Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

Abstract

Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.

Figure 1

Expression of fibroblast activation protein (FAP) mRNA. Synovial tissue samples were collected by orthopaedic surgery from 10 patients with end-stage osteoarthritis (OA) who underwent joint replacement or from 10 patients with destructive refractory rheumatoid arthritis (RA). (a) Quantification of FAP gene expression was determined by real-time polymerase chain reaction (PCR) as described in Materials and methods. The data are presented relative to HPRT and are graphically visualised as box-and-whisker plots. Expression of FAP gene is significantly higher in the synovial samples of patients with RA compared with OA synovial samples (p < 0.01). (b) Reverse transcription-PCR analysis of FAP, matrix metalloproteinase (MMP)-1, and MMP-13. Synovial tissue samples were collected from patients with destructive refractory RA (column A, lanes 1 to 5) or from patients with end-stage OA (column B, lanes 1 to 5). Extraction control was performed with GAPDH PCR. Positive controls (P) were fibroblasts for MMP-1, pooled breast cancer samples for amplification of MMP-13 and p53, and FAP-transfected HT1080 cells for FAP. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxanthine phosphoribosyltransferase; L, DNA ladder; N, negative controls.

Figure 2

Cell surface expression of fibroblast activation protein (FAP). Gene expression analysis is completed by immunohistochemical staining. FAP-specific staining was performed on synovial samples of five individuals of each entity (rheumatoid arthritis [RA] and osteoarthritis [OA]), demonstrating the stronger expression at the protein level in the inflamed synovia of patients with refractory RA. Magnification, ×100.

Figure 3

Immunohistochemical analysis of activation markers in synovial membranes of osteoarthritis. Phenotypic markers that are instrumental in extracellular matrix alteration are detectable only at a low level (MMP-1, CD44v7/8) in areas that are slightly FAP-positive. Magnification, ×400. FAP, fibroblast activation protein; MMP, matrix metalloproteinase.

Figure 4

Immunohistochemical analysis of activation markers in synovial membranes of rheumatoid arthritis. Expression of fibroblast activation protein (FAP) is accompanied by accumulation of activation markers (MMP-1 and MMP-13) and splice variants of CD44 (v3 and v7/8) that are known to correlate with the degree of inflammation and that are involved in extracellular matrix deposition. Magnification, ×400. MMP, matrix metalloproteinase.

Figure 5

Comparison of immunohistochemical detection of activation markers. Analysis of representative areas of synovial tissue samples taken from patients with end-stage osteoarthritis (OA) or refractory rheumatoid arthritis (RA) is shown. Expression pattern and staining intensity of RA samples represent the stronger intensity of synovial inflammation when compared with OA samples. Magnification, ×400. FAP, fibroblast activation protein; MMP, matrix metalloproteinase.

Figure 6

Immunohistochemical characterisation of fibroblast populations. Collected samples were snap-frozen, and sequential sections were stained for expression of Thy-1, fibroblast activation protein (FAP), or smooth muscle actin (SMA). Antigen-positive cells were identified by pink-brown colouration. Tissue samples of both entities, rheumatoid arthritis (RA) and osteoarthritis (OA), show distinct synovial fibroblast populations. Associated expression of SMA and FAP distinguishes myofibroblasts of the intimal synovial lining from Thy-1-positive fibroblasts or only SMA-expressing perivascular smooth muscle cells. Staining of anti-human FAP and anti-human SMA is more intense in RA tissue samples when compared with OA. Magnification, ×100.

Figure 7

Double-staining of fibroblast activation protein (FAP) and smooth muscle actin (SMA). Synovial tissue samples from patients with destructive rheumatoid arthritis were stained to demonstrate the simultaneous expression of both antigens. FAP-positive fibroblasts in the intimal synovial lining were coloured brown (DAB [3,3'-diaminobenzidin]). Coexpression of SMA is visualised in red (fast red). Magnification, ×400.