Enhanced induction of a 72 kDa heat shock protein in cultured retroocular fibroblasts

Fibroblasts in the retroocular connective tissue appear to play a central role in the pathogenesis of Graves' ophthalmopathy (GO). We hypothesize that specific attributes, differentiating normal from GO retroocular fibroblasts, may render the latter more vulnerable to the ongoing immunopathological process in GO. We investigated whether GO fibroblasts differ from normal fibroblasts with respect to their sensitivity and capacity to express the inducible 72 kDa heat shock protein (HSP) in response to stressful environmental stimuli. Cultured retroocular fibroblasts derived from patients with GO and normal individuals were exposed to various changes in the culture medium that may simulate conditions in the affected retroocular space in vivo. HSP 72 reactivity was determined using sodium dodecyl sulfate polyacrylamide-gel electrophoresis of cellular extracts, followed by immunoblotting with a mouse monoclonal anti-HSP 72 antibody and quantitative scanning densitometry. In addition, indirect immunofluorescence was performed on parallel fibroblast monolayers. Following exposure to heat and acidic pH, deprivation from nutrients, and high monolayer density, GO fibroblasts expressed HSP 72 with significantly greater sensitivity and in significantly higher abundance than did normal fibroblasts. These results demonstrate that changes in the physiological environment induce HSP 72 expression in cultured fibroblasts. The enhanced sensitivity and capacity of GO retroocular fibroblasts to express the inducible HSP 72 in response to stressful stimuli may play a role in the autoimmune process affecting the retroocular space in GO.