Allergic and irritant contact dermatitis mouse models

For the allergic contact hypersensitivity model, mice were sensitized via topical application of 0.5% (v/v) DNFB (Sigma-Aldrich, St. Louis, MO) in 4:1 acetone/olive oil on their shaved back (50µl) and were challenged 4–5 days later with 0.25% DNFB or vehicle control (5µl on the dorsal and 5µl on the ventral side of the ear). For the irritant contact dermatitis model, 2% (v/v) croton oil (Sigma-Aldrich, St. Louis, MO) in 4:1 acetone/olive oil or vehicle control was used. Ear thickness was measured using an engineer’s micrometer (Mitutoyo, Tokyo, Japan). For depletion of macrophages, a single dose of 100µl clophosome was injected intraperitoneally (i.p.) and a single dose of 20µL clophosome (FormuMax Scientific, Inc. Palo Alto, CA) was injected intradermal (i.d.) into mice 24 hrs prior to the sensitization or the elicitation phase of CHS. Injections of the same amount of empty liposomes (FormuMax Scientific, Inc. Palo Alto, CA) into mice served as control. The efficiency of clopohosomes to deplete macrophages was confirmed by flow cytometry analysis (data not shown). In some studies, a selective iNOS inhibitor, N6-(1-iminoethyl)-L-lysine, dihydrochloride (14) (L-NIL; Cayman Chemical Company, Ann Arbor, MI) was injected ip.6 mg/kg for the first dose and then 3 mg/kg twice daily or PBS were injected into animals during the time of the experiment.