LEUKOREGULIN INDUCTION OF PROSTAGLANDIN-ENDOPEROXIDE-H SYNTHASE-2 IN HUMAN ORBITAL FIBROBLASTS - AN IN-VITRO MODEL FOR CONNECTIVE-TISSUE INFLAMMATION

Several proinflammatory cytokines can increase prostaglandin E(2) (PGE (2)) synthesis in a variety of cell types, constituting an important c omponent of the inflammatory response. We demonstrate here that leukor egulin, a 50-kDa product of activated T lymphocytes, dramatically incr eases PGE, synthesis in cultured human orbital fibroblasts. This up-re gulation is mediated through an induction of prostaglandin-endoperoxid e H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Steady-state levels of PGHS-2 mRNA are increased within 1.5 h of leukoregulin addi tion and are near maximal. by 6 h, when they are 50-fold or higher abo ve basal levels, The increase in PGHS-2 mRNA levels is partially block ed by cycloheximide, suggesting de novo synthesis of an intermediate p rotein may be required for a maximal leukoregulin response. Nuclear ru n-on studies indicate PGHS-2 gene transcription is up-regulated by leu koregulin a-fold after 2 and 6 h. PGHS-2 protein, as assessed by Weste rn blotting and two-dimensional protein gel analysis, is increased dra matically in orbital fibroblasts, This lymphokine-dependent expression of PGHS-2 is blocked by dexamethasone, and the increase in PGE, and c AMP levels following leukoregulin treatment is also blocked by indomet hacin and by SC 58125, a newly developed PGHS-2-selective cyclooxygena se inhibitor. The dramatic increase in cAMP levels causes marked alter ation in orbital fibroblast morphology. PGHS-2 expression in dermal fi broblasts is also increased by leukoregulin; however, the response is considerably less robust, and these cells do not undergo a change in m orphology. Both orbital and dermal fibroblasts express high levels of PGHS-1 mRNA and protein, the other abundant form of cyclooxygenase. In contrast to its effects on PGHS-2 expression, leukoregulin fails to a lter PGHS-1 levels in either orbital or dermal fibroblasts, suggesting that PGHS-1 is not involved in cytokine-dependent prostanoid producti on in human fibroblasts. The increased PGHS-2 expression elicited by l eukoregulin in orbital fibroblasts may be a consequence of both transc riptional and post-transcriptional effects. These observations help cl arify the pathogenic mechanism relevant to the intense inflammation as sociated with Graves' ophthalmopathy. Lymphocytes trafficked to orbita l tissues have a putative role, through the cytokines they release, in the activation of fibroblasts in this autoimmune disease.