METHODS IN LABORATORY INVESTIGATION - IMMUNOFLUORESCENCE QUANTITATIONOF STROMELYSIN IN HUMAN SYNOVIAL FIBROBLASTS BY CONFOCAL LASER-SCANNING MICROSCOPY

BACKGROUND: Elevated levels of stromelysin have been reported in human s with osteoarthritis and rheumatoid arthritis, as well as in animal m odels of arthritis. However, a considerable amount of heterogeneity is observed in the expression of this enzyme in pathologic tissues as we ll as in in vitro systems. To analyze this variability, stromelysin ex pression was quantitated in individual human synovial fibroblasts (HSF ) obtained from osteoarthritis patients. EXPERIMENTAL DESIGN: HSF were incubated with interleukin-1 (40 units/ml), an agonist known to induc e stromelysin, in the presence or absence of dexamethasone (0.01 to 10 0 nM), an inhibitor of stromelysin transcription. With a stromelysin-s pecific antibody and a tetramethylrhodamine 5-isothiocyanate-labeled s econdary antibody, the enzyme was visualized and the fluorescence in i ndividual cells was quantified with an ACAS 570 laser cytometer in con focal mode. RESULTS: Stromelysin expression varied from one cell. to a nother; however, on the basis of the magnitude of expression of strome lysin by each cell, the ''nonresponders'' within each treatment were i dentified. Approximately 34% of the cells showed a higher level of str omelysin expression in IL-1-treated HSF compared with controls. A dose -dependent inhibition in the expression of stromelysin was observed in response to increasing concentrations of dexamethasone. The dose-depe ndent changes in the accumulation of stromelysin protein correlated we ll with the stromelysin mRNA expression. CONCLUSIONS: Confocal laser s canning microscopy can be effectively used to analyze cellular heterog eneity in stromelysin expression.