Nessy 1

After collecting the supernatant, all cells were pelleted, washed with cold phosphate-buffered saline (PBS), and lysed in RIPA buffer. Before denaturation with Laemmli buffer, protein concentration adjustment was conducted. Supernatants were resuspended in 6× Laemmli and boiled at 98°C for 10 min. Cell pellets and supernatants were separated on a 10% SDS-polyacrylamide gel. Membranes were blocked with 5% non-fat dry milk (Roth, Karlsruhe, Germany), probed with mouse anti-human IL-33 antibody (1:1,000; Nessy-1, Enzo Life Sciences, Lörrach, Germany), and peroxidase-labeled anti-mouse as secondary antibody (Dako, Denmark). Membranes were developed using the enhanced chemiluminescence method.

Human Nasal Epithelial Cells (HNEpCs) (Promocell, Rockville, MD, USA) were cultured on poly-L-lysine coated cover glasses (Matsunami, Osaka, Japan) in Airway Epithelial Cell Growth Medium (Promocell) and maintained in 5% CO₂ and 95% air at 37 °C. Twelve hours after stimulation with TDI-HSA, HNEpCs were fixed with 4% sucrose-containing 4% paraformaldehyde (Sigma Chemical Co.) for 20 min. The fixed cells were permeabilized with 0.2% Triton X-100/PBS (Sigma Chemical Co.) for 5 min and blocked with 10% goat serum/PBS for 1 h. Then, an anti-IL-33 antibody (Nessy-1, 1:250, Enzo Life Sciences, Farmingdale, NY, USA) was applied overnight at 4 °C. IL-33 was visualized by isotype-specific secondary antibody conjugated with Alexa 488 (1:200, Molecular Probes, Eugene, OR, USA). A fluorescent microscope (Axio Observer, Carl Zeiss, Oberkochen, Germany) was used to obtain fluorescent images.

Paraffin sections of lungs were deparaffinized in xylene, and hydrated through graded alcohols. To stain for IL-33, heat-induced epitope retrieval was performed using 10 mM citrate buffer and, then, sections were placed in 0.05 M Tris-HCl-Tween buffer. We blocked endogenous peroxidase with 3% H₂O₂ in distilled water, and proteins with 1% BSA. Sections were then incubated with anti-IL-33 Ab (1∶50; monoclonal biotinylated Ab, Nessy-1, Enzo Life Sciences) in Tris+Tween+saponin buffer for 1 h. Finally, we used streptavidin/peroxidase conjugate (Dako) and prepared DAB chromogenic substrate. To stain for pro-SPC, endogenous peroxidase was blocked with 3% H₂O₂ in methanol, with HCl added last. Proteinase K (Dako) was used to unmask antigens/epitopes, and proteins were blocked with 5% normal goat serum. Sections were incubated with anti-pro-SPC Ab (1∶2000; polyclonal Ab, Millipore) in Ultra Clean Diluent (Fisher Scientific) for 1 h. Finally, we used EnVision detection system with DAB+ (Dako). Mayer’s hematoxylin was used to counterstain. Negative controls were generated by omitting the primary Ab. For enumeration of IL-33⁺ cells, counts were made from five fields of view per mouse (2 from the upper lobe, 2 from the middle and 1 from the lower respiratory tract) at 400× and normalized per unit area (mm²).