Supporting Information
Diesel exhaust particles exacerbate allergic rhinitis in mice by disrupting nasal
epithelial barrier.
Materials and Methods
Quantitative PCR analysis
Total RNAs of nasal mucosa were extracted using Sepasol-RNA I Super G (Nacalai
Tesque, Kyoto, Japan) and reverse-transcripted into cDNA was synthesized using
ReverTra Ace (TOYOBO, Osaka, Japan) according to the manufacturer's instructions.
The gene expressions were quantified with TaqMan Gene expression assays (Applied
Biosystems) and Thermal cycler dice RT-PCR system (Takara Bio Inc., Otsu, Japan)
according to the manufacturer's instructions. Specific primers and probes used for
quantitative reverse transcription-PCR were TaqMan probes for Tjp1 and 18S rRNA
(Applied Biosystems). The values were normalized to 18S rRNA.
In vitro treatment of diesel exhaust particle plus N-acetyl-L-cysteine
RPMI 2650 cells (5×104 cells/cm2) were cultured on transwell 0.4-μm pore-size filters
for 12–14 days. Monolayer cells were treated with vehicle, NAC (10 mM), DEP (50
μg/ml), NAC plus DEP or NAC-treated DEP for 24 hours. DEP and NAC-treated DEP
were prepared by incubation of DEP with PBS and NAC (10 mM), respectively, for 15
minutes at room temperature and then washed with FBS-free medium by centrifugation
(15000 rpm, 60 minutes). To determine paracellular permeability, 40 kDa FITC-dextran
(2 mg/ml) was added to medium in upper wells. After 3 hours of incubation, culture
supernatants (100 μl) were collected from the bottom wells. Fluorescence levels of
culture supernatants were analyzed by a multi-mode microplate reader.
1
Supplementary figure legends
Fig S1. Dose-dependent effects of diesel exhaust particles on sneezing in
ragweed-pollen-induced allergic rhinitis.
Mice were sensitized and challenged as described in Fig. 1a. Diesel exhaust particles
(DEP) were used at various concentrations (1, 3, 10 μg/body). Number of sneezing
episodes was counted over a 10-minute period immediately after challenge. Graph
represents pooled data from two independent experiments (mean and SEM of n=5;
Vehicle and DEP group, n=6; RW and RW+DEP group).
Fig S2. Diesel exhaust particles do not augment Th2-type immune responses in
ragweed pollen-induced allergic rhinitis.
(a) Cervical lymph node (cLN) cells were cultured with ragweed extract for 5 days in
the presence of antigen-presenting cells and IL-2. Cytokine levels in the culture
supernatants were measured by ELISA. (b) Histological analysis of nasal mucosa after 4
days of challenge. V, vehicle. P, PBS. D, diesel exhaust particles. R, ragweed pollen.
Graphs represent pooled data from three independent experiments (mean and SEM of
n=9 mice per group).
Fig S3. Diesel-exhaust-particles-induced tight junction disruption is not mediated
by cell death in a cultured nasal epithelial cell line.
RPMI 2650 cells were treated with diesel exhaust particles (DEP) (0, 25, 50 or 100
μg/ml) for 24 hours. Dead cells were counted by trypan blue staining. Data are
representative of two independent experiments (mean and SEM).
2
Fig S4. Administration of diesel exhaust particles decrease the expression of tight
junction protein.
(a) Immunofluorescence staining of claudin-1. Claudin-1 (green) and DAPI (blue).
Scale bar=10 µm. Images are representative of two independent experiments (n=6 mice
per group). (b) qRT analysis of Tjp1 expression in nasal epithelial cells from mice 4
days after nasal administration of diesel exhaust particles (DEP). Graphs represent
pooled data from three independent experiments (mean and SEM of n=9 mice per
group).
Fig S5. Treatment of N-acetyl-L-cysteine prevents diesel exhaust particles-induced
increase in permeability of nasal epithelial cells in vitro.
Monolayer RPMI 2650 cells were incubated with vehicle, N-acetyl-L-cysteine (NAC;
10 mM), DEP (50 μg/ml), NAC+DEP or NAC-treated DEP for 24 hours, and then
treated with FITC-dextran (2 mg/ml) for 3 hours. Culture supernatants from bottom
wells were collected, and fluorescence levels were measured by fluorometer.
NAC-treated DEP was prepared by incubation with NAC (10 mM) for 15 minutes at
room temperature. Data are representative of three independent experiments (mean and
SD of triplicate cultures). *P < 0.05. **P < 0.01.
3