A novel approach to measure the contribution of matrix metalloproteinase in the overall net proteolytic activity present in synovial fluids of patients with arthritis

Abstract

Despite decades of research, only a very limited number of matrix metalloproteinase (MMP) inhibitors have been successful in clinical trials of arthritis. One of the central problems associated with this failure may be our inability to monitor the local activity of proteases in the joints since the integrity of the extracellular matrix results from an equilibrium between noncovalent, 1:1 stoichiometric binding of protease inhibitors to the catalytic site of the activated forms of the enzymes. In the present work, we have measured by flow cytometry the net proteolytic activity in synovial fluids (SF) collected from 95 patients with osteoarthritis and various forms of inflammatory arthritis, including rheumatoid arthritis, spondyloarthropathies, and chronic juvenile arthritis. We found that SF of patients with inflammatory arthritis had significantly higher levels of proteolytic activity than those of osteoarthritis patients. Moreover, the overall activity in inflammatory arthritis patients correlated positively with the number of infiltrated leukocytes and the serum level of C-reactive protein. No such correlations were found in osteoarthritis patients. Members of the MMP family contributed significantly to the proteolytic activity found in SF. Small-molecular-weight MMP inhibitors were indeed effective for inhibiting proteolytic activity in SF, but their effectiveness varied greatly among patients. Interestingly, the contribution of MMPs decreased in patients with very high proteolytic activity, and this was due both to a molar excess of tissue inhibitor of MMP-1 and to an increased contribution of other proteolytic enzymes. These results emphasize the diversity of the MMPs involved in arthritis and, from a clinical perspective, suggest an interesting alternative for testing the potential of new protease inhibitors for the treatment of arthritis.

Figure 1

Proteolytic activity in synovial fluid of patients with osteoarthritis and inflammatory arthropathies. (a) Typical two-parameter histogram (left) with forward-angle light scatter (FS) and side scatter (SS) (y and x axes, respectively), used to position the window on microspheres (one-parameter histogram, middle) and to minimize the interference with debris. To determine the proteolytic activity in synovial fluids (SF), fluorescein isothiocyanate-labelled polypeptides derived from denatured collagen (gelatin) were coated on polystyrene microspheres (shaded) and incubated with serial dilution of synovial fluid. LIN, Linear; LOG, Logarithmic. The mean fluorescent intensity at the top of each peak is then measured on 1,000–5,000 events (right). (b) Comparison of the level of proteolytic activity found in SF obtained from 26 osteoarthritis (OA) patients and 69 patients with inflammatory arthritis (IA), including patients with established rheumatoid arthritis (RA) or undifferentiated or recent-onset polyarthritis, patients with crystal-induced arthritis, or patients with other noninfectious IA, such as ankylosing spondylitis and reactive arthritis. FASC, fluorescent-activated substrate conversion. (c) Proteolytic activity in SF collected from the left and right knees of arthritic patients on the same day. The results are representative of at least two independent experiments. GA, Gout arthritis.

Figure 2

Correlation between the level of proteolytic activity found and the number of infiltrating leukocytes. Positive correlation between the net proteolytic activity (NPA) and the number of (a) infiltrating leukocytes (r_s = 0.710, P < 0.001) or (b) polymorphonuclear cells (PMN) (r_s = 0.696, P < 0.001) in synovial fluids of patients with inflammatory arthritis. (c) No such correlation between the NPA and the number of infiltrating leukocytes was observed in osteoarthritis (OA) patients. Each point represents one sample. The correlation was calculated using Spearman's rank correlation coefficient for each test. FASC, fluorescent-activated substrate conversion.

Figure 3

Zymographic analysis of MMP-2 and MMP-9 levels in arthritic synovial fluids. (a) Secretion of MMP-2 and MMP-9 was assessed by gelatin zymography using standard procedures. Aliquots of 10 μl synovial fluids (SF) (diluted 1:10) were loaded into each lane. OA, osteoarthritis; snPA, seronegative polyarthritis; rPA, seropositive rheumatoid polyarthritis; CPDD, calcium phosphate deposition disease; SpA, spondyloarthropathy; PA, polyarthritis; JCA, juvenile chronic arthritis; MA, microcrystalline arthritis. (b) Semiquantitative measures of the proMMP-9 level were carried out by densitometric analysis (scored as arbitrary units) and compared with the net proteolytic activity (NPA) found in the same SF. No correlations between the NPA and the level of the 225 kDa form of MMP-9 or with that of its active form (92 kDa) were observed (data not shown). FASC, fluorescent-activated substrate conversion. As a positive control, an aliquot of supernatant of HT1080 cell culture containing both MMP2 and MMP-9 gelatinases was used. The results were obtained using duplicates and are representative of two independent experiments. Error bars represent the mean ± standard error of the mean.

Figure 4

Effect of hydroxamate-based inhibitors on arthritic synovial fluids. (a) Detection of the inhibitory effect of the matrix metalloproteinase (MMP) inhibitors (inhibitor II and inhibitor III) at different doses by fluorescent-activated substrate conversion using recombinant human MMP-2 and MMP-9. Briefly, recombinant MMP-2 and MMP-9 were incubated with microspheres coated with fluorescein isothiocyanate-labelled polypeptides (derived from denatured collagen) at 37°C. The percentage of cleavage, measured by the loss of fluorescence, was measured by flow cytometry using standard optics for fluorescein (using a 525 nm bandpass filter). (b) Effect of the hydroxamate inhibitors (used at 10 μM) on rheumatoid arthritis (RA) synovial fluids. Each bar represents one sample. The results were obtained using duplicates and are representative of two independent experiments. Error bars represent the mean ± standard error of the mean.

Figure 5

Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients. (a) Inverse correlation between the matrix metalloproteinase (MMP)-dependent proteolytic activity and the level of proteolytic activity in synovial fluids of rheumatoid arthritis (RA) patients (r = -0.521, P = 0.046). (b) Correlation between the level of TIMP-1 and the net proteolytic activity (r = 0.592, P = 0.07). (c) Positive correlation between the level of TIMP-1 and the number of infiltrated leukocytes (r = 0.910, P < 0.001). The levels of TIMP-1 in synovial fluids were measured by commercial ELISA. The contribution of MMPs to the proteolytic activity was carried out using the hydroxamate inhibitor II, as described in Figure 4. Each point represents one sample. The correlation was calculated using Spearman's rank correlation coefficient for each test. FASC, fluorescent-activated substrate conversion.