The effect of cigarette smoke constituents on the expression of HLA-DR in orbital fibroblasts derived from patients with Graves ophthalmopathy

Purpose: Numerous studies have established a strong association between smo king and Graves disease, but the underlying mechanism of Graves ophthalmopa thy has not been elucidated. Recent studies of Graves ophthalmopathy have f ocused on the orbital fibroblast as an integral component in the pathogenes is of this disorder. This investigation focuses on the effect of cigarette smoke constituents, nicotine and tar, to alter the expression of human leuk ocyte antigen (HLA-DR) in cultured orbital fibroblasts from patients with G raves disease. Methods: HLA-DR expression was quantified by scanning densitometry of whole cell lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electr ophoresis with immunoblotting and also by direct immunofluorescence. Results: Cultured orbital fibroblasts, obtained from patients undergoing or bital decompression for severe Graves ophthalmopathy, failed to express HLA -DR as analyzed by both immunoblotting and direct immunofluorescence when t reated with nicotine alone (25-300 ng/ml). The expression of HLA-DR increas ed three-fold when nicotine (25 ng/ml) in combination with interferon-gamma (500 U/ml) was added to the cultured orbital fibroblasts (p < 0.0001). Cul tured orbital fibroblasts treated with tar alone (60-600 ng/ml) failed to e xhibit HLA-DR expression as assessed by direct immunofluorescence and immun oblotting. A greater than two-fold increase in HLA-DR expression occulted w hen tar (600 ng/ml) combined with interferon-gamma (500 U/ml) was added to the cultured orbital fibroblasts (p < 0.0001). Conclusion: The results suggest a possible molecular mechanism for the more severe ophthalmopathy observed in Graves patients who smoke cigarettes. Th ese findings could prove useful for possible medical interventions to decre ase or even inhibit the interaction between cigarette constituents, cytokin es, and orbital fibroblasts.