Nerve growth factor and receptor expression in rheumatoid arthritis and spondyloarthritis

Synovial fluid (SF) samples were collected from nine patients with rheumatoid arthritis (RA) fulfilling the American College of Rheumatology classification criteria and from 16 patients with SpA fulfilling the European Spondyloarthropathy Study Group classification criteria [8, 9]. Also, seven patients with osteoarthritis (OA) served as non-inflammatory controls. All patients had active synovitis of the knee. Patient characteristics and disease activity parameters are listed in Table 1. OA patients were graded according to the Kellgren and Lawrence classification [10]. The majority of patients had OA of the knee grade 2 (mean 2.3 ± standard deviation (SD) 1.0).

Synovial tissue (ST) biopsies were obtained from another panel of patients including 10 SpA and 10 RA patients with early and active arthritis of the knee (disease duration <6 months, only NSAID treatment, but no steroids, no disease-modifying anti-rheumatic drugs, or biologics).

All subjects gave their written informed consent before inclusion in the study, which was approved by the local ethics committee of the involved institutions.

SF was obtained by a conventional puncture of an actively inflamed knee joint. Samples were centrifuged for 15 minutes at 1000 g. Supernatants were removed, whole SF cell pellets were resuspended in RNAlater solution (Ambion, Austin, TX, USA), and frozen at -80°C until use. For intracellular fluorescence-activated cell sorting (FACS) analysis, mononuclear cells derived from SF samples (SFMC) were obtained by standard Ficoll histopaque procedure and conserved in FCS and 10% dimethyl sulfoxide. They were kept in liquid nitrogen until use. ST biopsies were obtained by a standard procedure as previously described [11]. The Ficoll procedure was also used in order to obtain peripheral blood mononuclear cells (PBMC) from four healthy individuals used as controls for PCR. Total RNA was extracted from SF cell pellets, ST biopsies, and PBMC using TRizol reagent (Invitrogen, Karlsruhe, Germany) and precipitation with isopropyl alcohol. All procedures were previously described in detail [12‐14].

Generation of cDNA by reverse transcription and utilization of TaqMan^® assay followed the protocol of the manufacturer (Applied Biosystems, Darmstadt, Germany). Briefly, 2 μg of total RNA were reverse transcribed using the MultiScribe^® (Applied Biosystems, Darmstadt, Germany) reverse transcriptase. Duplicate PCR reactions were performed using the TaqMan^® universal PCR master mix on ABI Prism^® 7000 sequence detection system (Applied Biosystems, Darmstadt, Germany). After denaturation at 50°C for two minutes and 95°C for 10 minutes, 45 PCR reaction cycles were performed each at 95°C for 9 seconds and 60°C for one minute. The following mRNA transcripts and assays were used (Applied Biosystems, Warrington, UK): assays-on-demand for NGF-beta (assay no. Hs00171458), [GenBank:X52599], BDNF (assay no. Hs00156058), [GenBank:M61176], and NT-3 (assay no. Hs00267375), [GenBank:M37763]; assay-by-design for NT-4 (cat. no. 4332078; [GenBank:M86528]); assays-on demand for the high-affinity receptors trkA (assay no. Hs00176787), [GenBank:X03541]; trkB (assay no. Hs00178811), [GenBank:U12140]; trkC (assay no. Hs00176797), [GenBank:U05012], and for the low-affinity receptor NGFRp75 (assay no. Hs00609976), [GenBank:M14764].

All PCR data were normalized to the expression of the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) housekeeping gene used as an internal control; SF data were also normalized with a set of four healthy PBMC used as an external control. The ST data were compared with each other. PCR data were obtained as cycle threshold (Ct) values. The Ct value is defined as the cycle with a fluorescence intensity significantly above the background fluorescence but within the exponential phase of the amplification [15, 16]. The mean of two Ct measurements of one sample was calculated for both the given target gene and the G3PDH gene. Delta Ct was determined as the mean of the duplicate Ct values for the target gene subtracted by the mean of the duplicate Ct values for the G3PDH gene. For each target gene, delta Ct measurements were performed separately for both the SF samples and the healthy PBMC. The delta delta Ct method represents the difference between the two cell types for a given target gene [17]. Expression levels are given as fold of expression and were compared between SpA, RA, and OA groups using the non-parametric Mann Whitney U test as appropriate.

SF mononuclear cells (SFMC) of three RA and three SpA patients from the panel described in Table 1, as well as PBMC from two healthy controls were prepared by density gradient centrifugation using biocoll (1.077 g/ml; Biochrom, Berlin, Germany). SFMC were harvested from the interphase and washed twice at 1000 g and 300 g, respectively. The cells were finally resuspended in PBS/BSA and stained for surface markers (CD3 PerCP, clone SK7; CD14 FITC, Leu-M3; CD56 APC, NCAM 16.2; all from Becton Dickinson, Heidelberg, Germany) for 20 minutes. After two washes with PBS/BSA (300 g/three minutes) the cells were fixed for 10 minutes at room temperature in PBS containing 4% paraformaldehyde. Cells were then washed once and resuspended in saponin buffer (PBS supplemented with 5 mM HEPES and 0.1% saponin) in order to perforate the cell membranes. Subsequently, aliquots were stained with monoclonal antibodies (mAb) against NGF (biotinylated anti-human β-NGF, catalogue number BAF256; R&D systems, Minneapolis, MN, USA). Unspecific binding of the mAb via Fc-receptors was discriminated by adding human IgG solution (Octagam; Octapharma, Langenfeld, Germany). After 30 minutes of incubation at 4°C, cells were washed three times with PBS/BSA and resuspended again in saponin buffer. PE-labeled streptavidin (SA-PE, Becton Dickinson, Heidelberg, Germany) for secondary staining of biotinylated NGF was added and cells were incubated for 30 minutes at 4°C. After three washes (300 g/three minutes) with PBS/BSA, cells were ready for FACS analysis.

Phenotypic analyses were performed as multicolor immunofluorescences. At least 10⁴ cells per appropriate lymphocyte or monocyte gate, respectively, according to forward scatter vs side scatter properties were analyzed using a dual-laser cytometer with Cell Quest Pro (FACSCalibur, Becton Dickinson, Heidelberg, Germany) and Summit 4.3 (Beckman Coulter, Krefeld, Germany) software.

Fibroblast-like synoviocytes (FLS) were isolated from synovial biopsies of one RA and one SpA patient as described previously [18]. After three passages, cells were resuspended at 10,000 cells/ml Dulbecco's modified eagle's medium (DMEM) with 10% FCS and plated at 2 ml/well in 24 well plates. Cells were grown for three days until confluence, then the normal medium (DMEM + 10% FCS) was replaced by starvation medium (DMEM + 1% FCS). After 24 hours, cells were stimulated with either medium alone (DMEM 1% FCS); TNF-alpha at 10 ng/ml; or IL-1 beta at 10 ng/ml, or lipopolysaccharide at 1 ug/ml. After 72 hours of culture with stimulation, supernatants were collected and used undiluted for measuring NGF by ELISA (R&D Systems, Minneapolis, MN, USA; lowest detection level: 30 pg/ml).