1 Sensitization and challenge protocols. Induction of AAI should be carried out as described elsewhere ??2??. Sensitise six to eight-week-old male mice with intraperitoneal injections of OVA (Grade V; Sigma, St. Louis, MO) (100 µg) plus 1 mg aluminum hydroxide (ALUM; Pierce, Rockford, IL) as adjuvant in 0.2 ml PBS on days 0 and 7. On days 14-17, give mice 50 µl of 2 mg/ml OVA in sterile PBS (i.n.). On day 18, 24 h after the last intranasal challenge, harvest bilateral BAL (2 aliquots of 1 ml PBS with 0.6 mM EDTA) and collect tissues for histological analysis. A second protocol can also be utilized as described by Kelly-Welch, et al. ??3??. In brief, mice are sensitized with 100 µg OVA and boosted after 14 days. Mice are then challenged via the intranasal route on days 19, 21, 24 and 26. Twenty-four hours after the last challenge animals are euthanized and BAL fluid collected for determination of cellular infiltration and cytokine levels. Perfused lungs are harvested for histological studies.
2 CD8+ T cells and NKT cells depletion. For depletion of CD8+ T cells or NKT cells from C57BL/6, BALB/c, CD1d--/--, or Kb--/--Db--/-- knockout mice, inject 100 µg/mouse/week of specific antibody for 4 weeks. Effectiveness can be confirmed by staining splenocytes with CD1d:Ig dimer and CD8-specific antibody.
3 Allergen-initiated respiratory inflammation. Measurement of inflammatory mediators can be determined in cell-free BAL fluid (2000 x g, 10 min) by sensitive and specific ELISA, in tandem, for IL-4, IL-5 and IL-13 (R&D Systems, Minneapolis, MN). Resuspend cells in HBSS, enumerate using a hemocytometer, and concentrate onto microscope slides by cytocentrifugation (STATspin, Norwood, MA) (265 x g). Stain cells with Wright–Giemsa stain (Sigma) to determine leukocyte differentials (after counting 200 cells).
4 Histology. Euthanize mice by CO2 inhalation. Remove lungs and embed in paraffin, then cut into 5 to 6 µm sections using a cryostat. Stain the sections with hematoxylin/eosin (H&E) and examine microscopically. Perform mucus staining with PAS. View the sections in a blind fashion, and score disease severity on the basis of cellular infiltration. Stain the paraffin sections with Alcian blue (AB)/periodic acid-Schiff (PAS) stain and observe using standard light microscopy. The mucus-producing cells should be stained dark brown.
5 Statistics. Assess data using the Student's t-test, ANOVA or Wilcoxon rank sum test, as appropriate. Data should be expressed as mean +/- SEM of evaluations of a minimum of six mice.