We investigated the effects of several cytokines on HLA-DR expression in cultured fibroblasts derived from retroocular connective tissue and pretibial and abdominal skin of patients with Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD), as well as from normal individuals. We hypothesized that differences in response to cytokines between fibroblasts from various anatomical areas might play a role in the site-selective involvement of the extrathyroidal manifestations of Graves' disease. HLA-DR expression in fibroblasts was quantitated by scanning densitometry of whole cell lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Direct immunofluorescence of cell monolayers was also performed. We hypothesize that unique characteristics of these fibroblasts may play a role in GO and PTD. Cultured retroocular, pretibial, and abdominal fibroblasts from patients with Graves' disease as well as from normal individuals did not express HLA-DR spontaneously. Treatment in vitro with interferon-gamma (IFN gamma; 100 U/mL) for 5 days induced HLA-DR by 50- to 80-fold (P less than 0.0001) in fibroblasts from all sites and subjects studied. However, IFN gamma-induced HLA-DR expression was significantly greater in retroocular (P less than 0.005) and pretibial (P less than 0.0005) fibroblasts from patients with GO and PTD than in fibroblasts obtained from the same anatomical sites of normal individuals. Further, retroocular and pretibial fibroblasts from patients with GO and PTD responded to IFN gamma more vigorously than did abdominal fibroblasts from these same patients (P less than 0.0001). IFN gamma-induced HLA-DR expression was enhanced by concomitant treatment with tumor necrosis factor-alpha (100 U/mL). In contrast, treatment of retroocular fibroblasts with transforming growth factor-beta (10 ng/mL), epidermal growth factor (1 ng/mL), or interleukin-6 (IL-6; 100 U/mL) significantly attenuated IFN gamma-induced HLA-DR reactivity by 40-59% (P less than 0.05). Incubation of retroocular fibroblasts with tumor necrosis factor-alpha, IL-1 alpha (10 U/mL), IL-2 (10 U/mL), IL-6, granulocyte-macrophage colony-stimulating factor (100 U/mL), epidermal growth factor, and transforming growth factor-beta alone did not affect HLA-DR expression. These results indicate that several cytokines can influence HLA-DR expression in cultured fibroblasts. The enhanced induction of HLA-DR by IFN gamma in retroocular and pretibial fibroblasts compared with that in abdominal fibroblasts may partially explain the selective involvement of the retroocular connective tissue and pretibial skin in fully expressed Graves disease.