(Received for publication, March 18, 1996, and in revised form, June 10, 1996)

Hwai-Shi Wang , H. James Cao ^¶ , Virginia D. Winn , Louis J. Rezanka ^'' , Yveline Frobert , Charles H. Evans ^'' , Daniela Sciaky , Donald A. Young and Terry J. Smith ^¶

From the  Division of Molecular and Cellular Medicine, Department of Medicine, ^¶ Department of Biochemistry and Molecular Biology, Albany Medical College and Samuel S. Stratton Veterans Affairs Medical Center, Albany, New York 12208,  E. Henry Keutmann Laboratories, Division of Endocrinology and Metabolism, Departments of Medicine and Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, ^'' Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20892, and  CEA, Service de Pharmacologie et d'Immunologie, DRM, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France

Several proinflammatory cytokines can increase prostaglandin E₂ (PGE₂) synthesis in a variety of cell types, constituting an important component of the inflammatory response. We demonstrate here that leukoregulin, a 50-kDa product of activated T lymphocytes, dramatically increases PGE₂ synthesis in cultured human orbital fibroblasts. This up-regulation is mediated through an induction of prostaglandin-endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Steady-state levels of PGHS-2 mRNA are increased within 1.5 h of leukoregulin addition and are near maximal by 6 h, when they are 50-fold or higher above basal levels. The increase in PGHS-2 mRNA levels is partially blocked by cycloheximide, suggesting de novo synthesis of an intermediate protein may be required for a maximal leukoregulin response. Nuclear run-on studies indicate PGHS-2 gene transcription is up-regulated by leukoregulin 2-fold after 2 and 6 h. PGHS-2 protein, as assessed by Western blotting and two-dimensional protein gel analysis, is increased dramatically in orbital fibroblasts. This lymphokine-dependent expression of PGHS-2 is blocked by dexamethasone, and the increase in PGE₂ and cAMP levels following leukoregulin treatment is also blocked by indomethacin and by SC 58125, a newly developed PGHS-2-selective cyclooxygenase inhibitor. The dramatic increase in cAMP levels causes marked alteration in orbital fibroblast morphology. PGHS-2 expression in dermal fibroblasts is also increased by leukoregulin; however, the response is considerably less robust, and these cells do not undergo a change in morphology. 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 alter PGHS-1 levels in either orbital or dermal fibroblasts, suggesting that PGHS-1 is not involved in cytokine-dependent prostanoid production in human fibroblasts. The increased PGHS-2 expression elicited by leukoregulin in orbital fibroblasts may be a consequence of both transcriptional and post-transcriptional effects. These observations help clarify the pathogenic mechanism relevant to the intense inflammation associated with Graves' ophthalmopathy. Lymphocytes trafficked to orbital tissues have a putative role, through the cytokines they release, in the activation of fibroblasts in this autoimmune disease.

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