Forty‐four patients with RA who fulfilled the revised American College of Rheumatology (ACR) criteria23
were consecutively recruited for the study when undergoing joint surgery. Synovial tissue from five patients with osteoarthritis was used as a control. The study protocol was approved by the Frankfurt University Hospital ethics committee and all patients gave their written informed consent. The characteristics of the patients with RA are summarised in table 1.
Table 1Characteristics of patients with RA
Peripheral blood and synovial fluid mononuclear cell preparation
Specimens for pairwise analyses were simultaneously obtained by vein puncture needle aspiration of the joint immediately before arthrotomy. Fluids were collected in NH4‐heparin containing S‐Monovettes (Sarstedt, Germany). Peripheral blood mononuclear cells and synovial fluid mononuclear cells were isolated by Ficoll Hypaque (Biochrom, Germany) density gradient centrifugation.
Preparation of synovial tissue for functional assays was done rapidly by injecting 1 mg/ml collagenase (Sigma‐Aldrich, Germany) into synovial tissue samples and incubating for 20 min at ambient temperature. The synovial tissue was subsequently minced and incubated for an additional 50 min at 37°C in collagenase 1%. The cell suspension was strained by 70 μm nylon filters (Falcon, USA), washed twice in phosphate‐buffered saline (PBS) and cultured in HAM F 10 medium (Invitrogen, Germany) containing 5% fetal calf serum and 1% penicillin/streptomycin at 37°C in 5% CO2 atmosphere over night.
Magnetic bead activated cell sorting
Synovial tissue cell cultures were CD3‐depleted using anti‐CD3 monoclonal antibody‐coated magnetic beads (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's instructions. A rosette kit (StemCell Technologies, USA) was applied for isolation of CD4+ peripheral blood T cells (fig 1) and CD25+ cells were enriched with an automated magnetic bead activated cell sorting system (autoMACS, Miltenyi Biotec, Germany). T cells were stained with 10 μl anti‐CD4‐PE/CY5, anti‐CD3‐ECD, anti‐CD45‐FITC (all BD Pharmingen, USA) or anti‐CD25‐PE (Miltenyi Biotec, Germany), incubated for 10 min at room temperature and measured by flow cytometry (Beckman Coulter, USA).
Figure 1Regulatory effect of autologous peripheral blood (PB) T cells on synovial tissue cells (STC) in patients with rheumatoid arthritis (RA). (A) Representative interferon γ (IFNγ) ELISPOT experiment, done in triplicate, with (more ...)
Synovial tissue cultures and peripheral blood mononuclear cell fractions were seeded in equal densities of 104 cells per well in sterile polyvinylidene fluoride microtitre plates (Millipore, USA) pre‐coated with anti‐IFNγ or anti‐IL‐10 antibody (Mabtech AB, Sweden) and cultured for 36 h at 37°C in a 5% CO2 atmosphere. Biotin‐labelled secondary monoclonal antibodies were added after removing the cell layer and thorough washing. Plates were dried for 2 h at ambient temperature and subsequently stained for 15 min with streptavidin alkaline phosphatase complexes and 5‐bromine‐4‐chlor‐3‐indoxylphos‐phate/nitroblue‐tetrazoliumchloride solution (BCIP/NBT, Mabtech, Sweden). Cytokine spots were quantified (A•EL•VIS GmbH, Hannover, Germany) and the mean spot number per well from duplicate or triplicate experiments was calculated. Net counts were established after background subtraction.
Acknowledging the relevance of transcription factors for gene expression and differentiation of lymphocytes, T‐bet and FoxP3 transcripts were quantified. FoxP3, the most specific Treg marker,25
is present in naïve natural and memory Tregs.6,7
T‐bet is mainly expressed in differentiated Th1 cells and closely linked to IFNγ expression.26
In brief, synovial tissue was immediately stored in RNAlater (QIAGEN, Hilden, Germany), homogenised after removal from RNAlater, peripheral blood mononuclear cells were disrupted and total RNA was isolated with RNeasy Mini Kit (QIAGEN) or TRIzol Reagent (Invitrogen, USA). First strand cDNA synthesis was performed using 1 μg total RNA by Thermoscript RT (Invitrogen, USA). Quantitative PCR was performed by pre‐designed TaqMan gene expression assays (Applied Biosystems, California, USA) for human T‐bet (Hs00203436), FoxP3 (Hs00203958), CD3ε transcripts (Hs00167894), a constitutively expressed gene in CD3+ T cells, and eukaryotic 18S rRNA (4333760T). Probes were labelled with FAM/MGB fluorescent dye and amplified with Absolute QPCR ROX Mix (ABgene, Epsorn, UK) in an ABI Prism 7700 sequence detector (Applied Biosystems, California, USA) in 25 μl reaction volume. The expression was defined by calculating the mean cycle number of triplicate experiments.
Immunohistochemistry for FoxP3 in RA synovial tissue
Specimens were immediately embedded in OCT compound (TissueTek TT 4583; Sakura Finetech, Torrance, California, USA) and snap frozen in liquid nitrogen. Sections 7 μm thick were prepared, fixed in acetone, washed in ddH2O and subsequently in PBS. Endogenous peroxidase activity was blocked using 0.1% H2O2. Slides were incubated for 30 min in blocking solution (2% horse serum in PBS) and incubated overnight at 4°C with 10 μg/ml mouse monoclonal anti‐FoxP3 antibody (AbCam, Cambridge, UK) or an isotype‐matched mouse IgG1 antibody (DakoCytomation, Glostrup, Denmark). Sections were incubated for 30 min with biotinylated goat anti‐mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA) secondary antibody in PBS. Immune complexes were stained with horseradish peroxidase‐conjugated streptavidin complex (Vectastain Elite ABC kit; Vector, Burlingame, California, USA) and aminoethylcarbazole chromogen substrate (DakoCytomation, Glostrup, Denmark). Nuclei were counterstained with haematoxylin.
The results are presented as mean (SE) unless otherwise stated. Group comparisons were calculated by the Mann‐Whitney U test for untailed groups and by the Wilcoxon signed rank test for tailed groups. The significance of correlations was estimated by the Pearson test. Statistical analyses were done with Graph Pad Prism 4.0 Software.