Fig. 6: Loss of endothelial and epithelial CXCR2 decreases transmigration of neutrophils through an endothelial-epithelial bilayer in vitro.

Primary murine lung microvascular endothelial cells (MLMVEC) and alveolar epithelial cells (AEC) were cultured on different sides of a Transwell™ insert. Transmigration of WT bone marrow-derived neutrophils towards a CXCL1 gradient was analyzed in case of different WT and CXCR2^−/− layer combinations (A). Confluency was assured via Phalloidin staining of (B) AECs and (C) MLMVECs on Transwell™ membranes. D To further confirm the development of confluent and tight cell layers a Blue Dextran solution was transferred into the upper Transwell™ chamber and allowed to distribute for 3 h at 37 °C. The amount of Blue Dextran in the lower chamber was assessed in a plate reader. E Reliable transmigration of neutrophils was evaluated by determining transmigration efficiency of WT and CXCR2^−/− neutrophils towards a CXCL1 gradient through either the Transwell membrane (w/o layer) or a bilayer consisting of WT AECs and MLMVECs (WT/WT bilayer). Transwells were equipped with WT/WT control layer or with CXCR2 deficient epithelial and endothelial layers, epithelial WT and endothelial KO layers or epithelial KO but endothelium WT layers (F). The percent of transmigrated neutrophils was assessed by the help of a Sysmex haemocytometer. Data are mean ± SD, n = 3–7 biologically independent samples, one-way ANOVA followed by Bonferroni correction, *p < 0.05.