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D. Sun, S. Nakao, S. Zandi, A. Hafezi-Moghadam; CD18-Mediated Leukocyte Transmigration Regulates IL-1β-Induced Corneal Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4973.
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© ARVO (1962-2015); The Authors (2016-present)
Inflammatory corneal angiogenesis is a significant cause of visual loss. Leukocyte transmigration into the stroma is an important step in inflammatory angiogenesis. However, contribution of adhesion molecules to inflammatory angiogenesis is not well understood. Recently, we introduced a novel technique for quantification of leukocyte transmigration rate in inflammatory corneal angiogenesis. Here, we investigate the contribution of CD18 to angiogenic leukocyte transmigration.
Angiogenesis was induced using the established corneal micropocket assay. Male C57Bl/6 or CD18-/- mice were anesthetized, and hydron pellets (0.3µl), containing mouse IL-1β (30ng), were implanted in 1 mm distance from the limbus. Two days after pellet implantation, 500µl acridine orange hemi (zinc chloride) salt (AO, 200µg/ml) was injected intravenously. Animals were perfused with PBS to wash out intravascular content and with rhodamine-labeled concanavalin A lectin (ConA, 10 µl/mL in PBS) at 0.5 and 2 h after AO injection to label endothelial cells. Immediately after perfusion, the corneas were removed, and flatmounts were prepared using a fluorescence anti-fading medium. Photomicrographs were obtained and leukocyte numbers were counted. Cornea’s topography was divided into three zones (1-3), as the area between the limbal vessel to 500 µm, 500-1000 µm, and 1000-1500 µm from the limbal vessels. Corneal angiogenesis was quantified using CD31 immunostaining.
IL-1β-induced corneal angiogenesis was reduced in CD18-/- (n=9, 0.92±0.13 mm2) compared with WT mice (n=9, 1.93±0.18 mm2) on day 6 (p=0.0005). Two days after pellet implantation the number of infiltrated leukocytes were 363±50.9 in zone 1, 162±110 in zone 2, and 119±96.6 in zone 3 in WT mice; and 31.8±13.4 in zone 1, 25±11.4 in zone 2, and 17±2.97 in zone 3 in CD18-/- mice, 0.5h after AO injection (p=0.0008, 0.2, and 0.3, respectively).2 h after AO injection 159±52.7 leukocytes were found in WT in zone 1, 250±54.6 in zone 2, 37±9 in zone 3, whereas 76±24.3 leukocytes in zone 1, 444±109 in zone 2, 714±215 in zone 3 in CD18-/-.
CD18-mediated leukocyte transmigration regulates IL-1β-induced corneal inflammatory angiogenesis. Our results suggest that CD18-mediated leukocyte transmigration is predominantly via inflamed limbal vessels. However, transmigration of some leukocytes appears CD18 independent.
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