Figure 1.
Study design for mouse asthma model.
Group 1: ovalbumin (OVA)-sensitized mice injected intraperitoneally with 10 µg of OVA plus 4 mg of Al (OH)3 on day 0 and 5 and saline-challenged on day 18 with 0.9% saline for 20 min; group 2: OVA-sensitized mice injected intraperitoneally with OVA on day 0 and 5 and OVA-challenged on day 18 with 5% OVA (w/v) in 0.9% saline for 20 min; group 3: ovalbumin (OVA)-sensitized mice injected intraperitoneally on day 0 and 5, treated with an intra-peritoneal injection of purified 500 µg of rat anti-mouse CD4 monoclonal antibody (mAb) (GK1.5, rat IgG2b, k) or rat IgG (Sigma–Aldrich Chemical) on day 17 and OVA-challenged on day 18, the airway hyperresponsiveness (AHR) and bronchoalveolar lavage fluid (BALF) were analyzed in all mice.
Figure 2.
Severe airway inflammation accompanied with CD4+ T cells and eosinophils in IL-18 Tg mice in a mouse asthma model.
(A) Histological evidence of airway inflammation in OVA-sensitized and challenged mice (group 1). Lung tissues were also obtained from OVA-sensitized and saline-challenged mice (group 2). Original magnification 400X, HE staining. (B) Cells in the BALFs were centrifuged onto glass slides, dried in air, and stained with Wright–Giemsa. Cell populations in the BALFs were calculated as described under Materials and methods. (n = 4 to 6 per each group) *: p<0.05 (C) Flow cytometric analysis was performed to examine CD4+ T cells and CD8+ T cells using a FC500® flow cytometer (Beckman Coulter, Palo Alto, CA). Anti-mouse CD16/CD32 mAb (2.4G2, PharMingen, San Diego, CA) was used to block the non-specific binding. Isolated BALF cells from mice were stained with PC5-anti-mouse CD4 mAb, FITC-anti-mouse CD8 mAb, and/or control isotype matched mAbs (eBioscience, San Diego, CA). Cell populations were calculated as described under Materials and methods. (n = 4 to 6 per each group) *: p<0.05.
Figure 3.
Overproduction of IL-18 in lungs increases Th1 and Th2 cytokines, and airway hyperresponsiveness.
(A) The concentrations of IFN-γ, IL-13, IL-1β, and eotaxin in the bronchoalveolar lavage fluids (BALFs) were measured by specific ELISA kits. (n = 4 to 6 per each group) *: p<0.05 (B) On day 19, BALF cells were isolated from OVA-sensitized and challenged mice. Three-color analysis was performed for intracellular cytokine staining. Isolated BALF cells were stained with FITC-anti-mouse CD4 mAb, PE-anti-mouse IL-13 mAb, PC7-anti-mouse IFN-γ mAb, and/or control isotype matched mAbs, as reported previously [14]. The lymphocyte population was gated for intracellular cytokine analysis. (C) Mice were anesthetized intraperitoneally with sodium pentobarbital, and their tracheas were cannulated via tracheostomy. The mice were ventilated mechanically (tidal volume, 325 µl; frequency, 120 breaths/minute). A paralytic agent (pancuronium bromide) was administered and airway opening pressure was measured with a differential pressure transducer and was recorded continuously. Stepwise increases of acetylcholine chloride (ACh, catalog no. A-6625, Sigma–Aldrich Chemical) in 0.9% saline (0.6 to 160 mg/ml) were given by an ultrasonic nebulizer (30 seconds). The data were expressed as the provocative concentration 200 (PC200); the concentration at which airway pressure was 200% of its baseline. PC200 was calculated by log-linear interpolation for individual mice. The data were also expressed as airway resistance changes from baseline in response to 9 different doses of Ach (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 mg/ml). (n = 6 to 10 per each group) *: p<0.05. (D) Serum Total IgE and OVA-specific IgE concentrations were measured. (n = 5 to 12 per each group) *: p<0.05.
Figure 4.
Treatment with anti-CD4 mAb decreased airway inflammation, Th1 and Th2 cytokines, and airway hyperresponsiveness in IL-18 Tg mice.
In group 3, OVA-sensitized mice were treated with 500 µg of rat anti-mouse CD4 mAb or rat IgG on day 17, and OVA-challenged on day 18. (A) Cell populations in the BALFs. (n = 5 to 7 per each group) *: p<0.05. (B) The concentrations of mouse IFN-γ and IL-13 in the BALFs were measured. (n = 5 to 7 per each group) *: p<0.05. (C) Airway responsiveness to aerosolized acetylcholine was examined as described above. The data were also expressed as airway resistance changes from baseline in response to 9 different doses of Ach (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 mg/ml). (n = 7 to 10 per each group) *: p<0.05.
Figure 5.
Deletion of IL-13 gene decreased airway inflammation, Th1 and Th2 cytokines, and airway hyperresponsiveness in IL-18 Tg mice.
WT Balb/c, IL-18 Tg, IL-13 gene deficient (KO), and IL-18 Tg/IL-13 gene deficient (TG/KO) mice were OVA-sensitized and challenged with OVA or saline. (A) Cell populations in the BALFs were calculated. (n = 14 to 20 per each group) *: p<0.05. (B) Airway responsiveness to aerosolized acetylcholine was examined as described above. The data were also expressed as airway resistance changes from baseline in response to 9 different doses of Ach (0, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 mg/ml). (n = 14 to 22 per each group) *: p<0.05.