Figure 1. CAR promotes transmigration of leukocytes across human bronchial epithelial cell monolayers.

(A) THP-1 cells were stained using cell-tracker orange before adding to the top well of transwell inserts containing WT or CAR-GFP HBEC monolayers and pre-treated with BSA (control) or recombinant fibre knob protein (FK). THP-1 cells in the bottom well were counted after 24 hours using FACS. Data is presented as relative fold change of THP-1 cell number transmigration (TEM) compared to WT control conditions from 4 independent experiments + /−SEM. (B) Quantification of THP-1 adhesion to WT or CAR-GFP HBEC. Experiments were set-up as in (A) and number of adhered THP-1 cells to the HBEC monolayer assessed after 24 hours. Data is representative of four independent experiments; mean values from n = 4 wells + /−SEM. (C) THP-1 cells were labelled using cell-tracker orange before adding to the top of transwell inserts containing monolayers of 16HBE14-o cells stably expressing control (scrambled) or CAR-targeted shRNA. THP-1 cells in the bottom well were counted after 24 hours using FACS. Data is presented as relative fold change of THP-1 cell number compared to WT control conditions from 4 independent experiments + /−SEM. (D) THP-1 cells were added to the underside of transwell inserts containing monolayers of WT or CAR-GFP-HBEC and allowed to adhere before inserts were inverted for 8 hours in the presence of a serum gradient to allow baso-lateral to apical migration of THP-1 cells. Data is representative of four independent experiments and presented as relative fold change of THP-1 cell number compared to WT control conditions from 4 wells + /−SEM. (E) Transmigration of primary human neutrophils and monocytes was analysed using Transwell chambers containing monolayers of WT and CAR-GFP-HBEC as in (A). Data is presented as relative fold change of THP-1 cell number compared to WT control conditions from 4 independent experiments + /−SEM.