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 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.
Wang
H S
HS
Department of Medicine, Albany Medical College, Albany, New York 12208, USA.
Cao
H J
HJ
Winn
V D
VD
Rezanka
L J
LJ
Frobert
Y
Y
Evans
C H
CH
Sciaky
D
D
Young
D A
DA
Smith
T J
TJ
D015152:Blotting, Northern; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D004790:Enzyme Induction / Q000187:drug effects; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D008222:Lymphokines / Q000494:pharmacology*; D008858:Microscopy, Phase-Contrast; D015735:Mifepristone / Q000494:pharmacology; D009915:Orbit / Q000166:cytology / Q000201:enzymology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
D008222:Lymphokines; D011720:Pyrazoles; D012333:RNA, Messenger; C044162:leukoregulin; C090859:1-((4-methylsulfonyl)phenyl)-3-trifluoromethyl-5-(4-fluorophenyl)pyrazole; D015735:Mifepristone; D003907:Dexamethasone; D003513:Cycloheximide; D000242:Cyclic AMP; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone