Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: Inhibition of the junctional protein VE-cadherin does not alter the expression level of IL-33 and knockdown of IL-33 does not alter the cellular distribution or expression level of junctional proteins CD31 or VE-cadherin. Immunocytochemical double staining for IL-33 (IL-33Nter, red) and VE-cadherin (mouse IgG1, green) in HUVECs preincubated with vehicle (PBS, A) or anti-VE-cadherin (IgG2a, B) from point of seeding until fixation. Pictures were obtained using identical exposure times and image-enhancement parameters. Arrowheads point to areas of intercellular contact. Note that cells in B with redistribution or lowered expression level of VE-cadherin maintained expression of IL-33. C and D: Immunocytochemical double staining for IL-33 (IL-33Nter, red) and CD31 (green) in HUVECs transfected with mock (C) or specific siRNA (D). E and F: Immunocytochemical double staining for IL-33 (red) and VE-cadherin (green) in HUVECs transfected with mock (E) or specific siRNA (F). Pictures were obtained using identical exposure times and image enhancement parameters. G: Western blot of IL-33 in cell lysates of HUVEC monolayers transfected with either scrambled siRNA controls (scr1 and scr2) or concentration-matched IL-33 targeting siRNA (spec1 and spec2). Shown are lanes from the same experiment, which was independently repeated twice with similar results. Scale bars = 10 μm.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Inhibition, Expressing, Double Staining, Transfection, Western Blot, Concentration Assay

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: IL-33 is globally expressed in nuclei of vascular endothelium in normal human tissues. Endothelial cells in vessels of human skin (A), small intestine (B), umbilical vein (C), and lung (D) were stained for IL-33 (Nessy-1, red) and with Ulex europaeus agglutinin I (A, C, and D; green) or CD31 (B, green) and Hoechst dye (blue) to visualize endothelial cells and cell nuclei, respectively. Three-color fluorescence micrograph of fixed and permeabilized tissue sections are shown. Note of presence of IL-33 positive single tissue resident cells in (B) and (C). Scale bars = 50 μm.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Staining, Fluorescence

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: Vascular IL-33 expression is lost in tumor vessels. Endothelial cells in vessels of human colon adenocarcinoma (A), ductal mammary carcinoma (B), renal clear cell carcinoma (C), and renal papillary carcinoma (D) were stained for IL-33 (Nessy-1, red) and with Ulex europaeus lectin (green) and Hoechst dye (blue) to visualize endothelial cells and cell nuclei, respectively. (For consistency throughout the figures, the red and green channels in Figure 4 were reversed.) The bottom left panels in A and B show high-power magnifications of the respective boxed areas. Note presence of autofluorescent intravascular red blood cells in A and B as well as lack of IL-33 signal in endothelial cell nuclei. The bottom right panels in A to D and bottom left panel in E show IL-33-positive vessels in tissue surrounding the tumor in the same section. E: Human colon adenocarcinoma (different sample from A) was stained for IL-33 (Nessy-1, red), Ki-67 (green), and Hoechst dye (blue). Autofluorescent erythrocytes indicate blood vessels. Right: High-power magnifications of the boxed area in E separating the different colors. Note the lack of IL-33 signal in Ki-67-positive endothelial nuclei. Scale bars = 50 μm.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Staining

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: IL-33 in HUVECs responds to cell density signals and is localized to the nucleus in nonproliferating, superconfluent cells. A–D: Monolayers of HUVECs stained for IL-33 (red) using IL-33Nterm (A–C) and Nessy-1 (D). CD31 is stained in green and cell nuclei are visualized by Hoechst dye (blue). A and B: HUVECs induce expression of nuclear IL-33 and enhance the localization of CD31 to the intercellular areas when cell density increases. C: Almost all cells are IL-33-positive in a superconfluent monolayer. D: Superconfluent HUVEC monolayer 4 hours after scratch wounding. The red signal in the cytoplasm cannot be distinguished from the signal obtained with irrelevant antibodies (data not shown). Note loss of nuclear IL-33 and reduction of CD31 (green) in cells that migrate into the open area (right half of image). E and F: Western blot for IL-33 detection in whole cell lysates of subconfluent (subconf) or superconfluent (supconf) cells (E) or nuclear versus cytoplasmic (cytopl) fraction of a superconfluent HUVEC monolayer (F). Monolayers of HUVECs stained for IL-33 (IL-33Nter in G, Nessy-1 in H–L; all red), Ki-67 (G--I, green), and ppRb (J–L, green). Endothelial nuclei are visualized by Hoechst dye (blue). Note the lack of IL-33 expression in serum and growth factor starved cells in I and L. Scale bars: 25 μm (A–D); 30 μm (G–L).

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Staining, Expressing, Western Blot

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: Vascular IL-33 expression is lost during wound healing. Biopsies from normal rat skin (A and B) and from wounds after 1 (C and D) or 2 days (E and F) were fixed and stained with H&E (A, C, and E) or immunostained (B, D, and F) for IL-33 (Nessy-1, red) and CD31 (green). A, C, and E show overviews of the wound area and the boxed areas indicate the position of vessels shown in B, D, and F, respectively. Cell nuclei are stained with Hoechst dye. Note presence of vessels without IL-33 and perivascular cell nuclei at days 1 and 2. Scale bars = 50 μm.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Staining

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: IL-33 expression is down-regulated by proinflammatory cytokines and VEGF. A--E: Response of superconfluent HUVEC monolayers exposed for 17 hours to medium alone (A), INF-γ (50 ng/ml) (B), IL-1β (0.5 ng/ml) (C), TNF-α (0.5 ng/ml) (D), or VEGF (10 ng/ml) (E). Immunostaining for IL-33 (IL-33Nter) after fixation of monolayers. Pictures were obtained using identical exposure times and image enhancement parameters. F: Quantitative PCR analysis of IL-33 expression in superconfluent HUVEC monolayers treated with cytokines for 7 hours at the same concentrations as given above. IL-33 transcript levels were obtained using primer pair IL-33 no. 2 and normalized to HPRT, data are given as ratio of transcript values in sample over those in medium-treated controls. Similar results were obtained when using a different primer pair for IL-33 detection or when normalizing against housekeeping gene GAPDH (data not shown). Scale bars = 25 μm.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Immunostaining, Real-time Polymerase Chain Reaction

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: Dose-response and time-course of IL-33 expression in HUVECs. Western blot detection of IL-33 protein after cytokine treatment of superconfluent HUVEC monolayers for 18 hours with the indicated concentration of cytokines (left column) or for the indicated time with INF-γ (50 ng/ml), IL-1β (500 pg/ml), TNF-α (500 pg/ml), or VEGF (10 ng/ml) (right column). Note the sensitivity of IL-33 levels to IL-1β and TNF-α whereas VEGF has a mild effect.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Western Blot, Concentration Assay

Journal:

Article Title: Nuclear Interleukin-33 Is Generally Expressed in Resting Endothelium but Rapidly Lost upon Angiogenic or Proinflammatory Activation

doi: 10.2353/ajpath.2008.080014

Figure Lengend Snippet: TNF-α and VEGF down-regulate IL-33 in vivo. Immunostaining for IL-33 (Nessy-1, red) and CD31 (green) of rat skin after subcutaneous injection of PBS (A, C) and cytokines (B, D). Cell nuclei are stained with Hoechst dye. The bottom left panels show the red channel and the bottom right panels the light microscopy images of the respective area. Twenty-four hours after the injection of 0.4 μg of rrTNF-α (B) or 8 hours after the injection of 100 ng of rrVEGF (D) the endothelial signal for IL-33 has decreased compared to the mock injection (A and C, respectively). Pictures were obtained using identical exposure times and image enhancement parameters for A and B as well as for C and D. Arrows indicate endothelial nuclei. Arrowheads indicate ink particles. E: Semiquantitative analysis of vascular IL-33 expression in rat skin after injection of vehicle (PBS), rrTNF-α, or rrVEGF. Endothelial cell nuclei were categorized as negative (0) or increasingly positive (levels 1 to 4) by a blinded observer, counting in each condition 20 nuclei. Scale bars = 50 μm.

Article Snippet: Slides were methanol-fixed and subsequently co-stained for VE-cadherin and IL-33 using another murine antibody to VE-cadherin (clone 4.8.G) and rabbit polyclonal antibody to IL-33 (IL-33Nter), followed by a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) and Cy3-conjugated goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) and Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories).

Techniques: In Vivo, Immunostaining, Injection, Staining, Light Microscopy, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Acid Stripping of Surface IgE Antibodies Bound to FcεRI Is Unsuitable for the Functional Assays That Require Long-Term Culture of Basophils and Entire Removal of Surface IgE

doi: 10.3390/ijms21020510

Figure Lengend Snippet: Response of the phosphate buffered saline (PBS)-treated human peripheral blood basophils to IL-3 stimulation. Basophils were incubated on ice for 5 min with PBS. Cells were then washed and cultured in serum-free X-VIVO 15 medium (0.1 × 10 6 cells/well per 200 µL) in 96-well U-bottomed plate along with IL-3 (100 ng/0.5 million cells) for 24 h. ( A ) The forward and side scatter plot of the basophils immediately following PBS treatment and following 24-h culture in the presence of IL-3. ( B ) Histogram overlays showing the expression of basophil activation markers CD69, ST2 and FcεRI in PBS-treated basophils cultured for 24 h in IL-3. Representative data from four donors are presented.

Article Snippet: Basophils were analyzed for the expression of FcεRIa (FcεRIa-FITC, clone CRA-1, Miltenyi Biotec, Paris, France), CD69 (CD69-APC/Cy7, Clone FN50, BD Biosciences, Le Pont de Claix, France), CD123 (CD123-BV421, clone 9F5, BD Biosciences), ST2 (ST2/IL-33R-PE polyclonal goat IgG, R&D Systems, Lille, France) and surface intensity of IgE (anti-IgE–APC, clone MB10-5C4, Miltenyi Biotec) by using LSR II flow cytometer (BD Biosciences) and data were analyzed by BD FACSDiva v8.0.1 (BD Biosciences) and FlowJo v10 softwares (FlowJo, LLC, Ashland, USA).

Techniques: Saline, Incubation, Cell Culture, Expressing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Acid Stripping of Surface IgE Antibodies Bound to FcεRI Is Unsuitable for the Functional Assays That Require Long-Term Culture of Basophils and Entire Removal of Surface IgE

doi: 10.3390/ijms21020510

Figure Lengend Snippet: Response of the acetic acid buffer (pH 4)-treated human basophils to IL-3 stimulation. Basophils were incubated on ice for 5 min either with phosphate buffered saline (PBS) or ice-cold acetic acid buffer pH 4 (AA pH 4). Cells were then washed and cultured in serum-free X-VIVO 15 medium (0.1 × 10 6 cells/well per 200 µL) in 96-well U-bottomed plate along with IL-3 (100 ng/0.5 million cells) for 24 h. ( A ) The forward and side scatter plot of AA pH 4-treated basophils following 24-h culture in IL-3. ( B ) The yield of basophils (mean ± SD, n = 4 donors) after 24 h culture of basophils in IL-3. The values were calculated based on the percentage of cells in the P1 gate of forward and side scatter plot. ( C ) Histogram overlays showing the expression of basophil activation markers CD69, ST2 and FcεRI in AA pH 4-treated cells after 24 h culture in IL-3. Representative data from four donors are presented. ( D ) The expression (mean ± SD, n = 4 donors) of various activation markers (% positive cells or median fluorescence intensity, MFI) after 24 h culture of basophils in IL-3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; two-sided Students t-test (panel B) or one-way ANOVA with Tukey’s multiple comparison test (panel D).

Article Snippet: Basophils were analyzed for the expression of FcεRIa (FcεRIa-FITC, clone CRA-1, Miltenyi Biotec, Paris, France), CD69 (CD69-APC/Cy7, Clone FN50, BD Biosciences, Le Pont de Claix, France), CD123 (CD123-BV421, clone 9F5, BD Biosciences), ST2 (ST2/IL-33R-PE polyclonal goat IgG, R&D Systems, Lille, France) and surface intensity of IgE (anti-IgE–APC, clone MB10-5C4, Miltenyi Biotec) by using LSR II flow cytometer (BD Biosciences) and data were analyzed by BD FACSDiva v8.0.1 (BD Biosciences) and FlowJo v10 softwares (FlowJo, LLC, Ashland, USA).

Techniques: Incubation, Saline, Cell Culture, Expressing, Activation Assay, Fluorescence, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Acid Stripping of Surface IgE Antibodies Bound to FcεRI Is Unsuitable for the Functional Assays That Require Long-Term Culture of Basophils and Entire Removal of Surface IgE

doi: 10.3390/ijms21020510

Figure Lengend Snippet: Lactic acid treatment does not affect viability, yield and IL-3 induced activation of basophils, but removes only a small fraction of the cell surface bound IgE. Basophils were incubated on ice either with phosphate buffered saline (PBS) or ice-cold lactic acid buffer pH 3.9 (LA). Cells were then washed and cultured in serum-free X-VIVO 15 medium (0.1 × 10 6 cells/well per 200 µL) in 96-well U-bottomed plate along with IL-3 (100 ng/0.5 million cells) for 24 h. ( A ) The viability of cells immediately following LA treatment as analyzed by fixable viable dye staining. ( B ) The forward and side scatter plot of LA-treated basophils following 24-h culture in IL-3. ( C ) The expression of basophil activation markers ST2, FcεRI and CD69 in LA-treated cells after 24 h culture in IL-3 (mean ± SD, n = 4 experiments from two donors). ( D ) Efficacy of stripping of basophil surface-bound IgE by LA as analyzed by surface staining of IgE and analyses by flow cytometry. The intensity of IgE on the surface of basophils was represented by MFI values (Median fluorescence intensity). ** p < 0.01; *** p < 0.001; **** p < 0.0001; one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Basophils were analyzed for the expression of FcεRIa (FcεRIa-FITC, clone CRA-1, Miltenyi Biotec, Paris, France), CD69 (CD69-APC/Cy7, Clone FN50, BD Biosciences, Le Pont de Claix, France), CD123 (CD123-BV421, clone 9F5, BD Biosciences), ST2 (ST2/IL-33R-PE polyclonal goat IgG, R&D Systems, Lille, France) and surface intensity of IgE (anti-IgE–APC, clone MB10-5C4, Miltenyi Biotec) by using LSR II flow cytometer (BD Biosciences) and data were analyzed by BD FACSDiva v8.0.1 (BD Biosciences) and FlowJo v10 softwares (FlowJo, LLC, Ashland, USA).

Techniques: Activation Assay, Incubation, Saline, Cell Culture, Staining, Expressing, Stripping Membranes, Flow Cytometry, Fluorescence, Comparison