Fig 1.
A-GalCer induces AHR, neutrophil influx and inflammation in lungs of WT mice.
A. Direct lung resistance (RL) was measured in WT mice after 24 hours after i.n. administration of vehicle or α-GalCer (αGC) using the ELAN system. B. Macrophage and neutrophil cell distributions were calculated (20x magnification) from cytospinned and MGG-stained BALF samples. C. Vehicle (upper figure) and α-GalCer—treated (lower figure), paraffin embedded lung sections were H&E stained, and D. Corresponding cryosections were stained with anti-CD3, anti-CD4 or with anti-CD8 mAbs to identify the number and localization of different T cell subsets. *P, 0.05; **P, 0.01; ***P, 0.001. n = 8 mice/group.
Fig 2.
NKT-cell-deficient mice have attenuated AHR, neutrophil influx and cytokine production as compared to WT mice.
A. AHR measured in WT, CD1d-/- and in Jα18-/- mice after 24 hours after i.n. administration of α-GalCer (αGC) using BUXCO system. B. Macrophage and neutrophil cell distributions of cytospinned and MGG-stained BALF samples, and C. mRNA expression of IL-4, IFN-γ, IL-10 and IL-13 cytokines in lung samples by TaqMan RT-pcr. *P, 0.05; **P, 0.01; ***P, 0.001. n = 8 mice/group.
Fig 3.
False color 2D-DIGE image of BALF protein spots.
WT BALB/c, CD1d-/- and Jα18-/- mice were exposed to α-GalCer or to a vehicle and their BALFs were used for 2D-DIGE. In the shown gel pair, purple spots represent decreased abundance and green spots represent increased protein abundance in the α-GalCer—treated WT samples as compared to vehicle-treated WT samples. Protein spots identified with LC-MS/MS are labelled with their UniProt entry name and are listed in Table 1.
Table 1.
List of identified proteins.
Fig 4.
Hierarchial clustering and VENN-diagram of identified proteins.
A. Hierarchical clustering of protein spots in groups. Two main clusters are found; one consisting of 12 α-GalCer-treated WT samples, the other containing 8 α-GalCer-treated Jα18-KO and CD1d-KO mice samples and 4 vehicle-treated WT samples, which all look alike. Up-regulated protein abundances are shown in red and decreased proteins levels are in green. B. As the VENN diagram of the identified proteins shows, most of the differentially expressed protein spots lie between the vehicle and α-GalCer-treated WT samples.
Table 2.
Functional annotation.
Fig 5.
Western blot abundance differences for proteins selected for validation.
Protein levels of all identified CLCA1 (A), CXCL15 (B) and complement C3 fragments (C) are shown as dotplots in the study groups as log standard abundance of the gel spots from the DeCyder EDA program. Differences in immunoblot band volumes are shown for CLCA1 (D), CXCL15 (E) and 9 kD C3 fragment called anaphylatoxin C3a (F). Student’s t-test p values for significant protein level differences between α-GalCer—treated (αGC) WT mice and other groups are marked. A BALF sample from ovalbumin-sensitized and challenged mouse was used as a positive control for asthma (Pos cntrl).