Ovalbumin-Induced Allergic Lung Inflammation

DN TNFR25-tg mice generated as described above and backcrossed at least seven generations into the C57BL/6J background were compared with WT C57BL/6J mice purchased from the National Cancer Institute. Mice were sensitized by i.p. injection of 66 μg ovalbumin (crystallized chicken egg albumin, grade V; Sigma-Aldrich) absorbed to 6.6 mg aluminum potassium sulfate (alum; Sigma-Aldrich) in 200 μl PBS on day 0. On day 5, mice were boosted intraperitoneally with the same dose of ovalbumin in alum. On day 12, mice were aerosol challenged with 0.5% ovalbumin in PBS for 1 h using an Ultrasonic Nebulizer (MABIS Healthcare, Inc.). Mice were assessed for allergic inflammation of the lungs 1–3 d (on day 13 to 15) after the aerosol exposure. Mice were killed by inhalation of CO_2. After cannulation of the trachea, the lung was lavaged 4 times with 1 ml of PBS. Cells recovered from the BAL fluid were counted and used for cytospin preparations (≤50,000 cells/slide). More than 200 cells were counted for each cytospin slide stained with Wright-Giemsa stain (Sigma-Aldrich) to determine differential cell counts for macrophages, eosinophils, lymphocytes, and neutrophils.