Immunofluorescence quantitation of stromelysin in human synovial fibroblasts by confocal laser scanning microscopy

Elevated levels of stromelysin have been reported in humans with osteoarthritis and rheumatoid arthritis, as well as in animal models of arthritis. However, a considerable amount of heterogeneity is observed in the expression of this enzyme in pathologic tissues as well as in in vitro systems. To analyze this variability, stromelysin expression was quantitated in individual human synovial fibroblasts (HSF) obtained from osteoarthritis patients.

HSF were incubated with interleukin-1 (40 units/ml), an agonist known to induce stromelysin, in the presence or absence of dexamethasone (0.01 to 100 nM), an inhibitor of stromelysin transcription. With a stromelysin-specific antibody and a tetramethyl-rhodamine 5-isothiocyanate-labeled secondary antibody, the enzyme was visualized and the fluorescence in individual cells was quantified with an ACAS 570 laser cytometer in confocal mode.

Stromelysin expression varied from one cell to another; however, on the basis of the magnitude of expression of stromelysin by each cell, the "nonresponders" within each treatment were identified. Approximately 34% of the cells showed a higher level of stromelysin 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-dependent changes in the accumulation of stromelysin protein correlated well with the stromelysin mRNA expression.

Confocal laser scanning microscopy can be effectively used to analyze cellular heterogeneity in stromelysin expression.