To the best of our knowledge, this is the first study assessing the NPA in SF from patients with different forms of arthritis. Levels of active MMPs in the joint are frequently quantified using immunological methods that do not discriminate between MMPs that are active, that are in their latent form, or that are bound to endogenous protease inhibitors. Our approach clearly showed several results. First, the NPA in patients with IA correlated positively with the number of infiltrated leukocytes. Also, although the expression level of MMP-9 is not indicative of the NPA found in SF, MMPs contribute to a large extent to the proteolytic activity. Third, small molecular weight inhibitors were very effective in inhibiting MMP activity in RA SF, but their effectiveness varied significantly among patients. Finally, and most interestingly, increased TIMP-1 secretion by leukocytes in patients with IA shifted the balance toward an MMP-independent proteolytic activity in those SF with high NPA. Overall, these results stress the importance of monitoring the repertoire of active proteases, not simply the presence of proteases, in patients during the course of their disease and under the influence of treatment. This may represent a sine qua non to achieve clinical success in the development of new generations of protease inhibitors with superior clinical efficacy.
Our findings that the NPA is higher in RA than in OA are consistent with previous reports that levels of MMPs and other proteases are higher in RA [2
]. This observation thus supports the idea that inflammation plays an important role in controlling the protease/inhibitor ratio in SF [31
], and is consistent with the recent observation of a direct correlation between tumour necrosis factor alpha/MMP production and collagen degradation [32
]. Indeed, we found that the NPA in SF correlates positively with the number of infiltrating leukocytes and with blood C-reactive protein levels. This finding suggests that leukocytes control, either directly or indirectly, the overall balance that results in the NPA found in SF – most probably by secreting proteases, or by inducing the secretion of proteases via the secretion of cytokines [33
Our results showing elevated levels of TIMP-1 in SF with the highest levels of NPA, both of which correlated with the levels of leukocytes, further emphasize the importance of infiltrating leukocytes in controlling the NPA of SF. Our results thus illustrate the need to measure the NPA resulting from the (dis)equilibrium between active proteases and their inhibitors. Other workers have also reported a molar excess of TIMP-1 in SF in animal models of OA, suggesting that matrix degeneration in osteoarthritic joints could be promoted by other proteases such as aggrecanase-1 and aggrecanase-2 [34
]. The use of other protease inhibitors specific for cathepsins, elastase, and other types of proteases previously found in SF of RA patients [29
] will be necessary to determine the contribution of these proteases to the NPA in various diseases.
We used the ability of the flow cytometer to accurately detect different classes of particles, such as microspheres, based upon a physical characteristic such as size and scattering, to develop a simple and reliable method that allows qualitative and quantitative measurements of specific enzymatic reaction using fluorochrome-labelled substrate coated onto polystyrene microspheres [8
]. The advantage of our approach is that the fluorochrome-labelled substrates are used in the context of laser flow cytometry, which increases the sensitivity of the assay. Using spheres of multiple sizes, one could also use this assay to perform real-time multiple determinations to determine the repertoire of different active proteases using selective substrates.
Flow cytometers hydrodynamically focus a fluid suspension of particles into a thin stream so that the microspheres flow down the stream in single file and pass through an examination zone. A focused laser light beam illuminates the spheres as they flow through the examination zone, allowing optical detectors within the flow cytometers to measure the fluorescence bound to the microspheres. Because the beads are analysed in a very small volume (about 6 pl) as they pass through the flow cytometers' laser beam, interference from free fluorescent molecules (cleaved substrate) does not interfere with the assay. This design is thus compatible with the use of highly specific and high-affinity inhibitors, such as MMP-specific monoclonal antibodies with neutralizing activity.
Such a measure of net enzymatic activity allows, in the case of proteases, one to specifically account for the presence of enzymatic inhibitors in SF. This is a key issue as the degradation of the tissue architecture and disease state depend on the biological activity of the enzymes; that is, on the ratio of free active enzymes to inactive (inhibitor-bound) enzymes.
In the present article we have provided evidence that this method constitutes a powerful tool to assess the performance of enzyme inhibitors for therapeutic applications. For example, our results with inhibitor II and inhibitor III suggest that MMP-2 and MMP-13 play a dominant role in the proteolytic activity found in the SF of RA patients. One could thus be able to determine which among the available protease inhibitors is best suited to inhibit the proteolytic activity in SF of a given patient. Since we found that MMP-2 is mostly found in its proform in SF, our results favour the implication of MMP-13 Interestingly, MMP-13, which is responsible for cleavage of type II collagen [36
], aggrecan [38
], and fibrinogen [39
], has been shown to be increased in RA SF [3
] and to be linked to synovial inflammation and bone destruction [40
]. Of course, one has to be careful with such conclusion, as it is probable that the repertoire of active proteases will vary with specific subgroups of inflammatory arthritis. For instance, Peake and colleagues recently showed that MMP-13 was not detected in SF or serum of patients with juvenile idiopathic arthritis [41
Future work is clearly required to establish with more precision the repertoire of active proteases in arthritic SF. The monitoring of activity on a patient may allow adjusting the dosage of a specific inhibitor or, alternatively, exploring whether additional combinatorial treatments are required. From a clinical point of view, therefore, this assay represents an ideal approach for testing the potential of new protease inhibitors aimed at inhibiting disease progression. Failure to do so may explain at least in part the current observation that MMP inhibitors failed in clinical trials. It is probable, however, that other factors may explain the failure of MMP inhibitors in clinical trials. For instance, some members of the MMP family were recently shown to exert an anti-inflammatory activity in some physiological processes or diseases. This hypothesis received strong support from data obtained using genetically engineered MMP-deficient mouse models [42
]. Moreover, cleavage of chemokines by MMPs has been shown to generate chemokine receptor antagonists that retain cellular binding affinity while inhibiting the biological activity of the receptor [43
]. SDF-1α and other cytokines/chemokines can also be degraded by several members of the MMP family that are constitutively expressed at high levels in a particular tissue, impairing their receptor-binding activity and their ability to mediate chemotaxis [44
]. In contrast, other chemokines, such as IL-8, can be proteolytically activated by MMP-9 and MT1-MMP [45
A more complete understanding of the joint destructive process and of the identity of the relevant proteases are keys to the future development of protease inhibitors in rheumatic diseases. Additional studies are needed to investigate the overall correlation between the types and levels of active MMPs that are found in SF and those MMPs that are found in the inflamed synovial tissues.