Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 1 | Secretory leukoprotease inhibitor (SLPI) is expressed in basophils and eosinophils but not in mast cells. (A) A quantitative RT-PCR of Slpi in s the bone marrow (BM)-derived basophils, eosinophils, mast cells, and BM cells. (B) Basophils and eosinophils were sorted from the spleen cells after the depletion of CD4+CD8+B220+ cells using magnetic separator. Eosinophils are also sorted from the peritoneal cavity (PEC). A quantitative RT-PCR of Slpi were shown in the indicated cells. (C) Immunoblotting of SLPI in the indicated cells. The data were normalized to the expression of β-actin and presented relative to the expression in BM cells. (D,E) Fluorescence microscopy and transmission electron microscopy (TEM) images of BM-derived basophils (BMBs) (D) and BM-derived eosinophils (BMEos). (E) from B6 and Slpi−/− mice. Bright field (BF), SLPI (red) DAPI (blue), Diff-Quick staining, alcian blue staining, and TEM images are shown (scale bar: 2 µm). (A,B) Data were normalized to the housekeeping Rps16 (mean ± SD). n = 4. **P < 0.01. (C–E) Data are representative of three independent experiments.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Quantitative RT-PCR, Derivative Assay, Western Blot, Expressing, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy, Diff-Quik, Staining

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 2 | Enhanced cytokine production and tryptase activity in Slpi−/− bone marrow-derived basophils (BMBs) after IgE stimulation. (A–D) B6 and Slpi−/− BMBs were incubated with TNP-OVA for 12 h at the indicated concentrations 1 h after the administration of 5 µg/ml anti-TNP-IgE. (A) The interleukin (IL)-4, 6, and 13 levels in supernatants were measured by an ELISA. (B) The enzyme activities of tryptase (left) and chymase (right) in supernatants were determined using MeOSuc-AAPV-pNA and N-Suc-AAPF-pNA substrate, respectively. (C) The percentages of β-HEX released after the administration of the indicated stimulators. (D) The histamine and cysteinyl leukotrienes (CysLT) production in supernatants was measured by an ELISA. (E) B6 and Slpi−/− BMBs were stimulated with TNP-OVA (1 ng/ml) at the indicated time, 1 h after the administration of 5 µg/ml anti-TNP-IgE. Representative immunoblots of the indicated proteins are shown. (A–D) Data are shown as the mean ± SEM of three different basophil cultures.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Activity Assay, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 3 | The depletion of basophil secretory leukoprotease inhibitor (SLPI) exacerbates IgE-mediated allergic responses. (A) The experimental protocol of IgE-mediated chronic allergic inflammation in 5-fluorouracil (5-FU)-treated Fcer1g−/− mice adaptively transferred with B6 and Slpi −/− DX5+ cells containing basophils from bone marrow (BM) cells. (B) The kinetics of the ear thickness after the antigen challenge are shown. (C) Ear specimens obtained 6 days after the antigen challenge were stained with HE. Data are representative of three separate experiments and are shown as the mean ± SD. n = 4–6. *P < 0.05, **P < 0.01.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Staining

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 4 | The absence of secretory leukoprotease inhibitor (SLPI) in BM-derived eosinophils (BMEos) increases interleukin (IL)-6 production and invasive activity. (A) I The production of IL-6 by B6 and Slpi−/− BMEos after lipopolysaccharide (LPS) stimulation for 12 h. (B) B6 and Slpi−/− BMEos were incubated with LPS (1 µg/ml) or IL-33 (0.1 µg/ml) for 12 h. BMEos were also incubated with TNP-OVA (1 ng/ml) for 12 h after the administration of 5 µg/ml anti-TNP-IgE. (A,B) IL-4, 6, and 13 levels in the supernatants of cells were measured by an ELISA. (C) The activities of tryptase and chymase prepared according to the methods described in Figure 2B. (D) The amounts of eosinophil peroxidase (EPO) in B6 and Slpi−/− BMEos after the administration of the indicated stimulators. (E) Chemotactic assays of B6 and Slpi−/− eosinophils by LTB4 (50 nM), CCL2 (50 nM), and CCL11 (10 nM). (F) Invasion assays using Matrigel in B6 and Slpi−/− eosinophils upon LPS (1 µg/ml) and CCL11 (10 nM) stimulation. All of the data are shown as the mean ± SEM of three different eosinophil cultures. * P < 0.05.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Derivative Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 6 | Secretory leukoprotease inhibitor (SLPI) interact with the JNK-interacting protein 3 (JIP3) scaffold protein, and negatively regulates Toll-like receptor (TLR) 4-mediated Elk-1 activation. (A) B6 and Slpi−/− bone marrow-derived eosinophils (BMEos) were stimulated with lipopolysaccharide (LPS) (1 µg/ml). Immunoblots of the indicated proteins are shown. The arrows indicate the p54 and P46 isoforms of JNK1. GAPDH was used as loading and internal monitoring controls. (B) The relative intensities of pJNK1/JNK1 and pSer383 Elk-1/Elk-1 in B6 and Slpi−/− BMEos were estimated by densitometric scanning with normalization to GAPDH (means ± SD). n = 3. *P < 0.05. **P < 0.01. (C) JIP3 and SLPI after the precipitation of anti-JIP3 Ab or control mouse IgG1 in B6 and Slpi−/− BMEos. The loading volumes (1/2 and 1/1) are shown. (A,C) Data are representative of three separate experiments.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Activation Assay, Derivative Assay, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 5 | Secretory leukoprotease inhibitor (SLPI) transcriptionally regulates the metalloproteinase (MMP)-9 expression in BM-derived eosinophils (BMEos). (A) A DNA microarray analysis of Slpi−/− BMEos before and 3 h after lipopolysaccharide (LPS) (1 µg/ml) stimulation. The relative expression to B6 BMEos is shown. (B) A qRT-PCR of Mmp9 in B6 and Slpi−/− BMEos after LPS (1 µg/ml) stimulation. (C) A qRT-PCR of Mmp9 and Slpi in Slpi−/− BMEos transfected with a plasmid carrying the Slpi gene. (D) Immunoblotting of MMP-9 in the indicated cells. (E) Immunoblotting of MMP-9 in B6 and Slpi−/− BMEos after LPS (1 µg/ml) or interleukin (IL)-5 (10 ng/ml) stimulation for 6 h. (B,C) Data were normalized to the housekeeping Rps16 (mean ± SD). n = 4. ** P < 0.01. (D,E) β-actin was used as a control. Data are representative of three separate experiments.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Expressing, Derivative Assay, Microarray, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

doi: 10.3389/fimmu.2017.01538

Figure Lengend Snippet: Figure 7 | The disruption of secretory leukoprotease inhibitor (SLPI) augments eosinophil-mediated airway inflammation. (A–C) The house-dust mite (HDM)- induced asthmatic model. Mice were intranasally sensitized with 1 µg of HDM on Day 0 and were challenged 7 days later with exposure to 1 µg of HDM for 5 consecutive days. (A) The percentages of eosinophils (Siglec-F+ Autofluorescence-) among bronchoalveolar lavage fluid (BALF) cells from B6 and Slpi−/− mice at 72 h after the last HDM challenge. (B) The number of eosinophils among the BALF cells on Day 14. (C) Lung sections from specimens obtained on Day 14 were stained with HE. (D,E) Chitin-induced airway inflammation using an eosinophil adaptive transfer system. CD45.2+ donor B6 or Slpi−/− BMEos were intravenously transferred into allergen-challenged CD45.1+ recipients. (D) The left panel shows the population of CD45.1+ recipient and CD45.2+ donor cells among BALF cells. The right panel shows donor Siglec-F+ F4/80+ eosinophils in CD45.1 recipient mice 1 day after the antigen challenge. (E) The numbers of total cells, recipient eosinophils (CD45.1+CD45.2− Siglec-F+ F4/80+cells), and donor eosinophils among BALF cells are shown. All data are representative of three separate experiments and are shown as the mean ± SD (A,B) n = 8–11, (D,E) n = 4–6. *P < 0.05, **P < 0.01.

Article Snippet: The cells were blocked with blocking reagent (Toyobo) for 1 h at room temperature, incubated with biotinylated goat anti-mouse SLPI (R&D Systems) in Can get signal solution A (Toyobo) overnight at 4°C, and then incubated with allophycocyanin-labeled streptavidin in Can get signal solution B (Toyobo) for another 1 h at room temperature.

Techniques: Disruption, Staining