The studies were carried out in accordance with the Guide for the Care and Use of Laboratory animals as adopted and promulgated by the National Institutes of Health. ICR mice (Japan Clea Co., Tokyo, Japan), were divided into three experimental groups, and intratracheally exposed to phosphate-buffered saline (PBS: Invitrogen Co., Carlsbad, CA) containing Tween 80 (Nakalai Tesque, Kyoto, Japan), ovalbumin (OVA; grade IV, Sigma Chemical, St. Louis, MO) dissolved in the same vehicle, or OVA plus DEP, suspended in the same vehicle, as described previously [³, ⁴]. Exposure to these solutions was given every 2 week for 6 weeks, followed by a rest for approximately 8 weeks, and the animals were then re-exposed every 2 weeks for 6 weeks according to the protocol by Fattouh et al. [⁵], with modification. Five h after the last exposure, mice were sacrificed and the lungs were removed. Five h after the last intratracheal administration, mice were sacrificed and the lungs were collected for ex vivo experiments.

Lung cells were isolated and resuspended in R10, which was RPMI 1640 medium (GIBCO BRL, Eggenstein, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Dainippon Sumitomo Pharma Co., Ltd, Osaka, Japan), 100?U/ml penicillin, 100??g/ml streptomycin (Sigma, St. Louis, MO). Lung cells (2.5???10⁶ cells/ml) were cultured in R10 with or without OVA (400??g/ml) for 5 d. Thereafter, supernatants were collected for cytokine measurement as previously conducted [³].

Levels of IL-4 (Amersham, Buckinghamshire, UK), IL-5 (Endogen, Cambridge, MA), IL-13 (R&D systems, Minneapolis, MN), and interferon (IFN)-? (R&D systems) were measured by enzyme-linked immunosorbent assay according to the manufacturer?s instructions. The secondary antibodies were conjugated to horseradish peroxidase. Subtractive reading of 550?nm from the reading at 450?nm was converted to?pg/ml using values obtained from standard curves generated with varying concentrations of recombinant IL-4, IL-5, IL-13, and IFN-? with limits of detection of 5?pg/ml, 5?pg/ml, 1.5?pg/ml, and 10?pg/ml, respectively (n?=?6 for IL-4, n?=?14?15 in each group, respectively).

Data were reported as mean???SEM. Differences were determined using analysis of variance (Stat view version 4.0; Abacus Concepts, Inc., Berkeley, CA). If differences between groups were significant (p<0.05), Bonferroni?s protected least significant difference test was used to distinguish between pairs of groups.

*p<0.05 vs vehicle-instilled mice, **p<0.01 vs vehicle-instilled mice; ^#p<0.05 vs ovalbumin (OVA)-instilled mice. Values are the mean???SEM.