) is as follows: 0 = no detectable pathology, 1 = synovial membrane hyperplasia, 2 = pannus and fibrous tissue formation and focal bone erosion, 3 =cartilage destruction and bone erosion, and 4 = extensive cartilage destruction and bone erosion.
Paraffin-embedded mouse joint tissue samples (4-µm-thick sections) were deparaffinized, and endogenous peroxidase activity was blocked by incubation in 1% H2O2 for 10 min. Sections were then blocked with 2% BSA for 30 min and incubated overnight at 4°C with an anti-ATX Ab (from Phoenix Pharmaceuticals unless otherwise indicated) or an IgG rabbit isotype control. Washing in PBS-T was followed by incubation with HRP-conjugated anti–rabbit IgG for 30 min at room temperature. Bound peroxidase activity was detected by staining with DAB. Sections were counterstained with hematoxylin, mounted, and photographed under an ECLIPSE E800 microscope (Nikon) using the ACT-I software. Expression levels were quantified by using a three-point scale semiquantitative intensity score: 0 = no staining, 1 = weak expression, 2 = moderate expression, and 3 = strong expression.
Formalin-fixed, paraffin-embedded sections of RA and OA synovial tissue slides were deparaffinized and treated at 80°C for 30 min with citrate buffer, pH 6.0. After washing with H2O, the endogenous peroxidase was blocked with 1% H2O2 for 10 min. The slides were blocked with 2% goat serum for 1 h and incubated overnight at 4°C with a rabbit polyclonal Ab against ATX (Cayman Chemical) or IgG1 rabbit isotype control (Dako). After washing twice in PBS, the slides were incubated with their respective biotinylated secondary Abs for 30 min. The signal was amplified with HRP conjugated with streptavidin Vectastain Elite ABC kit (Vector Laboratories). The slides were then developed with a chromogenic substrate for peroxidase and counterstained with hematoxylin. ATX expression was quantified semiquantitatively, blindly by three different reviewers, on a five-point scale: 0 = no staining; 1 = weak expression, single cells stained; 2 = mild expression, limited areas stained; 3 = moderate expression, weak overall expression; and 4 = strong expression, strong overall staining.
Joints were harvested as a block, and a paramedical patella incision was made on the joint to expose the inside. Harvested joints were fixed in formaldehyde/glutaraldehyde for 1 h at room temperature, washed three times in PBS/2 mM MgCl2, and incubated with 1 mg/ml 5-bromo-a-chloro-3-inodyl-β-d-galactopyranoside (X-gal) in 0.1 M Na phosphate buffer, pH 7.3 (2 mM MgCl2, 0.01% Na deoxycholate, 0.02% NP-40, 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6) at 37°C in the dark overnight. Tissues were rinsed three times in PBS/2 mM MgCl2 at room temperature and were then placed in decalcification buffer at 4°C for 6 d. After that, they were washed with PBS and embedded in paraffin. The resulting 4-µm sections were counterstained with eosin and visualized under an ECLIPSE E800 microscope as in the previous section.
Cell isolation and culture.
Primary SFs were isolated by enzymatic treatment of the joints of 6-wk-old mice as previously described (Aidinis et al., 2005
). Cells from two to three mice were pooled and cultured in standard conditions: DME supplemented with 10% FBS and 1% penicillin-streptomycin, 37°C, and 5% CO2
(Aidinis et al., 2005
); all experiments were performed after two to three passages with 70–80% confluent cells and confirmed with independent isolations. All in vitro experiments were performed after overnight serum starvation (supplemented with 0.2% fatty acid–free BSA). Cells were pretreated with the indicated concentrations of PTX and inhibitors of ERK, p38, JNK, and Rho kinase for 3 h before LPA stimulation. Brefeldin A (GolgiPlug; BD) was added to SF cultures for 4 h according to manufacturer’s instructions to prevent Golgi transport.
Cells were initially stained extracellularly with VCAM-1 (CD106) and subsequently with a secondary (PE) conjugated Ab. For intracellular stainings, cells were fixed and permeabilized with Cytofix/Cytoperm solution, followed by incubation with anti-ATX (1:200; Cayman Chemical) and antivimentin (1:50) Abs for 30 min. Primary Abs were detected after a 30-min incubation with anti–rabbit biotin Abs followed by PE-Cy5-streptavidin or with FITC-conjugated secondary Ab (all diluted 1:500). Analysis was performed using FACSCanto II and Diva software (BD).
SFs were grown in 24-well tissue-culture plates in DME. Preconfluent cell cultures were starved overnight, incubated for 24 h with LPA and the various compounds, and finally exposed to 0.5 µCi/ml [3H]thymidine. Cells were then washed with PBS, detached with 0.05% trypsin, and blotted on FilterMAT fiber paper using a cell harvester (Molecular Devices). The radioactivity of incorporated [3H]thymidine was determined using a liquid scintillation counter (LS 6500; Beckman Coulter). The experiments were performed in triplicate.
This assay was performed on plates coated with human fibronectin (10 µg/ml in PBS) for 2 h at 37°C. In brief, cells were allowed to adhere to the substrate for 4 h at 37°C, and unbound cells were removed by washing twice with PBS. Adhered cells were then stained with 0.2% crystal violet in 10% ethanol for 10 min at room temperature. After washing with water, cells were lysed with 33% acetic acid, and their absorbance was determined at 570 nm using a SpectraMax Plus photometer (Molecular Devices). The experiments were performed in triplicate.
Cell migration assay.
Modified Boyden chambers with 8-µm pores (Corning) were coated at the lower surface with 10 µg/ml human fibronectin for 2 h at 37°C. Cells were harvested with trypsin/EDTA from nonconfluent cultures, washed with PBS, and resuspended to 106 cells per ml. The cell suspension was then added to the upper chamber, and the cells were allowed to migrate at 37°C, 5% CO2, for 4 h. Nonmigratory cells were removed from the upper surface of the membrane, whereas migratory cells in the lower compartment of the chambers were washed with PBS and stained with 0.2% crystal violet in 10% ethanol for 10 min. After extensive washing in H2O, the cells were lysed, and absorbance was measured at 570 nm with a SpectraMax Plus photometer as in the previous section. The experiments were performed in triplicate.
Cells were cultured on glass coverslips, in the presence or absence of Brefeldin A, fixed in 4% paraformaldehyde, and stained with a primary Ab (1:300) against ATX (Cayman Chemical) overnight at 4°C. After incubation with a secondary anti–rabbit Ab (1:1,000) conjugated to Alexa Fluor 555 for 1 h at room temperature, cells were observed and photographed with an ECLIPSE E800 microscope as in Immunohistochemistry. F-actin (Sigma-Aldrich) was visualized with incubation with TRITC-conjugated phalloidin according to the manufacturer’s protocol, after permeabilization with 0.1% Triton X-100 in PBS.
The protein concentration of cell extracts or supernatants was determined with a Protein Assay kit (Bio-Rad Laboratories) using BSA as a standard. Supernatants or cellular extracts from cultured SFs were separated by 8% SDS-PAGE and transferred to Protran nitrocellulose membranes (GE Healthcare) using the Trans-Blot SD Semi-Dry Transfer system (Bio-Rad Laboratories). Primary anti-ATX Ab incubation (monoclonal 4F1, 1:1,000, unless otherwise specified) was performed overnight in 2.5% (wt/vol) nonfat milk at 4°C. The membranes were then washed three times with TBS-Tween 0.05% and incubated with an anti–rat HRP-conjugated secondary Ab (1:1,000) for 1 h at room temperature. Membranes were washed three times with TBS-Tween 0.05%, and Ab–antigen complexes were revealed using luminol as a chemiluminescent reagent.
Plates were coated overnight with standards and diluted samples, blocked with 2% BSA, and then incubated with an anti-ATX Ab (Cayman Chemical). For mouse plasma, dilution was 1:16, for human serum 1:40, and for human synovial fluids 1:120, selected from the linear range of serial dilutions. ATX antigen was detected after incubation with an HRP-labeled secondary Ab and development with TMB substrate. Readings were obtained at 450 nm with a SpectraMax Plus photometer. Cytokine levels in mouse cell culture supernatants were determined using the multiplex LINCOplex assay kit (Millipore), according to the manufacturer’s instructions.
RNA extraction and RT-PCR analysis.
RNA extraction and RT-PCR analysis were performed as previously described (Aidinis et al., 2005
). Immune cell RNAs were provided by P. Stamou and D.L. Kontoyiannis (Alexander Fleming Biomedical Sciences Research Center, Athens, Greece). Primer sequences (designated as s, sense; and as, antisense) and product sizes were as follows: Enpp2 (s, 5′-GTGAAATATTCTTAATGCCTCTCTG-3′; as, 5′-GCCTTCCACATACTGTTTAATTCC-3′; 410 bp), b2m (s, 5′-TTCTGGTGCTTGTCTCACTGA-3′; as, 5′-CAGTATGTTCGGCTTCCCATTC-3′; 104 bp), MMP-9 (s, 5′-CAGATGATGGGAGAGAAGCA-3′; as, 5′-CGGCAAGTCTTCAGAGTAGT-3′; 222 bp), LPA1 (s, 5′-GAGGAATCGGGACA-3′; as, 5′-TGAAGGTGGCGCTC-3′; 227 bp), LPA2 (s, 5′-GACCACACTCAGCCTAGTCAAGAC-3′; as, 5′-CAGCATCTCGGCAGGAAT-3′; 200 bp), LPA3 (s, 5′-GCTCCCATGAAGCTAATGAAGACA-3′; as, 5′-TACGAGTAGATGATGGGG-3′; 188 bp), LPA4 (s, 5′-AGTGCCTCCCTGTTTGTCTTC-3′; as, 5′-GCCAGTGGCGATTAAAGTTGTAA-3′; 142 bp), and LPA5 (s, 5′-ACCCTGGAGGTGAAAGTC-3′; as, 5′-GACCACCATATGCAAACG-3′; 176 bp).
Unless otherwise indicated, statistical significance was assessed in pairwise comparisons with control values by using a paired Student’s t test, after confirmation of normal distributions with SigmaPlot 11.0 (Systat Software). Unless otherwise indicated, all values are expressed as means ± SE, and p-values <0.05 were considered significant (*).
Online supplemental material.
Fig. S1 contains various controls supporting an increased ATX expression in arthritic joints, as well as supporting that TNF treatment of the transgenic animal model of arthritis attenuates ATX expression. Fig. S2 contains various controls on the effects of the conditional ATX deletion in modeled disease development. Fig. S3 shows that systemic fluctuations of ATX serum levels do not affect disease development in the hTNF+/−
animal model. Tables S1 and S2 contain personal and clinical data of patients used for determination of ATX levels in sera and synovial fluids, respectively. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20112012/DC1