Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: Interleukin-3 (IL-3) induces CD69 expression on human basophils and eosinophils. Flow cytometry analysis data are shown of CD69 expression in basophils and eosinophils after 18 hr of culture in medium alone or in medium containing 300 pm IL-3. The figure shows representative histograms of three independent experiments. The percentage of positive cells is indicated.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: Expressing, Flow Cytometry

Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: The phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (LY) blocks interleukin-3 (IL-3)-induced cell survival and CD69 expression in human basophils. (a) LY inhibits the IL-3-induced basophil survival. Freshly isolated human basophils were cultured in medium alone or with 300 pm IL-3, or in the presence or absence of LY294002 (25 µm or 50 µm), or 50 µm PD98059. At 48 hr, basophils were collected and stained with annexin-V and propidium iodide (prop. iod.), and analysed by flow cytometry. Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide negativity indicates cell viability (% cell survival). *P < 0·05 when compared with IL-3 alone. (b) LY inhibits CD69 expression on basophils. The experimental condition was the same as in (a), except that the cells were cultured for 18 hr and then stained with anti-CD69 or isotype-control antibody (IgG2b). A representative histogram is shown of human basophils stained with anti-CD69 or control antibody after 18 hr of culture with IL-3 alone and IL-3 plus 50 µm LY. Values in parenthesis indicate the mean fluorescence intensity. The figure is representative of three independent experiments.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: Expressing, Isolation, Cell Culture, Staining, Flow Cytometry, Control, Fluorescence

Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: Lactacystin (LC) inhibits interleukin-3 (IL-3)-induced cell survival and CD69 surface expression in human basophils. (a) LC inhibits IL-3-induced cell survival. Freshly isolated human basophils were cultured in medium alone, or in medium containing IL-3 or IL-3 + LC, for 48 hr. Basophils were then collected and stained with annexin-V and propidium iodide, and analysed by using flow cytometry. Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (prop. iod.) negativity indicates cell viability (% cell survival). These data presented here are representative of three experiments. (b) LC inhibits IL-3-induced cell-surface expression of CD69. A representative histogram is shown of human basophils stained with anti-CD69 or control antibody after 18 hr of culture with IL-3 alone or IL-3 + 10 µm LC. The experimental conditions were the same as in (a), except that the cells were cultured for 18 hr and then stained with anti-CD69 or isotype-control antibody (IgG2b). Values in parenthesis indicate the mean fluorescence intensity. The figure is representative of three independent experiments.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: Expressing, Isolation, Cell Culture, Staining, Flow Cytometry, Control, Fluorescence

Journal:

Article Title: Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-?B-dependent and -independent pathways

doi: 10.1046/j.1365-2567.2002.01517.x

Figure Lengend Snippet: A schema presenting our hypothesis that interleukin-3 (IL-3) inactivates phosphatidylinositol 3-kinase (PI3-K), initiating signalling cascades that lead to the generation of survival genes through both an NF-κB pathway, as well as through an alternate mechanism. In summary, we demonstrated that the in vitro spontaneous rate of apoptosis of human basophils is higher than that of eosinophils, and that IL-3 inhibits basophil apoptosis as well as up-regulating basophil CD69 surface expression via a PI3-K-dependent mechanism(s) that entails new RNA and protein synthesis, partially via NF-κB signalling. As basophils are active participants in allergic reactions, and IL-3 is one of the most abundant proinflammatory cytokines in the secretions from allergic tissue, IL-3-mediated inhibition of basophil apoptosis may exacerbate the inflammation associated with allergic disorders.

Article Snippet: Specific mouse anti-human CD69 (MCA 736) and isotype-matched control antibody [mouse immunoglobulin G2b (IgG2b) negative control, MCA 691] were obtained from Serotec (Toronto, Ontario, Canada).

Techniques: In Vitro, Expressing, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: A critical role for the autophagy gene Atg5 in T cell survival and proliferation

doi: 10.1084/jem.20061303

Figure Lengend Snippet: Impaired proliferation of Atg5 −/− T cells upon anti-CD3 stimulation. (A) Proliferation of Atg5 −/− and wild-type CD4 + T cells upon TCR stimulation. Splenocytes from control and Atg5 −/− chimeras were labeled with CFSE and stimulated under the following conditions for 3 d: 5 μg/ml of plate-bound anti-CD3, or plus 1 μg/ml plate-bound anti-CD28, or plus 100 U/ml of recombinant IL-2, or 10 ng/ml PMA plus 300 ng/ml of ionomycin. Shown are the percentage of cells with diluted CFSE staining and the mean fluorescence intensity of the proliferating population. (B) Up-regulation of CD25 and CD69 on Atg5 −/− and wild-type T cells after overnight stimulation with plate-bound anti-CD3. (C) Apoptosis rates of Atg5 −/− and wild-type T cells after TCR stimulation. Total splenocytes were cultured either in media alone or with 5 μg/ml of plate bound anti-CD3 for the indicated times. Apoptosis was measured by annexin V staining. Data are representative of two experiments.

Article Snippet: Antibodies used included anti-CD4, -CD8, -TCR-β, -B220, -Gr1, -Mac1, -CD25, -CD69, and -CD45.2 (eBioscience, BioLegend, and BD Biosciences).

Techniques: Control, Labeling, Recombinant, Staining, Fluorescence, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response

doi: 10.1084/jem.20042155

Figure Lengend Snippet: The monovalent and multivalent TCR complexes are differentially sensitive to antigen stimulation. (A and C) T cells respond differentially over a wide range of antigen concentrations. The HA peptide–specific T cell line CH7C17 and the PCC peptide–specific T cell hybridoma 2B4 were stimulated for 24 h with varying concentrations of peptide loaded onto either DAP-DR1 or DCEK APCs. Surface staining of the T cells with anti-CD69 antibodies was measured using a flow cytometer and the percentage of positive cells is depicted in semilogarithmic scale. Values are mean ± SD. (B and D) At low antigen doses, only multivalent TCRs become tyrosine phosphorylated. CH7C17 and 2B4 cells were antigen stimulated for 5 min as in A and C or stimulated with pervanadate. Cells were lysed in Brij96, and phosphorylated TCRs were affinity purifed with immobilized antiphosphotyrosine antibody and resolved by two-dimensional BN/reducing SDS-PAGE. Immunoblotting was performed using an antiphosphotyrosine antibody (B) or anti-ζ (D). The position of phosphorylated ζ (p-ζ) is indicated.

Article Snippet: The antiphosphotyrosine (4G10) and anti-LAT antibodies were purchased from Upstate Biotechnology, the anti-Flag (M2) was purchased from Sigma-Aldrich, the anti-CD3 OKT3 hybridoma was purchased from the American Type Culture Collection, and the anti-CD69–PE was purchased from Caltag.

Techniques: Staining, Flow Cytometry, SDS Page, Western Blot