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Sally S. Twining, Debra J. Warejcka; Thrombin Increases Human Corneal Fibroblast Expression Of The Chemokines, CXCL1, CCL2, IL-6, IL-8, CXCL10 And CCL13. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1088.
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Proteins of the coagulation cascade are synthesized by corneal cells and result in the activation of prothrombin to thrombin after injury to the epithelial layer or the stroma. Thrombin increases proinflammatory chemokines and cytokines such as CXCL1, CCL2, IL-6 and IL-8 in many tissues but has not been studied in the corneal fibroblasts. The purpose of this study was to determine whether thrombin increases human corneal stromal fibroblast expression of these chemokines plus CXCL10, an antiangiogenic chemokine, and CCL13, a proinflammatory chemokine which have not been previously studied in the presence of thrombin.
Stromal cells were isolated from three pairs of human donor corneas and were expanded in culture and treated with 1 ng/ml fibroblast growth factor (FGF). After seven days, cells were treated with 0, 0.1 or 1 unit/ml α-thrombin for 1 hour (for mRNA) or 12 hours (for ELISA). RNA was purified and primers for CXCL1, CCL2, IL-6, IL-8, CXCL10, CCL13 and GAPDH were used in real time PCR. Twelve hour supernatants were assayed by ELISA for protein levels of the various chemokines.
One unit/ml thrombin increased mRNA expression in stromal fibroblasts for IL-6 (15 fold) , IL-8 (7 fold), CXCL1 (6 fold), and CCL2 (7 fold) over untreated cells Expression of message for CXCL10 was increased (10 fold) and CCL13 mRNA increased only 2 fold when cells were treated with 1 unit/ml thrombin. Treatment of cells with thrombin increased protein expression of IL-6 (from 5 to 520 pg/ml), IL-8 (from 7 to 30 pg.ml), CXCL1 (from 112 to 1904 pg/ml), CCL2 (from 1586 to 9888 pg/ml), CXCL10 (from 3 to 17 pg/ml) and CCL13 (from 0 to 14 pg/ml).
As expected, thrombin treatment of corneal fibroblasts increased expression of proinflammatory chemokines such as CXCL1, CCL2, IL-6 and IL-8. Expression of CXCL10 and CCL13 mRNA and proteins were also increased by thrombin treatment of stromal fibroblasts. Regulation of CXCL10 and CCL13 by thrombin has not been previously described. These results suggest that thrombin generated in response to corneal injury can regulate the immune response.
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