In this investigation, we included 10 patients with OA and seven patients with RA. Diagnosis of RA was based on the established criteria according to the American College of Rheumatology (formerly, the American Rheumatism Association) [26
]. The characteristics of patients are given in Table . Erythrocyte sedimentation rate and serum levels of C-reactive protein were measured by using standard techniques.
The study was approved by the Ethics Committee of the University of Regensburg. Patients were informed about the purpose of the study and gave written consent.
Synovial tissue preparation, isolation, and culture of primary mixed synovial cells
OA and RA patients underwent elective knee-joint replacement surgery. Synovial tissue samples were obtained immediately after opening the knee-joint capsule, preparation of which was described [24
]. In brief, a piece of synovial tissue of ≤9 cm2
was dissected. A larger piece of the synovial tissue was used to isolate mixed synovial cells (for culture experiments, see later). Approximately eight pieces of the same synovial area were used for immunohistochemistry and immunofluorescence, which were fixed for 12 to 24 hours in phosphate-buffered saline (PBS) containing 4% formaldehyde and then incubated in PBS with 20% sucrose for 12 to 24 hours. Thereafter, they were placed in protective freezing medium and quick-frozen (Tissue Tek; Sakura Finetek, Zoeterwoude, The Netherlands). All tissue samples were stored at 80°C.
For culture experiments, mixed synovial cells were isolated during the morning hours by enzymatic digestion for 1 to 2 hours at 37°C by using Liberase (Roche Applied Science, Mannheim, Germany). At approximately 2 to 4 p.m., the synovial cells were resuspended in RPMI 1640 medium (Sigma, Taufkirchen, Germany), supplemented with 1% penicillin/streptomycin (Life Technologies, Inc., Paisley, U.K.) and 0.1% amphotericin B (Bristol-Myers Squibb, Munich, Germany). In total, 2.5 × 105 isolated synovial cells of OA or RA patients were incubated for 24 hours in the presence of 200 μM L-ascorbic acid, together with norepinephrine, TNF, cortisol, or norepinephrine plus cortisol in indicated concentrations (all substances from Sigma, Steinheim, Germany).
The percentage of different types of synovial cells was tested by specific antibodies against prolyl 4 hydroxylase (for the synoviocyte type B, fibroblasts; Calbiochem, Bad Soden, Germany), CD163 (synoviocyte type A, macrophages; Dako, Hamburg, Germany), CD3 (T cells; Dako), CD19 (B lymphocytes; Dako) neutrophils (elastase; Fitzgerald Industries Int. Inc., Acton, MA, USA), and mast cells (tryptase; Abcam, Cambridge, UK). In preliminary experiments with primary early-culture mixed synoviocytes, we detected that ~37% were positive for prolyl 4 hydroxylase, 26% for CD163, 12% for CD3, 5% for CD19, 10% for elastase, and <1% for tryptase.
Immunohistochemistry and double immunofluorescence
Approximately ten 5-μm sections were cut from the frozen tissue blocks. For immunohistochemistry with alkaline phosphatase as the enzyme system, sections were blocked with 20% acetic acid for 20 minutes at 4°C. Sections were further blocked with 10% bovine serum albumin, 10% fetal calf serum, and 10% chicken serum for 45 minutes (all from Sigma). Then, sections were incubated with either monoclonal mouse anti-human antibodies against HNP1-3 (BMA Biomedicals, Augst, Switzerland, no. T 1034; dilution, 1:1,000; these antibodies recognize HNP-1 to -3) or polyclonal rabbit anti-human antibodies against HBD-2 (Biologo, Kronshagen, Germany, no. DEF002; dilution, 1:100). Both primary antibodies were incubated overnight at 4°C. After intensive washing with phosphate-buffered saline, sections were incubated with secondary antibodies for 1 hour at room temperature (for HNP1-3, goat anti-mouse coupled to alkaline phosphatase; Dako, no. D0486; dilution, 1:100; for HBD-2, goat anti-rabbit coupled to alkaline phosphatase, Dako, no. D0487; dilution, 1:100). The sections were developed by using BCIP/NBT substrate (Dako, K0598). We controlled specific staining by using irrelevant primary antibodies or serum or by omitting the primary antibody.
Double immunofluorescence was carried out with fixed frozen tissue and a similar blocking procedure to that mentioned earlier. For HNP1-3 and HBD-2, the primary antibodies of BMA Biomedicals and Biologo were used in the same dilution as given earlier. For double staining, respective antibodies were used against activated macrophages (CD163, Dako), T lymphocytes (CD3, Dako), fibroblasts (prolyl-4 hydroxylase, Dako), B lymphocytes (CD19, Dako), mast cells (tryptase; Abcam, Cambridge, UK), and neutrophils (elastase; Fitzgerald Industries International; and Lab Vision NeoMarkers via Thermo Scientific, Dreieich, Germany). Fluorescent staining of positive cells was achieved by incubating the sections with respective secondary Alexa Fluor 488 and 546 antibodies or F(ab')2 fragments (Molecular Probes via Invitrogen, Karlsruhe, Germany). Nuclei were stained with Vectashield mounting medium with DAPI (Vector Laboratories via Biozol, Eching, Germany). We controlled specific staining by using irrelevant primary antibodies or serum or by omitting the primary antibody.
Determination of HNP1-3 and HBD-2 in supernatants of synovial cells
HNP1-3 were measured with commercially available ELISA (HyCult Biotechnology, Uden, The Netherlands; this ELISA recognizes HNP-1 to -3). The detection limit of this assay was 20 pg/ml. Intra- and interassay coefficient of variation were <10%.
With respect to HBD-2, a new ELISA was established by using two commercially available antibodies (capture: Biologo, no. DEF002, polyclonal rabbit anti-human antibodies; dilution, 1:1,000; detection, R&D Systems, Wiesbaden, Germany; AF2758, polyclonal goat anti-human antibodies; dilution, 1:200). After overnight coating with the capture antibody at 4°C, extensive washing, and blocking with 10% fetal calf serum, 100 μl of standard (recombinant HBD-2; Dianova, Hamburg, Germany, no. CYT-26732) or 100 μl supernatant of synovial cells was incubated for 2 hours at room temperature. After extensive washing, the detection antibody was added for another hour (at room temperature). After extensive washing, a rabbit anti-goat tertiary antibody was used, coupled to biotin (Dako, Hamburg, Germany; no. E0466). By using streptavidin coupled to horseradish peroxidase and tetramethylbenzidine (TMB) as the substrate, the ELISA was developed. The detection limit of this assay was ~8 pg/ml. Intra- and interassay coefficients of variation were <15%.
Superfusion technique of synovial tissue
As described in detail earlier [24
], we used a microsuperfusion-chamber apparatus to superfuse pieces of synovial tissue with culture medium. This technique allows the determination of spontaneous defensin release directly from fresh synovial tissue. The superfusion chambers had a volume of ~80 μl. Super-fusion was performed for 6 hours at a tempera-ture of 37°C and a flow rate of 66 μl/min. Synovial tissue pieces had a standard size of 5 μm in diameter with a precision biopsy punch (Stiefel, Offenbach, Germany). Every hour, superfusate was collected to measure HNP1-3 and HBD-2, as described earlier.
Presentation of the data and statistical analysis
All data are given as mean ± SEM. Box plots give the 10th, 75th, 50th (median), 25th, and 10th percentile. Group medians were compared by using the nonparametric Mann-Whitney test (SPSS/PC, Advanced Statistics, V15.0, SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was the significance level.