4 ± 2.3 pg/mL; mean ± SD; n= 9) fraction were around the basal level; and there was no additional effect after mixing either of them with the lymphocyte-rich fraction (data not shown). On the other hand, bulk cells from mice

that had been injected once i.n. with a mixture of allergen and complete Freund’s adjuvant (Fig. 9b) produced almost no IL-4 (18.4 ± 6.9 pg/mL; mean ± SD; n= 9). The cells in their 2 + 3 fractions (macrophage-rich and lymphocyte-rich; 15.9 ± 6.9 pg/mL; mean ± SD; n= 9) or single (6.5–12.5 pg/mL; n= 9) fractions were also inactive, revealing that the cytokine IL-4 is crucial for class switching to IgE. Of particular interest, a combination of the lymphocyte-rich population (for IgG production) with the macrophage-rich population (for IgE production) produced see more a large amount of IL-4 (73.3 ± 14.2 pg/mL; mean ± SD; n= 12). In contrast, a mixture of the lymphocyte-rich population (for IgE production) with the macrophage-rich population (for IgG production) produced a small amount of IL-4 (21.1 ± 6.1 pg/mL; mean ± SD; n= 12)(Fig. 8c), suggesting that macrophage-rich fraction (for IgE production) plays a crucial role in production of IL-4. We next buy BIBW2992 studied which type of cells expresses IL-4 mRNA in submandibular lymph nodes. We obtained bulk cells of submandibular lymph nodes from BALB/c mice (day 10) that had been sensitized i.n. once with allergen alone, stained

them with a panel of fluorescein-labeled Abs, and isolated CD3+ cells (47.1±3.8%; mean ± SD; n= 5), B220+ cells (50.6±4.2%; mean ± SD; n= 5), and Mac-1+ cells (1.8±0.6%; mean ± SD; n= 5) by FACS. A PCR product of approximately 300 bp was clearly obtained from the RNA of the bulk Benzatropine or CD3+ cells, but not from that of the B220+ or Mac-1+ cells (Fig. 10). However, no PCR product was detected in the RNA of the CD3+ cells of submandibular lymph nodes from BALB/c mice (day 0 or 3) that had been sensitized once with allergen (data not shown). In contrast, the numbers of other types of cells, including mast cells, basophils, and eosinophils, in the submandibular lymph nodes on days 0–10 after sensitization with cedar pollen i.n. once were too small (each less than 0.1%) to be analyzed

by RT-PCR. These results indicate that IL-4 is essential for IgE Ab production and is produced mainly in CD3+ T lymphocytes. In most previous animal models of pollen-induced allergic rhinitis, the allergic reactions were induced by repetitive pollen inhalation challenges to animals that had been sensitized by repeated instillation of the pollen extract plus adjuvant into their nostrils (19–21). Under these conditions, because leukocytes, especially eosinophils, migrate into the nasal cavity and induce edema in the mucosa; it has not been possible to determine precisely which reaction of the immune system to the allergen occurs first. Recently, it was reported that sensitization of mice by i.n. application of nine serial doses of Cry j 1 (0.