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D. Gagen, A. Tawil, Z. Li, C. W. Smith, M. Petrescu, A. R. Burns; In vitro Observations of Neutrophil (PMN) Surface Interactions With Keratocytes: A Role for CD18 Integrins. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4317.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, using a mouse model of corneal epithelial injury, transmission electron microscopy revealed that extravasated PMNs form close surface associations with keratocytes. While 40% of the surface of wild type PMNs is in close contact with keratocytes, this value decreases to 10% in CD18 -/- mice. These data suggest that leukocyte CD18 integrins mediate PMN contact with keratocytes during corneal wound healing. In the present report, we developed an in vitro model to further investigate the role of CD18 integrins in regulating PMN motility on keratocytes.
Keratocytes, isolated from the corneas of C57BL/6 wild type mice, were cultured in keratinocyte serum free medium (KSFM) to maintain the keratocyte phenotype and prevent fibroblastic conversion. Extravasated PMNs were isolated from the peritoneal cavity of C57BL/6 mice 3h after thioglycollate challenge. Extravasated PMNs, rather than PMNs isolated from peripheral blood, were used because these cells mimic the in vivo situation where extravascular (not intravascular) PMNs interact with interstitial keratocytes. Cultured keratocytes were treated with IL-1 (10 U/ml) for 4h prior to the addition of PMNs and PMN adhesion-dependent motility was digitally recorded on a Delta Vision Spectris inverted microscope and analyzed using SoftWorx software.
PMNs adhered to and migrated over the surface of IL-1-treated keratocytes. PMN motility was markedly inhibited in the presence of an anti-CD18 antibody (GAME 46). Immunostaining and qrtPCR analysis confirmed that keratocytes express ICAM-1, as well as JAM-C, two potential CD18 ligands.
The in vitro observations are consistent with our previous in vivo observations and strengthen the concept that CD18 integrins regulate PMN motility on keratocytes. Keratocyte ICAM-1 can potentially interact with both CD11a/CD18 and CD11b/CD18, whereas JAM-C potentially interacts with CD11b/CD18.
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