Plasminogen activation in synovial tissues: differences between normal, osteoarthritis, and rheumatoid arthritis joints

Abstract

Objective: To analyse the functional activity of the plasminogen activators urokinase (uPA) and tissue type plasminogen activator (tPA) in human synovial membrane, and to compare the pattern of expression between normal, osteoarthritic, and rheumatoid synovium. The molecular mechanisms underlying differences in PA activities between normal and pathological synovial tissues have been further examined.

Methods: Synovial membranes from seven normal (N) subjects, 14 osteoarthritis (OA), and 10 rheumatoid arthritis (RA) patients were analysed for plasminogen activator activity by conventional zymography and in situ zymography on tissue sections. The tissue distribution of uPA, tPA, uPA receptor (uPAR), and plasminogen activator inhibitor type-1 (PAI-1) was studied by immunohistochemistry. uPA, tPA, uPAR, and PAI-1 mRNA values and mRNA distribution were assessed by northern blot and in situ hybridisations respectively.

Results: All normal and most OA synovial tissues expressed predominantly tPA catalysed proteolytic activity mainly associated to the synovial vasculature. In some OA, tPA activity was expressed together with variable amounts of uPA mediated activity. By contrast, most RA synovial tissues exhibited considerably increased uPA activity over the proliferative lining areas, while tPA activity was reduced when compared with N and OA synovial tissues. This increase in uPA activity was associated with increased levels of uPA antigen and its corresponding mRNA, which were localised over the synovial proliferative lining areas. In addition, in RA tissues, expression of the specific uPA receptor (uPAR) and of the plasminogen activator inhibitor-type 1

Figure 1

PA activities expressed by N, OA, and RA synovial tissues. (A) Zymographic analysis. Equal amounts of proteins (5 µg), from N synovial tissue extracts or from synovial tissue extracts of OA and RA patients were analysed by SDS-PAGE zymographies as outlined in the Methods section. Two representative samples from two different subjects are shown for each group. (B) Histological zymograms on sections of normal synovial tissue and of synovial tissue from OA and RA patients. No lysis was observed in the absence of plasminogen (negative control, first column). Caseinolytic areas appear, in the presence of plasminogen, under dark ground illumination, black on the figure (uPA +tPA activities, second column). Lysis zones on N and OA synovial tissue correspond to tPA, as they were not inhibited by amiloride (tPA activity, third column), but by antibodies against tPA (uPA activity, fourth column). RA tissues displayed caseinolytic areas that were almost totally inhibited by amiloride, but only slightly inhibited by anti-tPA antibodies. Arrows denote the localisation of the synovial lining layer, facing the synovial fluid. Photographs of zymograms were taken after two to three hours' incubation at 37°C. Original magnification × 3.

Figure 2

Immunohistochemical localisation of uPA and uPAR antigens in N, OA, and RA synovial tissues. Cryostat sections of N (A, B), OA (C, D), and RA (E, F) synovial tissues were incubated with monoclonal antibodies against uPA (left panels, A, C, E) or uPAR (right panels, B, D, F). Bound antibodies were visualised by the avidin-biotin-peroxidase complex method and colour was developed by 3-3'-diaminobenzidine, giving a dark brown signal over the blue-purple counterstaining colour. Original magnification × 100.

Figure 3

Comparison of uPA, uPAR, tPA, and PAI-1 mRNA concentrations in N and pathological synovial tissues. (A) Northern blot analysis. Total RNA (12 µg/lane) extracted from normal synovial tissue or from synovial tissue of OA and RA patients was hybridised with the corresponding ³²P-cRNA probes. Three representative different samples from three different subjects are shown in each group. A positive control, total mRNA from the human fibrosarcoma HT1080, known to produce uPA, uPAR, tPA, and PAI-1 was included as a control in all northern blots (C, last lane). Exposure times were four hours for the uPA probe, 16 hours for the uPAR probe, and 40 hours for tPA and PAI-1 probes. (B) Densitometric analysis. Laser densitometry was used to quantify mRNA levels seen on northern blots. Results are expressed as % of mRNA concentration measured in normal synovia (mean (SEM), n=6-11). * p<0.05 as determined by Mann-Whitney test.

Figure 4

Visualisation of PA activities and mRNAs on tissue sections of synovial membranes from OA and RA patients. (A) OA synovial tissue. Top row: in situ zymographies. OA histological zymograms show amiloride resistant PA enzymatic activity corresponding to tPA. Bottom row: in situ hybridisations. Hybridisation of consecutive sections show an accumulation of uPA mRNA mainly in the synovial lining layer whereas no uPAR mRNA is detected. (B) RA synovial tissue. Top row: in situ zymographies. RA histological zymograms show both amiloride resistant PA activity resulting from tPA, in the deep lining layer, and amiloride sensitive PA activity corresponding to uPA, associated to the lining border and the synovial proliferative lining area. Bottom row: in situ hybridisations. Hybridisation of consecutive sections show an accumulation of uPA and uPAR mRNAs mainly in the synovial lining layer. Control hybridisation was performed with the uPA sense probe. Autoradiograms of in situ hybridisations were exposed for two weeks at room temperature. Arrows denote the localisation of the synovial lining layer, facing the synovial fluid. Original magnification × 2.