Hfl 1

Human fetal lung fibroblasts (HFL-1; ATCC, Rockville, USA) were used between passages 17 and 20. HFL-1 (10000 cells/well) and co-cultures of HFL-1 (10,000 cells/well) and LAD2 cells (7500 cells/well), were seeded in 4-well glass chamber slides (154526; Thermo Scientific, Waltham, MA) and incubated overnight at 37 °C, 5% CO₂. The cells were then fixed in 4% paraformaldehyde for 15 min and blocked in 2% BSA-TBS containing 5% goat serum (Vector laboratories, Burlingame, CA) and 0.2% Triton-X for 30 min, followed by washing twice in tris-buffered saline (TBS). Cells were incubated for 60 min with monoclonal tryptase antibody (M7052, Dako, Glostrup, Denmark) and monoclonal PAR2 antibody (cat.nr: 35–2300, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The cells were then washed in TBS and incubated for 45 min with secondary antibodies (Thermo Fisher Scientific), goat anti-mouse IgG2a (Alexa Fluor® 647, A21241), goat anti-mouse IgG1 (Alexa Fluor® 647, A21240) or goat anti-mouse IgG1 (Alexa Fluor® 555, A21127), followed by washing in TBS. Nuclei were stained by using DAPI containing mounting medium (Dako). Cells were imaged using a VS120 slide scanner with XV image processor L100 VS-ASW (Olympus, Tokyo, Japan). Image viewer software VS-OlyVIA (version 2.9) (Olympus Soft Imaging solutions GmbH; Münster, Germany) was used for image visualization.

Human monocytic leukemia THP-1 cells (TIB-202) and human lung fibroblast cells (HFL-1; CCL-153) were obtained from the American Type Culture Collection (ATCC; USA). THP-1 cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), whereas HFL-1 cells were cultured in F-12K medium (Invitrogen, USA) containing 10% FBS. Cells were maintained at 37°C and 5% CO₂ in a humidified incubator.
THP-1 cells were treated with 150 ng/ml phorbol 12-myristate 13-acetate (PMA; P8139, Sigma-Aldrich, USA) for 24 h to obtain macrophage-like M0 cells [31 (link)], followed by exposure to hypoxia (atmosphere of 5% CO₂ and 95% N₂).
For cocultivation, a transwell (Corning, USA) coculture system was employed as described previously [32 (link), 33 (link)]. Briefly, the differentiated M0 macrophages and the polarized M1 macrophages as described above were seeded in the upper chamber, and the HFL-1 cells were plated into the bottom chamber for cocultivation for 48 h.