Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

Synovial tissues were collected during routine orthopaedic surgery from 10 consecutive patients with end-stage OA and 10 consecutive patients with refractory destructive RA who underwent joint replacement. The latter patient population fulfilled the American College of Rheumatology criteria for RA [22]. The mean age of patients with RA was 62 years (eight females and two males). Mean laboratory parameters were erythrocyte sedimentation rate of 41 mm/hour and leucocyte count of 8.95 × 10³ per microlitre, and 90% of patients were rheumatoid factor-positive. All patients gave informed consent, and the study protocol was approved by the local ethics committees.

RPMI 1640 medium (supplemented with 10% [vol/vol] heat-inactivated foetal calf serum, penicillin [100 units per millilitre], streptomycin [0.1 mg/ml], and glutamine [0.3 mg/ml]) (all from Gibco, now part of Invitrogen Corporation, Carlsbad, CA, USA) was used as standard medium. HT1080 FAP-transfected (HT1080 FAP) and mock-transfected (HT1080 par) cells were previously described [23]. The following antibodies were obtained from the following sources: Murine F19 (mouse anti-human FAP monoclonal antibody) was obtained from the Ludwig Institute for Cancer Research (New York, NY, USA). Anti-human CD90 (Thy-1; clone AS02) and biotin-SP-conjugated rabbit anti-mouse immunoglobulin G (IgG) were purchased from Dianova GmbH (Hamburg, Germany). Mouse anti-human CD44v3 (clone VFF-327) and CD44v7/8 (clone VFF-17) were purchased from Serotec Ltd. (Oxford, UK), and mouse anti-human smooth muscle actin (SMA) (clone 1A4) was obtained from Dako Denmark A/S (Glostrup, Denmark). Goat anti-human MMP-1 (C-18) and MMP-13 (D-17) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Biotinylated rabbit anti-goat IgG was purchased from Vector Laboratories (Burlingame, CA, USA).

FAP- or mock-transfected HT1080 cells, tumour samples from patients with breast cancer, or a part of collected synovial tissue was homogenised immediately after surgery by using a tissue tearer, and total RNA was extracted (RNeasy; Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions. Total amount of RNA was quantified by spectrophotometry, and 500 ng was used for first-strand cDNA synthesis using RevertAid™ H Kit (Fermentas GmbH, St. Leon-Rot, Germany). The following primers were used for amplification of specific cDNA fragments as described previously [24]: The FAP polymerase chain reaction (PCR) product was generated with 5'-GTTATTGCCTATTCCTATTATG-3' and 5'-GTCCATCATGAAGGGTGGAAA-3'. The MMP-1 PCR product was generated with 5'-CTGAAGGTGATGAAGCAGCC-3' and 5'-AGTCCAAGAGAATGGCCGAG-3'. The MMP-13 PCR product was generated with 5'-CTATGGTCCAGGAGATGAAG-3' and 5'-AGAGTCTTGCCTGTATCCTC-3'. The GADPH (glyceraldehyde-3-phosphate dehydrogenase) PCR product was generated with 5'-GTGAAGGTCGGAGTCAACGGATTT-3' and 5'-CTCCTTGGAGGCCATGTGGGCCAT-3'. cDNA was amplified with 1.25 units of Taq DNA polymerase with 2.5 ml of 10× standard taq buffer (Thermopol; New England Biolabs, Ipswich, MA, USA) in a final volume of 25 μl containing 0.5 μl of nucleosidtriphosphate (10 mM), 0.5 μl of each primer (10 μM), and Aqua dest. PCR amplification was carried out on a PTC-200 thermocycler (MJ Research, now part of Bio-Rad, Hercules, CA, USA) with the following profile: 95°C for 5 minutes, annealing for 60 seconds, and extension at 72°C for 1 minute. The number of cycles and the annealing temperature depended on the primers used: (a) FAP (55°C, 30 cycles), (b) MMP-1, (c) MMP-13 (62°C, 40 cycles), and (d) GADPH (62°C, 25 cycles). Aliquots (20 μl) of each PCR product were separated on a 1.5% agarose gel and visualised by ethidium bromide staining (Sigma-Aldrich, St. Louis, MO, USA). Cell lines or breast cancer samples served as controls.

Measurement was performed on LightCycler detection systems (Roche, Heidelberg, Germany) using the relative quantification method with external standard (LightCycler technical note no. LC 10/update 2003; Roche). The endogenous control gene hypoxanthine phosphoribosyltransferase 1 (HPRT1; NM_000194) and the FAP gene (NM_004460) were measured by quantitative reverse transcription-PCR using the GenGlobe™ technology from Qiagen GmbH and an HPRT quantification kit (Roche). Design of specific HPRT and human FAP primers was executed by Qiagen GmbH (QuantiTect Primer Assay, QT00074963) and Roche. Assay conditions for the real-time PCR were adjusted using an optimised PCR protocol with the following profile: initial activating of Taq-polymerase at 95°C for 15 minutes, followed by 35 cycles with denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 20 seconds. Samples from all patients (10 patients with RA and 10 patients with OA), controls, and standards were run in triplicate. The amount of the housekeeping gene HRPT was used to correlate FAP expression employing LightCycler analysis software.

Remaining portions of collected samples were embedded in Tissue-Tek OCT medium (Miles Diagnostics, Elkhart, IN, USA), snap-frozen in liquid nitrogen, and then stored at -80°C for immunohistochemical analysis. Sequential 5-μm sections were placed on SuperFrost^®Plus microscope slides (Menzel-Gläser, Braunschweig, Germany). Slides were fixed in cold acetone (4°C for 10 minutes), air-dried, rehydrated in phosphate-buffered saline, and then blocked using a Biotin Blocking System (Dako North America, Inc., Carpinteria, CA, USA). Before the primary antibody (anti-human FAP, anti-human Thy-1, or irrelevant isotype-matched IgG, 1:200, incubated for 60 minutes at room temperature; anti-human CD44v3, 1:50; anti-human CD44v7/8, 1:200; anti-human SMA, 1:50; and anti-human MMP-1, anti-human MMP-13, or irrelevant isotype-matched IgG, 1:100, incubated overnight at 4°C) was added, slides were blocked with rabbit serum from the Vectastain^® ABC Kit (Vector Laboratories). The biotinylated secondary antibody (rabbit anti-mouse IgG [1:500] or rabbit anti-goat IgG [2 μg/ml]) and the preformed avidin-biotinylated horseradish peroxidase P-complex (ABC reagent) were used according to the manufacturer's instructions. The antibody-ABC complex was visualised with a 3-amino-9 ethylcarbazole (AEC) (Sigma-Aldrich)-based chromagen, resulting in a pink-brown colouration of antigen-positive cells. Simultaneous staining of FAP and SMA was performed using the DakoCytomation EnVision Doublestain-Kit (code K1395; Dako North America, Inc.) according to the manufacturer's instructions. FAP was stained first with DAB (3,3'-diaminobenzidin) followed by colouration of SMA with fast red. All slides were counterstained for approximately 2 to 5 minutes with Meyer's haematoxylin. Final slide adjustment was performed using Adobe Photoshop Elements (Adobe Systems GmbH, Unterschleissheim, Munich, Germany).

Comparison of FAP gene expression measured by real-time PCR between synovial tissue samples taken from patients with end-stage OA or severe RA was performed with Mann-Whitney test (U test). P values < 0.01 were considered statistically significant.

Synovial tissue was collected from 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA. mRNA extraction and cDNA synthesis were performed immediately after surgery. Quantitative real-time PCR analysis was established to compare the levels of FAP gene expression in synovial samples of both entities. FAP gene expression was significantly higher in RA synovial samples (p < 0.01; Figure 1a). High specificity of PCR is demonstrated by agarose gel electrophoresis and melting curve analysis (data not shown). Furthermore, all samples were analysed for mRNA transcripts coding for fragments of FAP (741 bp), MMP-1 (428 bp), and MMP-13 (390 bp). Amplification of a GADPH fragment with the expected size of 995 bp served as control. Positive PCR controls were obtained from cDNA derived from FAP-transfected HT1080 cells (FAP) or breast cancer tumour samples (MMP-1 and MMP-13). Amplification of cDNA derived from mock-transfected HT1080 cells was negative for specific fragments. Analysis of synovial samples from patients with severe RA (Figure 1b, column A) or end-stage OA (Figure 1b, column B) demonstrates ubiquitary expression of MMP-1, MMP-13, and FAP. However, expression was more pronounced in refractory RA samples. Differences in transcription of FAP gene were confirmed by immunohistochemistry. Corresponding to the significant difference in gene expression analysis, FAP positivity is much more pronounced in rheumatoid synovial samples than in the osteoarthritic counterparts (Figure 2).

The histomorphological characterisation of the synovial lining layer by FAP expression is accompanied by the accumulation of other activation markers in this area. The phenotypic markers analysed in this context were MMP-1 and MMP-13, together with the v3 and v7/8 splice variants of CD44, which are instrumental in ECM alteration in malignancies and already known to be expressed in the synovial membranes of diseased joints (Figures 3 and 4). The expression signature characterises the area of FAP-expressing cells as the centre of high inflammatory activity in the rheumatoid synovium (Figures 4 and 5). In contrast to the synovial lining layer of rheumatoid joints, osteoarthritic joints show limited staining for MMPs and CD44 variants, with only minor expression of MMP-13 and CD44v7/8 (Figures 3 and 5).

RA and OA synovial fibroblasts express CD90 (Thy-1) on their surface. However, there is a distinct fibroblast population in the lining layer of the synovium from both patient groups, completely lacking Thy-1 surface expression. This distinct synovial cell population is clearly characterised by cell surface expression of FAP (Figure 6) which is paralleled by cell surface expression of activation markers (Figure 5). Fibroblasts with myofibroblastic phenotype are a known source of proteolytic enzymes that are involved in ECM deposition. A reliable marker for identifying this subpopulation is anti-SMA. In addition to staining of perivascular smooth muscle cells, immunohistochemistry revealed the expression of SMA by FAP-positive FLSs in the intimal lining layer (Figure 6), and the simultaneous expression of both antigens could be demonstrated by double-staining technique (Figure 7). As a result, expression pattern and staining intensity of SMA are considerably different between the rheumatoid and the osteoarthritic synovium. Furthermore, either the expression density of activation markers or their staining intensity differs between the samples of osteoarthritic synovial tissue from different patients. With regard to the degree of inflammation, immunohistochemical analysis of synovial samples from patients with refractory RA showed homogenous expression pattern and staining intensity.