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.