Evaluation of the net proteolytic
activity, by flow cytometry, in synovial fluid and serum from patients
suffering from inflammatory joint diseases.
Pierre Tremblay(1), Marcel Desrosiers(1), Liesbet Paemen(2),
Bernard Grillet(3), Ghislain Opdenakker(2) and Yves St-Pierre(1).
(1) Centre de Recherche en Immunologie, Institut Armand-Frappier, Universite
du Quebec, Laval, Canada.
(2) Rega Institute for Medical Research, Laboratory of Molecular Immunology
and (3) University
Hospital, Division of Rheumatololgy, University of Leuven, Leuven,
The contribution of the metalloproteinases (MMPs) in the degradation
of matrix components in degenerative diseases is well established. Various
members of this family have bveen found in excessive concentration in serum
and synovial fluids of patients suffering from these diseases, suggesting
that degradation of extracellular matrix proteins results from a positive
balance toward excessive proteolytic activity. The measurement of excess
activity in serum and synovial fluids, however, has not been documented
to date since most conventional assays that monitor the presence of proteases
are unsuitable for measuring the net proteolytic activity in biological
fluids. We describe here the development of the fluorescent activated-substrate
conversion (FASC) assay (patent pending 2,189,486) based on the degradation
of fluorochrome-labeled substrates coated on microspheres. Our approach
could be used to determine the repertoire of proteolytic activities in
synovial fluids during the treatment of joint diseases and to monitor the
efficacy of treatments that are directed toward diminishing MMP activity.
Figure 1: In figures 1a and 1b, the principles of the FASC assay is
depicted. The methodology has been describe previously by us (St-Pierre
et al. (1996), Cytometry 25:374-380).
Figure 2: We used a member of the matrix metalloproteinase, gelatinase
B (MMP-9) to construct a dose-response curve for two incubation periods
(90min and 18h). The total reaction volume was 100µl. For each concentration
(done in triplicates) the residual fluorescence of the beads was evaluated
with an Coulter XL-MCL. The fluorescence intensity (in log scale)
for the different concentration of gelatinase B after an incubation of
18h is shown.
Figure 3: We evaluated the potential of our technique to determine the
net proteolytic activity contained in a biological fluids. We obtained
synovial fluids (SF's) from patients suffering from inflammatory joint
diseases, with known levels of MMP-9. We could evaluate the titer of the
net proteolytic activtities of the SF's by performing a FASC assay on serial
dilutions of the samples and the fluorescence evaluated after an incubation
Figure 4: We performed a kinetics experiment to determined the minimum
time required to obtained a significant response with a biological fluid
(ex. SF's). With most SF's a response could be obtained within 30 min.
Figure 5: We compare our assay with the zymography, which is the usual
assay for gelatinases. This assay is based of the degradation of the substrate
(eg. gelatin) which is copolimerized in the seperating gel of a SDS-PAGE.
The sample are run, renatured and the reaction proceed for 18h. Subsequently
the gel is stained, and the presence of enzyme, and its levels of activity,
is revealed by a clear zone on a blue background. When we compare the SF's
with known levels of gelatinase B determined by zymography and the FASC
titer for the same sample, we determined that no correlation exist between
the presence of the enzyme and the net proteolytic activity.
Note: These data have been provided by colleagues
worldwide. Appropriate acknowledgement should be made when material is