Epithelial Rho GTPases and the transepithelial migration of lymphocytes

Tissue injury and inflammation lead to leukocyte recruitment from the bloodstream into the inflamed organ. Because leukocytes in excessive numbers and over prolonged periods can cause tissue damage, it is important that the trafficking of leukocytes is regulated. Although much attention has been focused on leukocyte recruitment, much less is known about the resolution of inflammation. Hollow organs, such as the lung and the gut, are unique in that tissue accumulation of leukocytes is determined by the recruitment of leukocytes from the blood; survival of tissue leukocytes; and migration of leukocytes from the interstitial space, either to the lymphatics or into the lumen of the organ, so-called egression. It has been shown that preventing egression of peribronchial leukocytes in a murine model of bronchial inflammation was fatal. This has led to an interest in the molecular mechanisms underlying egression from the lung. We have used a human bronchial cell line, 16HBE14(0-), in vitro to analyze transepithelial migration and to investigate the role of Rho GTPases in this process. This chapter describes methods used to establish monolayers of bronchial epithelial cells either the correct way up or inverted on Transwell filters and describes an assay of transepithelial migration of primary human T lymphocytes across this monolayer. This chapter shows how this system can be used to dissect out the molecular events that are required for successful egression. In particular, pretreatment of either the lymphocytes or the epithelium with blocking antibodies against cell surface receptors or with cell-permeable inhibitors directed against signaling molecules allows an analysis of the individual roles played by the T lymphocytes and the epithelial monolayer.