Graves' ophthalmopathy is an autoimmune thyroid disease where orbital connective tissues become infiltrated with hyaluronan and immunocompetent cells such as lymphocytes in the setting of sometimes intense inflammation. Our studies thus far show orbital fibroblasts are phenotypically distinct from those outside the orbit, especially in their susceptibility to certain actions of inflammatory cytokines. Of particular importance is an exaggerated upregulation of hyaluronan synthesis in response to leukoregulin, a product of activated T lymphocytes. This response we believe is due to an activation of lipoxygenase (LO) pathways. Another distinction is a low basal PGE2 production despite high levels of the constitutive cyclooxygenase, PGHS-1 in orbital fibroblasts. Yet in response to leukoregulin, orbital fibroblasts synthesize extremely high levels of PGE2 as a consequence of a massive induction of the inflammatory cyclooxygenase, PGHS-2. Our findings represent substantial progress in defining the basis for both the excessive accumulation of hyaluronan and the inflammation in Graves' ophthalmopathy and set the stage for our continuing studies proposed in this application. We hypothesize that the site-selective vulnerability of the eicosanoid pathways in orbital fibroblasts is the pathogenic basis for Graves' ophthalmopathy. To identify the particular lipoxygenase pathway activated in the upregulation of hyaluronan, we propose to assess the expression and response to leukoregulin of LO proteins by Western blot and their encoding mRNAs by Northern analysis and to measure major LO metabolites by HPLC. With regard to inflammation, we will measure the expression of PGHS-1 and PGHS-2 in several strains of orbital fibroblasts from both Graves' disease and normals to determine whether responses differ as a function of the disease. To determine why basal PGE2 is low in spite of high levels of PGHS-1 protein, we will sequence the open reading frame of PGHS-1, measure microsomal cyclooxygenase activity, and analyze PGHS-1 for differences in post-translational processing. We also propose to assess whether the induction of PGHS-2 by leukoregulin is a consequence of increased gene transcription or altered mRNA turnover. We propose to identify the prostanoid receptors expressed by orbital fibroblasts and to define the signal transduction pathways activated by leukoregulin. We believe that the insights gained from these studies will be helpful in developing specific therapies.
Showing the most recent 10 out of 138 publications
