ril 25  (R&D Systems)

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

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: Age and sex matched wild-type (WT), ( n = 4) and Mmp7 −/− mice ( n = 4) were immunized intranasally with CAA or PBS for every 4 days for a total of five times and 24 h after the last immunization, mice were assessed for (a,b) AHR is shown as dose response curve to acetylcholine and using PC 200, and (c) Glycoproteins in BAL detected by ELISA. (d) Eosinophil cell counts from BAL were determined from the same groups of mice. * P < 0.05 relative to PBS challenged WT and ** P < 0.05 relative to CAA immunized WT mice using one way ANOVA and t -test. Data represent mean values ± SD (e) Representative photomicrographs of bronchovascular bundles (n=4 per group) stained with H&E. Bar indicates 100 μm. (f,g) WT and MMP7 null mice were given intranasal PBS or rIL-25 (5 μg) twice daily for 3 days, and 24 h following the last dose, total cell and eosinophil count (f) and IL-4, and IL-5 (g) were assessed in the BAL fluid. (Data represent mean values ± SD; representative of 3 independent experiments; n=4 per group; * P < 0.05 relative to WT mice treated with rIL-25).

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: Enzyme-linked Immunosorbent Assay, Staining