Figure 6.

IL-13 and natural helper cells cause AHR. (a,b) Change in lung resistance (a) and cells in BAL fluid (b) in 8-week-old wild-type and Il13^−/− mice after infection with H3N1 or mock infection (n = 5–6 per group; assessed as in Fig. 1a,b). *P ≤ 0.05 and **P < 0.001 (two-way ANOVA (a) or Student’s two-tailed t-test (b)). (c) Quantitative RT-PCR analysis of IL-13 mRNA (top; relative to expression at day 0) and enzyme-linked immunosorbent assay of IL-13 protein (bottom) in homogenates of H3N1-infected BALB/c lungs (n = 3 per group). *P < 0.001, compared with day 0 (Student’s two-tailed t-test). (d) Flow cytometry analysis of intracellular IL-13 and Sca-1 in CD45⁺Lin⁻ST2⁺c-kit⁺Sca-1⁺ lung cells left unstimulated (US) or stimulated for 5 h with the phorbol DC ester PMA plus ionomycin (PMA + iono), assessed after gating (as in top row) on Lin⁺ST2⁺ cells (middle row) or Lin⁻ST2⁺ cells (bottom row). (e) Intracellular IL-13 and Sca-1 in lung cells obtained from wild-type or Rag2^−/− mice IL-13 on day 5 d after infection with H3N1 or mock infection and stimulated for 5 h with PMA plus ionomycin (bottom), assessed after gating on Lin⁻ST2⁺c-Kit⁺ cells (top). (f) Absolute number of Lin⁻ST2⁺c-Kit⁺Sca-1⁺ cells (left) or Lin⁻ST2⁺c-Kit⁺Sca-1⁺IL-13⁺ cells (right) in the lungs of mice in e. *P ≤ 0.001 (Student’s two-tailed t-test). (g) Flow cytometry (top) of lung interstitial macrophages (F4/80⁺CD11c⁻), alveolar macrophages (F4/80⁺CD11c⁺) and DCs (F4/80⁻CD11c⁺) among lung leukocytes (CD45⁺) from the mice in e; below, intracellular IL-13 expression in single cells. Data are representative of three experiments (a–d; mean and s.e.m. in c) or three independent experiments (e–g; mean and s.e.m. in f).