Journal: Nature immunology
Article Title: Divergent Roles for Airway Epithelial MMP7 and Retinoic Acid in Experimental Asthma
doi: 10.1038/ni.1719
Figure Lengend Snippet: ( a ) Detection of MMP7 in lung of PBS treated (C57BL/6), and mice immunized with CAA by immunohistochemistry (arrows indicate MMP-7 expression; insets show 40× magnification of airway). Bars indicate 100 μm and 25 μm bar. ( b ) Recombinant (r)IL-25 (5 μg), rIL-13 (5 μg) and rTSLP (5 μg) were incubated for 2 h at 37 °C with APMA-activated MMP7 (0.5 μg), and were resolved on 16.5% Tricine gel followed by detection using silver stain (arrows indicate bands corresponding to fragments of rIL-25 that were cleaved by MMP7). ( c ) Naïve lymph node cells (left) and spleen cells (right) isolated from C57BL/6 mice, were stimulated with 2 μg/ml of plate-bound anti-CD3 in the presence of equal amount of native rIL-25 (250 ng/ml) or MMP7 cleaved rIL-25 (IL-25′C; 250 ng/ml). On day 2, cell culture-supernatants from each condition were examined for the concentration of IL-4, IL-5, IL-13 and IFN-γ by ELISA. Media alone and APMA containing buffer that was used to activate MMP-7 (vehicle) were used as controls (n=3; data represent mean values ± SD; representative of three different experiments).* P < 0.05 relative to rIL-25, using the one-way ANOVA test. ( d ) Representative flow cytometry data of lymphocytes, following three days of culture under the same conditions as described in ( c ). Cells were restimulated with 500 ng/ml ionomycin and 50 ng/ml PMA in the presence of GolgiStop for 5 h, and the percentage of cells expressing IL-4 and IL-5 were detected by intracytoplasmic staining. Data is representative of 3 independent experiments.
Article Snippet: Lymph node cells and splenocytes were isolated from C57BL/6 mice and were stimulated with 2 μg/ml of plate-bound anti-CD3 and 50 U/ml of human IL-2 in the presence of 250 ng/ml of mouse rIL-25 (R&D) or 250 ng/ml of MMP7 cleaved mouse rIL-25 (IL-25′C).
Techniques: Immunohistochemistry, Expressing, Recombinant, Incubation, Silver Staining, Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining