Synovial tissue was obtained during open joint replacement surgery or arthroscopic synovectomy from a total of 16 patients with the clinical diagnosis of RA (13 knee joints, one hip joint, one wrist, one metacarpo-phalangeal joint) as well as 21 patients with the clinical diagnosis of OA (all knee joints; Table ) from the Department of Orthopedics, University of Leipzig, Germany, the Clinic of Orthopedics, Bad Düben, Germany, and the Clinic of Orthopedics, Eisenberg, Germany, as well as the Department of Orthopedic Surgery, University of Michigan, Ann Arbor, MI, USA. All RA patients fulfilled the American Rheumatism Association criteria for RA [8
]. The study was approved by the Ethics Committees of the University of Leipzig and the University of Jena, Germany, and the University of Michigan, MI, USA. One portion of each sample was immediately frozen in isopentane (Merck, Darmstadt, Germany), cooled in liquid nitrogen and stored at –70°C for immunohistochemistry. The remaining tissue was placed in cell culture medium at ambient temperature and subjected to tissue digestion within 2 h.
Clinical characteristics of the patients at the time of synovectomy
Histochemical detection of non-specific esterase
Non-specific esterase activity was analyzed in methanol-fixed (10 min at room temperature) first-passage RA-SFB and OA-SFB on chamber slides, and in air-dried, unfixed rat kidney sections or rat peritoneal macrophages, by incubation for 1 h at 37°C with a reaction medium containing 0.02% 5-bromo-4-chloro-3-indoxylacetate (initially dissolved in dimethylformamide), 1 mM potassiumferricyanide, and 1 mM potassiumferrocyanide (final concentrations) (all from Sigma) in 2 mM Tris7#150;HCl buffer (pH 6.8). The positive controls (rat kidney or peritoneal macrophages) were stained in all experiments, while negative control cell preparations/kidney sections incubated with substrate-free medium did not show any positive reaction (respective data not shown). The cells/sections were then washed in PBS (see later) and mounted in glycerin gelatin.
Immunohistochemical analysis was performed on cryostat sections of RASM (fixed with acetone for 10 min at 4°C) or chamber slides (fixed with 100% methanol for 10 min at room temperature; Nunc, Wiesbaden, Germany) using the antibodies presented in Table . The mouse mAbs, diluted in Tris–buffered saline (TBS)/1% bovine serum albumin for per-oxidase/alkaline phosphatase detection, were added for 30 min in a humid chamber at room temperature following pre-incubation of the sections/chambers with 20% normal human serum in TBS for 20 min (pH 7.4). A peroxidase-coupled rabbit anti-mouse antibody (Dako) and a peroxi-dase-coupled swine anti-rabbit antibody (Dako; both 1:30 in TBS/20% human serum) were added for 30 min each. The peroxidase was revealed using diaminobenzidine (5 min, 0.5 mg diaminobenzidine in 1 ml PBS [pH 7.4] containing 30 μl H2O2). Blocking of endogenous peroxidase (0.03% H2O2) before immunohistochemical analysis, as performed in some experiments, did not alter the staining results.
Antibodies used in the present study
Alternatively, either the indirect alkaline phosphatase or the alkaline phosphatase anti-alkaline phosphatase (APAAP) techniques were used for detection of the monoclonal or polyclonal primary antibodies [35
]. An alkaline phosphatase-coupled goat-anti-mouse antibody (Dako) was applied for 30 min (1:30 in TBS/20% human serum; pH 7.4) in the case of the indirect alkaline phosphatase, and the alkaline phosphatase was revealed in substrate buffer (TBS; pH 9.5) containing 0.16 mg/ml 5-chromo-4-chloro-3-indoxylphosphate and 0.15 mg/ml nitroblue tetra-zolium chloride (both from Boehringer Mannheim). In the case of the APAAP, a rabbit anti-mouse antibody (Dako) was applied for 45 min (1:100 in TBS/20% human serum; pH 7.4). The sections/chamber slides were washed, incubated for 45 min with APAAP complex (1:80 in TBS/1% bovine serum albumin; Dianova), and washed again with TBS. To amplify the staining, incubation with the rabbit anti-mouse mAb and the APAAP was repeated once. The alkaline phosphatase was revealed in Tris–HCl buffer (pH 8.6) containing 0.3 mg/ml Naphthol AS-MX phosphate (first dissolved at 15 mg/ml in dimethylformamide; both from Sigma), 1 mg/ml Fast Red TR salt or Fast Blue BB salt, and 0.24 mg/ml levamisole (all from Sigma). The solution was filtered and added to the sections/chamber slides for 30 min in a humid chamber. For the APAAP technique, polyclonal primary antibodies (against procollagen I and III) were incubated with a secondary mouse anti-rabbit antibody (Dako) before using the secondary detection systems.
Double-staining experiments on cryostat sections of the RA SM were performed by combining the peroxidase technique with the APAAP technique. The sections were blocked with 20% normal human serum in TBS for 30 min following completion of peroxidase staining. Isotype-matched mouse mAbs, rabbit sera, or purified rabbit immunoglobulins at identical concentrations to the primary antibodies were used as controls for all immunohistochemical analyses (Table ), which gave no positive results. Cross-reactivity of the peroxidase and the alkaline phosphatase detection (theoretically possible on the basis of employing mouse primary mAbs and anti-mouse secondary antibodies in both systems) was never observed when using control Ig or when omitting one or both primary mAbs, presumably because the peroxidase reaction with diaminobenzidine eliminates all immunoreactive epitopes from this step.
For comparative immunohistochemical and FACS analyses, the percentage of cells stained for the individual markers following immunohistochemistry on chamber slides was determined (total of 100 cells analyzed) and compared with the percentage of positive cells in FACS analysis (see later).
Tissue digestion and cell culture
Samples of RA or OA SM were finely minced with scissors (tissue pieces of approximately 30 mm3) and digested for 30 min at 37°C in 20 ml PBS containing 0.1% trypsin (Sigma). After removal of trypsin/PBS, the samples were digested in 20 ml 0.1% collagenase P (Boehringer Mannheim) in DMEM/10% FCS for 2 h at 37°C, 5% CO2. The cell suspension was then filtered through a sterile sieve (Sigma), the cells collected by centrifugation, washed twice with serum-free DMEM medium, and subsequently cultured for 7 days in DMEM/10% FCS, 25 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B (all from Gibco BRL, Karlsruhe, Germany). Primary-culture skin-FB from normal donors (kindly provided by Dr Sauer, Leipzig, Germany) were prepared by first incubating skin samples with 0.5 U/ml Dispase II (Boehringer Mannheim) overnight at 4°C and, following removal of the epidermis, by digesting with 0.25% collagenase P (Boehringer Mannheim) in DMEM/1% FCS at 37°C and 5% CO2 for 4 h. The resulting cell suspension was then cultured for 14 days as already described. Repeated-passage HUVEC (kindly provided by Dr C. Syring, Institute of Clinical Immunology and Transfusion Medicine, University of Leipzig) were cultured in DMEM/10% FCS/25 mM HEPES containing 10 U/ml heparin, 10 ng/ml basic fibroblast growth factor (Sigma), and 100 μg/ml recombinant endothelial growth factor (Sigma). The medium was changed every 2-3 days in all cases. Adherent RA-SFB or OA-SFB were subjected to very short trypsinization (2 min, 0.25% trypsin/0.2% EDTA; Gibco), removed from the culture dish by mechanical dislocation, washed in PBS/2% FCS, and used for negative isolation. The samples were randomly tested to exclude Mycoplasma contamination.
Negative isolation from primary culture
Trypsinized and washed RA and OA synovial cells from primary culture (107/ml) were incubated with 4 × 107/ml Dynabeads® M-450 CD14 (clone RMO52; Dynal) in PBS/2% FCS for 1 h at 4°C under bidirectional rotation. Nine milliliters of PBS/2% FCS were then added and the conjugated cells collected using the Dynal magnetic particle concentrator®. Magneto-bead-conjugated cells and unconjugated cells were collected and washed twice in PBS/2% FCS; cell composition and phenotype were analyzed by flow cytometry using the antibodies presented in Table . For the purpose of comparison with fourth-passage cells obtained by conventional isolation (ie repeated passaging; see later), negatively isolated RA-SFB and OA-SFB were then passaged four times by culture in complete DMEM/10% FCS (see earlier) with a 1:3 split of confluent cells in each passage.
Conventional isolation of RA-SFB and OA-SFB by repeated passage
Synovial cells were obtained by trypsin/collagenase digestion of the RASM as already described. The cells were subsequently cultured for four passages in DMEM/10% FCS containing the aforementioned additives by splitting confluent cells in each passage at a ratio of 1:3.
The antibodies presented in Table were used for FACS analyses of primary-culture RA and OA synovial cells (following 7 days of culture), isolated RA-SFB and OA-SFB (immediately following isolation or at fourth-passage), conventional fourth-passage RA-SFB and OA-SFB, normal skin-FB (primary-culture or fourth-passage), or repeated-passage endothelial cells (HUVEC). Primary antibodies were used at concentrations of 10–20 μg/ml. Standard single and double-staining procedures for surface molecules were performed as previously described [10
]. The specificity of staining was confirmed using isotype-matched control mAbs, rabbit serum, or rabbit Ig at identical concentrations (Table ). The FACS analysis in RA-SFB and OA-SFB was not always performed with the complete set of antibodies due to the initial establishment of the isolation procedure.
Trypsinized cells were washed twice with serum-free medium and fixed for 10 min at 4°C in 4% paraformalde-hyde (Fluka, Deisenhofen, Germany) for detection of antigens in cytoplasm and/or nucleus (eg prolyl-4-hydroxylase; Table ). After washing twice in PBS/2% FCS, the pellet was resuspended in permeabilization buffer (PBS/1% FCS, 0.01% NaN3, and 0.5% saponine; Serva, Heidelberg, Germany) and incubated for 10 min at room temperature. Unlabeled primary mAbs were added at saturating concentrations and detected with a secondary phycoerythrine-labeled goat anti-mouse antibody (Dako), both for 45 min at 4°C in permeabilization buffer. Polyclonal antibodies were incubated with a secondary mouse anti-rabbit antibody (Dako) before using the phycoerythrine-labeled goat anti-mouse antibody. After washing twice in permeabilization buffer, the cells were incubated for 10 min in PBS/5% FCS and 0.1% NaN3 without saponine. In the special case of the CD68 epitope recognized by the mAb PG-M1, present on the cell surface and in the cytoplasm, FACS analysis was performed in both non-permeabilized and permeabilized cells. For double-staining analyses, fluoresceine isothiocyanate (FITC)-labeled Anti-Thy-1 mAb AS02 (20 μg/ml in PBS/2% FCS) or a matched concentration of the FITC-labeled IgG1 isotype control mAb were added for 30 min at 4°C.
Analyses were performed on a FACSCAN® using the software Cell Quest (Becton Dickinson, San Jose, CA, USA). Forward and side scatter gates were set to include all viable cells. In single-staining experiments, a gate was set to exclude 99% of the cells stained with control Ig. The gates for control Ig in double-staining experiments were placed to limit, to less than 1%, not only the individual percentages in the upper left, upper right, and lower right quadrants, but also the sum of the percentages in the upper left and upper right quadrant, or in the upper right and lower right quadrant.
The proliferation rates of normal skin-FB as well as OA-SFB and RA-SFB, derived/negatively isolated from primary culture, from the fourth passage of the isolated cells (in the case of OA-SFB and RA-SFB), or from conventional fourth passage, were assessed by seeding the cells at a density of 1.7 × 103/well in 96-well plates and subsequent culture in DMEM/10% FCS with the aforementioned additives for 24 h at 37°C and 5% CO2. The cells were then starved in DMEM/1% FCS for 72 h and subsequently stimulated for 18 h by addition of 50, 100, or 150 U/ml IL-1β (Genzyme, Rüsselsheim, Germany) or 2.5, 5, or 10 U/ml PDGF-BB (R&D Systems, Wiesbaden, Germany). A total of 1 μCi [3H]-thymidine (Amersham, Braunschweig, Germany) was added to each well, the cells cultured for an additional 18 h, harvested, and the incorporated radioactivity determined in a β-counter (Canberra-Packard, Frankfurt/Main, Germany).
The data were first subjected to the multi-group Kruskal-Wallis test because of multiple comparisons. Only those parameters showing significant differences (P ≤ 0.05) underwent further analysis. The non-parametric Mann–Whitney U test was then applied for analysis of the phenotypic and functional features. The Spearman Rank correlation test was used to analyze correlations among phenotypic features of SFB and between these features and the clinical status/treatment of individual patients. Differences were considered statistically significant in all cases for P ≤ 0.05. Analyses were performed using the SPSS 9.0™ program (SPSS Inc., Chicago, IL, USA).