Induction of joint inflammation
In 17 10-week-old female Lewis rats (Charles River, Sulzfeld, Germany), an inflammation was induced in the right knee joint. In the first step the rats received a subcutaneous injection of 500 μg antigen (methylated BSA; Sigma, Deisenhofen, Germany) in 500 μl saline emulsified with 500 μl complete Freund's adjuvant (Sigma; supplemented to 2 mg/ml heat-killed Mycobacterium tuberculosis strain H37RA, Difco, Detroit, MI, USA). In addition, an intraperitoneal injection of 2 × 109 heat-inactivated Bordetella pertussis (Chiron Behring, Marburg, Germany) was performed on the same day. The same immunisation procedure was repeated 7 days later. After a further 14 days, a sterile solution of antigen (methylated BSA), 500 μg in 50 μl saline, was injected into the right knee joint cavity (day 0). Either 3 days (acute AIA) or 20 to 28 days (chronic AIA) after induction of inflammation in the knee joint, the rats were killed by cervical dislocation during ether anaesthesia. In total, 17 untreated rats of the same age and sex were used as normal control animals. All rats were used for the preparation of FLS cells. All procedures complied with the regulations of the Thuringian Commission for Animal Protection.
Preparation of fibroblast-like synovial cells
Explant cultures of FLS cells were prepared from the knee joints of normal rats or from rats 3 days (acute phase) or 20 to 28 days (chronic phase of inflammation) after induction of AIA. The patella and the menisci of the joints with adjacent synovial tissue were separated and cultured in 24-well plates in DMEM (Gibco, BRL, Eggenstein-Leopoldshafen, Germany) containing 20% fetal calf serum (FCS, [Gibco]), 0.1 mg/ml streptomycin (Grünenthal, Aachen, Germany), 100 U/ml penicillin (Jenapharm, Jena, Germany), 2 mmol/l glutamine (Gibco), 10 mM Hepes (Gibco), and 1 mmol/l sodium pyruvate (Gibco) for 7 days at 37°C in a humidified incubator gassed with 5% CO2 in air. During this time, out-growing FLS cells emerged from the tissue. In the first 7 days the medium was replaced daily. After 7 days the residual tissue was removed and 2 days later the cells were transferred to new plates. For this purpose the cells were washed with PBS and incubated for 2 to 4 minutes in PBS containing 0.25% trypsin and 0.02% EDTA (Gibco). Thereafter, the cells were collected, washed with DMEM containing 20% FCS and disseminated. After another 3 to 6 days the cells were transferred into new plates. For the co-culture, cells were used after the third to fifth passage. FLS cells were slowly cooled down with isopropanol to -70°C in DMEM containing 10% dimethylsulfoxide and stored at -192°C over liquid nitrogen until co-culturing.
Primary culture of dorsal root ganglion neurones
Normal male Wistar rats, 60 days old, were sacrificed with a lethal dose of ether. DRGs from all segments of the spinal cord were dissected. Ganglia were incubated at 37°C with 215 U/ml collagenase type II (Paesel and Lorei, Hanau, Germany) dissolved in Ham's F12 medium (Gibco) for 100 minutes. After washing with Ca2+- and Mg2+-free PBS, the ganglia were placed in DMEM (Gibco) containing 10,000 U/ml trypsin (Sigma) for 11 minutes at 37°C. The cells were dispersed by gentle agitation and aspiration with a fire polished Pasteur-pipette. The dispersed cells were collected by centrifugation (500 × g, 5 minutes), washed 3 times in DMEM and centrifuged. The cell pellets were suspended in Ham's F-12 medium containing 10% heat-inactivated horse serum (Gibco), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), and 100 ng/ml nerve growth factor (NGF; Paesel and Lorei). On average, 200 to 300 DRG neurones were plated on poly-L-lysine- (200 μg/ml) coated glass cover slips (diameter 13 mm) situated in 35/10 mm dishes and maintained for 1 night at 37°C in a humidified incubator gassed with 3.5% CO2 and air. After this overnight setting period, the co-cultures of DRG neurones and the FLS cells were prepared.
Co-culture of FLS cells and DRG neurones
Two days before co-culturing, the FLS cells (from normal rats or from rats with acute or chronic AIA) were thawed and cultured in DMEM containing 20% FCS in a concentration of 2 × 100 cells/well in 24-well dishes. After 2 days, the FLS cells were incubated with 0.25% trypsin (Gibco) and 0.02% EDTA (Sigma) in DMEM for 2 to 4 minutes at 37°C. Thereafter, the cells were washed 3 times with DMEM (containing 100 ng/ml NGF) and added to the glass cover slips with the cultured DRG neurones (see above) in a concentration of 105/ml. The two cell types were co-cultured for 24 hours in DMEM containing 100 ng/ml NGF maintained at 37°C in a humidified incubator gassed with 5% CO2 in air. As a control, either only DRG neurones or only FLS cells were cultured and handled in the same way as the co-cultures. After culturing for 24 hours, all cells on the glass cover slips were fixed and used for immunocytochemical labelling of the NK1, the BK 2 (B2) and the TRPV1 receptors.
In addition, in some experiments, DRG neurones were cultured in medium containing only the supernatant of FLS cells. These FLS cells were isolated from either normal knee joints or knee joints at the acute (day 3) or chonic state (20 to 28 days) of AIA and then cultured for 2 days. The supernatants of these FLS cells were added to the DRG neurones, which were kept for 24 hours at 37°C in a humidified incubator gassed with 5% CO2 in air, and, in addition, 100 ng/ml NGF were administered.
Furthermore, co-cultures of DRG neurones and FLS cells from normal or acutely and chronically inflamed knee joints were made in a medium containing either the cyclooxygenase (COX) inhibitor indomethacin (1 μmol/l; Calbiochem, Bad Soden/Ts, Germany), an antibody against IL-6 (1 μmol/l; BioTrend, Köln, Germany) or IgG from normal rat (1 μmol/l; BioTrend). These co-cultures were kept at 37°C in a humidified incubator gassed with 5% CO2 in air for 24 hours in DMEM containing 100 ng/ml NGF.
Detection of bradykinin 2 receptors
Because a reliable B2 receptor antibody was not available, we used BK-gold conjugates for labelling of B2 receptors. The BK-gold conjugates were prepared as described earlier . In brief, 1 μmol BK (Bachem, Heidelberg, Germany) was dissolved in 500 μl HEPES (20 mmol/l, pH 7.5). This solution was added to 6 nmol sulfo-N-hydroxy-succinimido Nanogold reagent (BioTrend), dissolved in 500 μl ddH2O, and incubated for 1 hour at room temperature. To separate BK-gold conjugates from unbound BK, a membrane centrifugation (Amicon microcon-10 system) was used. The BK-gold conjugate was dissolved in PBS containing 0.1% BSA (Sigma), 0.2 mol/l sucrose (Sigma), 4 μg/ml leupeptin (Sigma) and 10 mmol/l sodium azide (Sigma). This solution was aliquoted and stored at -20°C for a maximum of three months.
The cells were fixed with 2% paraformaldehyde in 0.1 mol phosphate buffer (pH 7.2) for 30 minutes. After washing with PBS (20 mmol/l, pH 7.4), the cells were pre-treated with 50 mmol/l glycine in PBS and, thereafter, with 5% BSA and 0.1% gelatine in PBS for 30 minutes. Then the cells were washed with 0.1% acetylated BSA (BSA-C; BioTrend) and incubated overnight with 0.3 nmol/ml BK-gold in PBS containing 0.1% BSA-C, bacitracin (40 μg/ml; Sigma), leupeptin (4 μg/ml; Sigma) and chymostatin (2 μg/ml; Sigma) at 4°C in a moist chamber. Following washing with PBS plus 0.1% BSA-C and, thereafter, with PBS to remove unbound BK-gold, cells were postfixed with 2% glutaraldehyde in PBS for 10 minutes. After extensive washing with PBS and ddH2O, the gold particles were intensified with silver enhancer (R-Gent, pH 5.5; BioTrend) for 15 minutes at 22°C. The reaction was stopped by washing in ddH2O. To examine whether the binding was related to B2 receptors, 3 nmol/ml BK-gold was incubated in parallel control dishes in the presence of 1 μmol/ml D-Arg [Hyp3-Thi5,8-D-Phe7]-BK (Sigma), a BK analogue that specifically binds to the B2 receptor. This analogue produces a complete displacement of BK-gold.
Immunocytochemical labelling of NK1 and TRPV1 receptors
The cover slips were transferred to 2% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.2) plus 0.3% Triton X-100 (TX-100) for 30 minutes. After washing with PBS plus 0.3% Triton X-100 (PBS TX-100), cells were incubated with 50 mmol/l glycine in PBS TX-100 and, thereafter, with 5% BSA and 0.1% gelatine in PBS TX-100 for 30 minutes. Then the cells were washed with PBS TX-100 and incubated for 30 minutes in PBS TX-100 containing 2% normal goat serum (BioTrend). Thereafter, the cells were washed with PBS TX-100 containing 0.1% BSA-C and incubated overnight with an anti-NK1 antibody diluted 1:100 in PBS TX-100 plus 1% normal goat serum (the antibody was raised in rabbit against amino acids 393 to 407 of the rat NK1 receptor; Sigma) or with an antibody to the TRPV1 receptor (VR11-A, 1:75; Alpha Diagnostics, San Antonio, USA) at 4°C in a moist chamber. The cover slips were extensively rinsed in PBS TX-100 plus 0.1% BSA-C and, thereafter, in PBS TX-100. After washing, the cells were incubated for 4 hours at 20°C with a gold-labelled (10 nm) anti-rabbit antibody developed in goat (BioTrend), diluted 1:100 in PBS TX-100 plus 1% normal goat serum. After washing with PBS TX-100, PBS and ddH2O, the gold particles were intensified with silver enhancer (R-Gent, pH 5.5) for 20 minutes at 21°C. The reaction was stopped by washing in ddH2O. To test for unspecificity of the detection system, cells were incubated only with the secondary antibody. In these cultures no cells were labelled.
Double-labelling with an antibody against neurone-specific enolase
To identify the neurones in the cultures, double-staining with an anti-neurone-specific enolase (NSE) antibody was used in all experiments in which B2, NK1, and TRPV1 receptor labelling was performed. After washing with PBS, the cover-slips were incubated overnight at 4°C with the anti-NSE-antibody (Sigma) developed in rabbit (diluted 1:100). Then the cover-slips were incubated with a goat anti-rabbit antibody labelled with Cy3 (diluted 1:200; Jackson ImmunoResearch, Cambridgeshire, UK) for 2 hours. After washing with PBS, all cover slips were embedded in Vectashield (Vector, Burlingame, England). In control experiments in which the primary antibody (anti-NSE) was omitted, no fluorescence signal was detectable.
Prostaglandin E2and IL-6 measurement in the supernatant of the cultures
The supernatants of cultured DRG neurones, cultured FLS cells and co-cultures of DRG neurones and FLS cells (from normal and acutely or chronically inflamed joints) were analysed for the production of prostaglandin E2 (PGE2) and IL-6. The samples were stored at -20°C until analysis. For each substance, four independent cultures were used. All samples were measured twice. The supernatants were analysed with commercial ELISA kits for PGE2 (Cayman Chemicals, Ann Arbor, Michigan, USA) and for rat IL-6 (OptEIA; BD Biosciences, Heidelberg, Germany).
From each cover slip, 100 structurally intact and NSE-labelled neurones were examined with a light microscope (Axioplan 2, Zeiss, Germany) coupled to a CCD video camera and an image analysing system (KS 300, Zeiss, Germany). The mean area and mean grey value were determined for each neuronal soma. To take into account differences in the basal grey values on each coated cover slip, a relative grey value of each neurone was calculated by dividing the mean grey value of the neurone by the grey value of the cover slip background. For an unbiased discrimination of cells with or without positive labelling with the antibodies against NK1 and TRPV1 or BK-gold, neurones were considered positive if their relative grey value was above that of neurones from the control incubations, which were not treated with the antibodies against NK1 or TRPV1 receptors. In experiments with BK-gold labelling, neurones were considered positive if their relative grey value was above that of neurones from the displacement control incubations with a BK analogue that specifically binds to the B2 receptor. This value was 0.16; thus, all neurones with grey density >0.16 were considered as positive for the antibodies or B2 receptor BK-gold binding. Proportions of labelled neurones are expressed as the mean ± standard deviation. For statistical analysis we used χ2-tests taking into account multiple comparisons. Significant differences were acknowledged if p < 0.05.