Confocal Laser Scanning Immunofluorescence Imaging for Quantitation of Stromelysin in Human Synovial Fibroblasts

Abstract

Cartilage degradation associated with osteoarthritis is mediated, in part, by stromelysin produced by chondrocytes and synovial fibroblasts.' Human synovial fibroblasts (HSF) express elevated levels of stromelysin when incubated in the presence of interleukin-1 (IL-1) in vitro.* Elevated levels of stromelysin have been reported in humans with osteoarthritis as well as in animal models of a r t h r i t i~.~.~ The arthritic lesions are generally focal, and chondrocytes from these lesions express higher levels of stromelysin than those present in surrounding t i~s u e .~ Variability in the expression of stromelysin in these cells is also observed in vitro with IL-1 stimulation? Procedures such as measurement of enzyme activity, ELISA-or immunocytochemistrybased protein detection have been used to quantitate stromelysin from normal and arthritic tissues. Nevertheless, these procedures do not take into account variability among cell populations. Therefore, to obtain information on spatial distribution and to quantitate stromelysin expressed by individual cells, HSF were treated with IL-1 in the presence of different concentrations of dexamethasone and analyzed by confocal laser scanning cytometry.As shown in FIGURE 1, stromelysin protein-associated fluorescence was detected as discrete deposits in the perinuclear cytoplasmic region in the form of an envelope. However, not all the cells showed increased levels of stromelysin synthesis in response to stimulation with IL-1 in vitro. Approximately 34% of the cells showed a higher level of stromelysin expression in IL-1-treated HSF (FIG. 2). A dosedependent inhibition (ICso = 0.38 nM) in the expression of stromelysin was observed when HSF were stimulated with IL-1 in the presence of increasing concentrations of dexamethasone. The dose-dependent changes in stromelysin mRNA expression correlated well with the accumulation of stromelysin (data not shown). These results suggest that de novo biosynthesis of stromelysin by individual cells could be quantitated by fluorescence confocal imaging, and therefore this technique is useful in analyzing heterogeneity in the expression of the enzymes by cells in culture.On the basis of fluorescence intensity, the present study suggests that increased numbers of cells are involved in the synthesis of stromelysin in HSF treated with IL-1 and that dexamethasone decreased the number of these cells in a dose-dependent bAddress correspondence to Vijaykurnar M