Prostaglandin E , Alters Human Orbital Fibroblast Shape Through a Mechanism Involving the Generation of Cyclic Adenosine Monophosphate *

Orbital fibroblasts from patients with Graves’ ophthalmopathy, when treated with prostaglandin E, (PGE,), become stellate and develop prominent cellular processes. In this paper, we describe results of studies designed to characterize the action of PGE, on orbital fibroblast shape changes in vitro. Orbital and dermal fibroblasts were incubated with PGE,, one of several prostanoid analogues, 8-br-CAMP or forskolin and were then visualized by phase-contrast microscopy. Other studies involved seeding cells in special chambers equipped with electrodes for cell sensing using electric cell-substratein$edante sensing (ECIS) to detect changes in shane. PGE, (lo-’ mol/L) elicited a rapid and dramatic alteration in the shape 02 orbital fibroblasts but not those derived from the skin. Cells became stellate and developed prominent cytoplasmic processes that extended out from the central area containing the cell nucleus. The effects were stereoselective in that a number of structurally related compounds, including Sulprostone, PGI,, PGF,,, thromboxane A,, thromboxane B,, and 11 deoxy,l6,16 dimethyl PGE, failed to elicit a similar shape change. Butapr&t (10m5 mol&, a specific EP, agonist, elicited-a similar shape-change as that observed with PGE,. 16,16-dimethyl PGE,, a nonselective agonist, could mimic the action of PGE,. The effect of PGE, was apparent at lo-‘mom, maximal at a concentration of 10m7 mol/L and took 4-8 hr to evolve completely. Cycloheximide (10 pg/ mL) and actinomycin D (1 pg/mL) failed to block the shape change. The morphologic change could be reproduced by addition of 8-br-CAMP (3 mmol/L) and by forskolin (5 wmol/L). Moreover, PGE, and Butaprost treatment elicited in orbital cultures a massive increase in endogenous CAMP production while analogues not affecting cell shape failed to influence cyclic nucleotide generation. Three strains of orbital fibroblasts from patients with Graves’ ophthalmopathy and three from normal orbits were tested and all responded to PGE, (10m7 mol/L). Four strains of dermal fibroblasts failed to respond to PGE,. The changes in orbital fibroblast morphology were accompanied by a marked decrease in monolayer impedance as assessed by electric cell-substrate impedance sensing. The earliest effects were apparent within 30 min using this sensitive technique. The widely recognized roles of PGE, and related compounds in the mediation of the inflammatory response make our current findings in orbital fibroblasts of potential importance to the pathogenesis of Graves’ ophthalmopathy. (J Clin Endocrinol Metab 80: 35533560, 1995) P ROSTAGLANDIN E, (PGE,) is an important component of the inflammatory cascade and has a diverse set of actions on many target cell types (1, 2). The vast array of physiological roles played by this and related compounds is only now becoming apparent. Prostanoids elicit their biological effects by associating with plasma membrane bound receptors, of which several subtypes have thus far been identified (3-6). Prostanoid receptors have been shown to activate second messenger pathways (7). Human dermal fibroblasts maintained in primary cell culture synthesize and respond to PGE, (8). Moreover, the rate of PGE, synthesis in fibroblasts is regulated by cytokines and serum through the induction of a cyclooxygenase (9). Fibroblasts emanating from the human orbit engender phenotypic characteristics in vitro that set them apart from those derived from the integument (10-12). These differReceived April 1,1995. Revised May 26,1995. Re-revised June 8,1995. Accepted June 13, 1995. Address all correspondence and reprint requests to: Terry J. Smith, M.D., Division of Molecular and Cellular Medicine (A-1751, Albany Medical College, Albany, New York 12208. *This work was supported in part by National Institutes of Health Grant R01 EY08976 (T.J.S) and by the Research Service of the Department of Veterans Affairs (T.J.S.). ences exist despite similar morphologies as determined on the light (10) as well as transmission electron microscopic levels (13). In particular, the response domain for interferon gamma differs substantially in orbital and nonorbital fibroblasts (14-16). Even when cultures from distant areas of the skin of individual donors are compared, consistent, distinct patterns of protein synthesis and cytokine response emerge (17-18). We have reported recently that PGE, can alter dramatically the morphology of cultured orbital fibroblasts derived from patients with severe Graves’ ophthalmopathy (19). These cells were changed from fusiform shapes that resemble most primary human fibroblasts to a stellate morphology. They develop multiple cellular processes that commingle with the bodies and processes of adjacent cells. The changes occurred rapidly, within 3-6 hr of prostanoid addition to the culture medium and were transient in that there was spontaneous reversion toward the native morphology despite the continued presence in the medium of the compound. Readdition of fresh PGE, failed to influence this reversion. The shape-changes were preceded and then accompanied by a substantial reduction in culture monolayer impedance, as assessed by electric cell-substrate impedance sensing (ECIS) (20-22). These noninvasive measurements