Figure 4
Suppression of house dust mite (HDM)- and T-helper 2 (Th2) cell-induced eosinophilic inflammation in the airways by lipopolysaccharide (LPS). (a) Diagram of the treatment protocol. HDM (100 μg per mouse)±LPS (10 μg LPS per mouse) was administered intratracheally (i.t.) at the indicated time points. Lung-draining lymph nodes (LNs) were harvested from one group 7 days after one HDM instillation±LPS to assess CD4+ T-cell priming. The rest of the animals were challenged with HDM±LPS and at 72 h after the final instillation, bronchoalveolar lavage (BAL) fluid and lung tissue samples were obtained. (b) Enzyme-linked immunosorbent spot (ELISPOT) assay of cells harvested from lung-draining LNs. Naive mice were used as control. For all the groups, the total cell population was used for ELISPOT assay and the number of cytokine-expressing Th cells was estimated based upon the percentage of CD4+ T cells determined by flow cytometry. For the HDM and HDM+LPS groups, CD4+ T cells were also purified by magnetic bead selection before being subjected to the assay to confirm that the cytokines were being produced by CD4+ T cells. In all cases, cells were stimulated for 8–10 h with phorbol-12-myristate-13-acetate (PMA; 25 ng ml−1) plus ionomycin (500 ng ml−1). Results are expressed as the number of cytokine-producing cells per sample. Data shown are mean±s.d. **P<0.01. The data shown are representative of two independent experiments. (c) Counts of cells recovered in the BAL fluid. Values are mean+s.e.m. ***P<0.005. (d) The concentrations of interleukin (IL)-5, IL-13, and interferon-γ (IFN-γ) in lung homogenates were measured by multiplex assay and are presented as mean±s.e.m. *P<0.05 and **P<0.01. Histological examination of lung sections stained with periodic acid-Schiff (PAS) for assessment of inflammation and mucus production. Lung infiltrates around bronchovascular bundles in which eosinophilic response is most pronounced in disease were of +4 grade in animals that received HDM and +1/2 in those that received LPS together with HDM. Arrows indicate mucus staining. The data shown are representative of three independent experiments. (e) T-helper 2 (Th2) cells were generated in vitro using CD4+ T cells from DO11.10 mice and were injected i.v. into either LPS-treated mice or naive recipient mice (5 × 106 cells per mouse). The mice were then exposed to aerosolized ovalbumin (OVA) daily for 7 days and were analyzed 24 h after the last exposure. Total and differential counts of cells recovered in the BAL fluid (upper panel) and hematoxylin and eosin (H&E) staining of lung sections (lower panels) of both groups of mice were performed. Values are mean±s.e.m. *P<0.01 and **P<0.001. The data shown are representative of two independent experiments.
