Histamine

Polarized Th9 cells (2 × 10⁷) were intravenously injected in each BALB/c mouse on day 0. From 6 hours after the cell transfer, these mice were challenged with intranasal administration of 20-μL OVA (30 mg/mL; Sigma, Sigma, St. Louis, MO, USA) or phosphate buffered saline (PBS) once a day on days 0–3 (Fig. 1). NHR was assessed 6 hours after the last challenge by counting the number of sneezes for 5 minutes just after the administration of 10-μL histamine (100 mM; Nacalai tesque, Kyoto, Japan) [3 (link)]. Inflammatory cells in the nasal lavage fluid were classified by means of morphological criteria as described previously [3 (link)]. The number of transferred Th9 cells in nasal-associated lymphoid tissue (NALT) were determined by flow cytometry upon staining with anti-CD4-APC-Cy7 (BioLegend) and anti-DO11.10 T-cell receptor (TCR)-FITC (eBioscience). This procedure did not induce any inflammatory responses in the lower airways [10 (link)]. In some experiments, 10-mg/kg dexamethasone (Dex: Tokyo Kasei, Tokyo, Japan) suspended in PBS containing 0.5% Tween-20 (Sigma) was subcutaneously injected in mice 30 minutes before each OVA challenge.

In the allergen-immunized model, mice were sensitized by an intraperitoneal injection of 20 μg OVA (Sigma-Aldrich) emulsified with 2.25-mg alum (Thermo Fisher Scientific, Waltham, MA, USA) on days 0, 7, 14, and 21 of the experiment. Each day on days 35–39 and 42–46, mice were exposed to ETS, and 30 minutes later, they were challenged with an intranasal (i.n.) administration of 600-μg OVA dissolved in 20-μL saline or saline alone (Fig. 1A). In the Th2 cell transfer model, polarized Th2 cells (2 × 10⁷) were intravenously injected in each BALB/c mouse on day 0, and these mice were exposed to ETS and challenged with OVA or saline each day on days 1–5 and 8–12 (Fig. 1B). In these models, NHR was assessed 6 hours after the last challenge by counting the number of sneezes for 5 minutes just after the administration of 10-μL histamine (100 mM; Nacalai tesque, Kyoto, Japan) [14 (link)]. Inflammatory cells in the nasal lavage fluid were classified by means of morphological criteria as described previously [20 (link)]. These models did not exhibit any inflammatory features in the lower airways [21 (link)].