Specified pathogen-free female BALB/c mice, aged 6 to 8 weeks, were provided by the Chinese Academy of Medical Sciences (Beijing, China). The animal experiment was approved by Nanjing Medical University according to the guidelines of the Institutional Animal Care and Use Committee. A murine asthma model was established as described previously [14
] with minor modifications.
On days 0 and 7, mice received intraperitoneal injection of 20 μg of chicken ovalbumin (OVA, Grade V, Sigma-Aldrich, St. Louis, MO) adsorbed to 20 mg of aluminum hydroperoxide gel (Pierce, Rockford, IL). On days 14, mice were randomized to receive aerosol challenge with either 6% OVA in phosphate-buffered saline (PBS) or PBS alone via a nebula (1–5 μM particles, Bohringer Ingelheim, Germany) for 40 min per day up to 7 days. During the treatment period, As2O3 (Yida Pharmaceutics, Harbin, China) at dose of 0.5–4.5 mg/kg, dexamethasone (Dex, Phoenix Pharmaceutics, Belmont, CA) at dose of 2.5 mg/kg or PBS alone was injected into the peritoneum 30 min before each airway challenge. After the last aerosol exposure, mice were sacrificed at designated timepoints.
Baseline resistance and AHR induced by nebulized methacholine (Sigma-Aldrich, St. Louis, MO) at dose of 12.5–100 mg/ml in conscious unrestrained-mice were assessed using barometric whole-body plethysmography (Buxco Electronics Inc., Troy, NY) as described previously [15
]. Airway resistance is expressed as: Penh
)-1] × [2 Pef
], where Penh
= enhanced pause, Te
= expiratory time (sec), Tr
= relaxation time (sec), Pef
= peak expiratory flow (ml/sec), and Pif
= peak inspiratory flow (ml/sec).
Four hours after the last airway challenge, mice underwent euthanasia and were cannulated in the trachea. The lungs were washed twice with 1 ml aliquots of PBS to collect the bronchoalveolar lavage fluid (BALF). Subsequently, the lungs were removed, quickly frozen in liquid nitrogen, and stored at -70°C. Additionally, the lungs were collected at 1, 12, and 24 hrs post the last airway challenge to study the kinetics of pulmonary NF-κB activation.
Paraffin embedded lung sections (5 μm) collected 24 hrs after airway challenge were stained with hemotoxylin & eosin (Sigma-Aldrich, St. Louis, MO) for examination of histology.
Diff-Quick staining is a modified Wright's staining [16
]. Centrifuged at 300 × g
for 10 min, the pelleted cells of BALF were suspended in a serum-free RPMI 1640 medium. The cell viability, evaluated by the trypan blue exclusion method, was over 95%. Total and differential cell counts were enumerated on cytospins (Thermo Shandon, Pittsburgh, PA) in compliance with the Diff-Quick staining profile (Merck, Germany) by counting at least 200 to 500 cells in cross-section.
Enzyme-linked immunosorbant assay (ELISA)
Serum levels of OVA-specific immunoglobulin E (IgE) were analyzed by ELISA using samples collected 24 hrs after the last OVA challenge. Briefly, 96-well plates were coated with either purified anti-mouse IgE (5 μg/ml, BD PharMingen, San Diego, CA) or OVA (100 μg/ml). After addition of serum samples, OVA-specific IgE was detected using horseradish peroxidase (HRP)-conjugated sheep anti-IgG (Calbiochem, La Jolla, CA). Arbitrary units (AU) were calculated according to OD50 of the standard curve.
Murine chemokines, eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted), in the BALF samples were measured by utilizing paired antibodies following the manufacturer's recommendations. The ELISA kits were purchased from R&D Systems (Minneapolis, MN) with a minimum detectable levels of 3 and 5 pg/ml for eotaxin and RANTES, respectively.
EOS chemotaxis assay (ECA)
Interleukin (IL)-5 transgenic mice (CBA/CaH-TnN) were provided by the Institute of Chemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). EOS (~98% purity) were derived from spleen of IL-5 transgenic mice with depletion of B, T, and antigen-presenting cells using anti-B220, anti-CD4, anti-CD8 and anti-class II, as well as rat anti-mouse Ig-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) as described previously [17
]. EOS were seeded at 5 × 104
density in triplicate and preincubated for 15 min at room temperature with 0.25–2 μM of As2
prior to chemotaxis measurement.
Chemotaxis was assessed in 48-well micro-Boyden chambers using polyvinylpyrrolidone-free polycarbonate membranes (NeuroProbe, Bethesda, MD). Cell suspension and diluted chemokines of eotaxin or RANTES (PeproTech, London, UK) were added into the chamber with RPMI 1640 containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acids (HEPES, pH 7.4) and 0.05% bovine serum albumin. The plates were incubated for 60 min at 37°C under 5% CO2. The migrated cells were counted in five randomly selected high-power fields (magnification was × 1,000). Spontaneous migration was evaluated in the absence of chemoattractant.
Extraction of nuclear and total proteins
Nuclear and total proteins of lung tissue were collected as described previously [18
]. Briefly, aliquots of liquid nitrogen-frozen tissue were pulverized and lysed in 200 μl of cold Buffer A [10 mM Tris-HCl (pH7.5), 150 mM NaCl, 1.5 mM MgCl2
, 0.65% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM dithiothreitol (DTT)] for 3 min. After centrifugation at 10,000 × g
for 1 min at 4°C, the nuclear pellets were extracted with 20 μl of Buffer B [20 mM HEPES (pH7.9), 1.5 mM MgCl2
, 420 mM NaCl, 0.5 mM DTT, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM PMSF and 25% glycerol] for 30 min with intermittent mixing on ice. The supernatant containing nuclear proteins was collected by centrifugation at 12,000 × g
for 5 min.
The total proteins were prepared by addition of Buffer A to the lung powder and subjected to two freeze/thaw cycles to fracture the nuclear membranes. After centrifugation, the supernatant was collected. The nuclear and total proteins were quantitated using the Bradford assay (BioRad, Hercules, CA), aliquoted and stored at -70°C until use.
Electrophoretic mobility shift assay (EMSA)
EMSA analysis was performed using a commercial kit (Promega, Madison, WI). Double-stranded oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') containing a consensus NF-κB sequence (underlined) was end-labelled with [γ-32P]-adenosine triphosphate (Furui Biotechnology, Beijing, China) by T4 polynucleotide kinase and purified by chromatography. The binding reaction was conducted in a final volume of 20 μl containing 5 μg of nuclear proteins and 30 fmol of 32P-labelled oligonucleotide probe. Protein-DNA complexes were separated by electrophoresis on a 5% native polyacrylamide gel (37:1 acrylamide:bis-acrylamide) in a 0.5 × Tris-borate-EDTA running buffer. The dried gel was exposed to PhosphorImager (Molecular Dynamics) using ImageQuant software (Amersham Life Science, Arlington Heights, IL).
For competition assay, a 100-fold excess of unlabelled NF-κB or activator protein 1 (AP-1) oligonucleotide probe was added to the reaction mixture 10 min before addition of the labelled probe. For supershift assay, a 0.5 μg of anti-p50 or anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the reaction mixture prior to the labelled probe for 30 min.
Western blot analysis
Denatured samples (100 μg of total proteins) were fractionated by 10% sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blots were blocked with 5% milk containing 1 × TBST [40 mM Tris-HCl (pH7.6), 300 mM NaCl and 0.1% Tween-20] at 4°C overnight. Thereafter the blot was probed with primary antibodies of anti-IκBα (1:1,000 dilution) or anti-β-actin antibody (1:800 dilution) for 1 hr. After an HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) incubation, the immunoblots were visualized by an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL) according to the manufacture's instructions.
Statistical analysis was performed by one-way analysis of variance (ANOVA) and q test with SPSS 11.0 software package (SPSS Inc., Chicago, IL). The negative relationship was evaluated by Pearson correlation analysis. Data were expressed as mean ± SEM, and p < 0.05 was considered statistically significant.