Abbreviations
SC : Subcutaneous
IP : Intraperitoneal
SCIT : Subcutaneous immunotherapy
AHR : Airway hyperresponsiveness
H.scandens : Humulus scandens
HSE : H. scanden s pollen extract
Penh : enhanced pause
Mch : Methacholine
SHS : Splenocyte homogenate supernatants
Ig : Immunoglobulin
sIgE : specific serum immunoglobulin E
sIgG2a : specific serum IgG2a
Figure 1. Overview of experimental design. Establishment of
murine asthma model(A). Specific SCIT for murine asthma model(B). A.
Mice were sensitized by the SC or IP routes on days 1, 8, and 15 with
HSE or PBS. Seven days later the last sensitization, all animals were
challenged with aerosolized HSE (1% in saline) or saline (control
group) for 30 min daily for 7 consecutive days. On day 28, AHR to Mch
was assessed. On day 29, the mice were sacrificed for further
experiments. B, mice were sensitized on days 1, 8, and 15 by SC
injection with 25μg of HSE or PBS. Seven days after the last injection,
all animals were placed in a plastic box and challenged with aerosolized
HSE (1% in saline) or an equal volume of saline (control group) for 30
min daily for 3 consecutive days. On days 25–39, mice in the treatment
group received eight SC injections of 300μg of HSE every other day; the
control and vehicle group animals received PBS. Mice were re-challenged
with a 1% HSE aerosol on days 43–49 and on day 50, AHR to Mch was
assessed. One day later, the mice were sacrificed for further analysis.
Figure 2. The effect of SC route or IP route sensitization on
AHR, airway inflammation and immunological response. Mice were
sensitized by SC route or IP route and aerosol challenged as described
in Fig.1A. AHR to Mch(A): Penh dose-response curves to Mch were
determined 24 h after the last challenge(on day 28).The mice were
sacrificed 1 day later for analysis of HSE-specific IgE(B) in serum,
inflammatory cell recruitment (C1) and percentages of eosinophils(C2) in
BALF, Cytokine production of IL-4 (D1), IL-13 (D2), IFN-γ (D3), and
IL-10 (D4) in BALF and SHS. Data are expressed as mean ± SEM of n=10
mice per group. **P<0.01, SC.HSE vs. SC.PBS group, or IP.HSE
vs. IP.PBS group; *P<0.05, SC.HSE vs. SC.PBS group, or IP.HSE
vs. IP.PBS group; ## P<0.01, SC.HSE vs. IP.HSE group; #
P<0.05, SC.HSE vs. IP.HSE group.
Figure 3. Inflammatory cell infiltration(A), mucus production(B)
in lung tissue and scoring of inflammatory cells and goblet cells(C).Lung tissues were harvested 24 h after the last HSE challenge. H&E
staining was performed to examine cell infiltration and Alcian
blue-periodic acid-Schiff staining was performed to evaluate mucus
production. Representative images for inflammatory cell infiltration
(A1–A4) and goblet hyperplasia are shown (B1–B4): A1 and B1, IP.PBS
group; A2 and B2, IP.HSE group; A3 and B3, SC.PBS group; A4 and B4,
SC.HSE group. The scoring of inflammatory cells and goblet cells (C) was
performed in at least three different fields for each lung section, and
the data are expressed as mean±SEM of n = 10 mice per
group.**P<0.01, SC.HSE vs. SC.PBS group, or IP.HSE vs. IP.PBS
group.
Figure 4. SDS-PAGE(A) and Western blotting analysis(B). A.
Representative image of SDS-PAGE. Lane HSE: 20μg protein profiles ofH. scandens pollen extract. Lane M, molecular mass markers. B.
Western blotting analysis of the antigen-binding characteristics of
serum specific IgE antibodies with H. scandens pollen extract.
Lane M, molecular mass markers; lane N, pooled sera from both control
groups; lanes 1–10, sera from SC.HSE group animals; lanes 11–20, sera
from IP.HSE group animals.
Figure 5. Effects of SCIT on HSE-induced AHR, airway
inflammation and immunological response. Mice were sensitized and
challenged as described in Fig.1 B. AHR to Mch(A): Penh dose-response
curves to Mch were determined 24 h after the last challenge(on day
50).The mice were sacrificed 1 day later for analysis of inflammatory
cell recruitment (B1) and percentages of eosinophils(B2) in
BALF,HSE-specific IgE(C1) and IgG2a(C2) in serum and cytokine production
of IL-4 (D1), IL-13 (D2), IFN-γ(D3), and IL-10 (D4) in BALF and SHS.
Data are expressed as mean ± SEM of n=10 mice per group.
**P<0.01, vehicle vs. control group; *P<0.05,
vehicle vs. control group; ##P<0.01, treatment vs. vehicle
group; #P<0.05, treatment vs. vehicle group.
Figure 6. Effects of HSE SCIT on Inflammatory cell infiltration
and goblet hyperplasia in the lungs of allergic mice. The experimental
procedure is described in Fig. 1B. H&E staining was performed to
examine cell infiltration and Alcian blue-periodic acid-Schiff staining
was performed to evaluate mucus production. Representative images for
inflammatory cell infiltration (A) and goblet hyperplasia (B) are shown:
A1 and B1, control group; A2 and B2, vehicle group; A3 and B3, treatment
group. Inflammatory cell infiltration and goblet cell hyperplasia was
blindly semi-quantified (C). Scoring of inflammatory cells and goblet
cells was performed in at least three different fields for each lung
section, and the data were presented as mean ± SEM of n = 10 mice per
group. **, P<0.01 vehicle vs. control group; ##,
P<0.01, treatment vs. vehicle group, #, P<0.05,
treatment vs. vehicle group.