FIGURE 6.

Priming with interleukin (IL)-4 sensitises human lung microvascular endothelial cells (HMVECs) and leads to increased loss of barrier function and increased polymorphonuclear leukocyte (PMNL) transmigration upon pirfenidone treatment. a) Electric Cell-substrate Impedance Sensing (ECIS) measurement of HMVEC barrier resistance in response to pirfenidone. Dimethyl sulfoxide (DMSO) and basal medium served as vehicle and negative control, respectively. b) ECIS measurements of HMVEC barrier resistance in response to IL-4 alone compared to basal medium (vehicle control distilled water). c) ECIS measurements of HMVEC barrier resistance in response to IL-4 in combination with pirfenidone or DMSO vehicle control. d and e) Detailed analysis of b) and c) at d) 10 min and e) 120 min post-treatment. Statistical analysis was performed using two-way ANOVA with multiple comparison testing. IL-4 effect: *: p<0.05, **: p<0.01, ***: p<0.001; pirfenidone effect: ^#: p<0.05. f) Schematic representation of the transendothelial migration experimental setup. Endothelial cells (ECs) were cultured on 3 µm transmembrane inserts and PMNLs (consisting of eosinophils and neutrophils) were allowed to migrate to 0% or 10% fetal bovine serum, respectively, in the presence or absence of IL4 and/or pirfenidone. g) Transendothelial migration of PMNLs through 3 µm transwell insets with and without HMVEC monolayers in basal medium with 0% serum. h) Transendothelial migration of PMNLs through established HMVEC monolayers in the presence of IL-4 and/or pirfenidone and corresponding vehicle controls. Statistical analysis was performed by one-way ANOVA with Dunnett's post-test using 0% serum with DMSO as control. *: p<0.05, **: p<0.01; t-test was used to compare IL-4 with and without pirfenidone treatment. ^#: p<0.05.