Journal: PLoS ONE

Article Title: The Alarmin Concept Applied to Human Renal Transplantation: Evidence for a Differential Implication of HMGB1 and IL-33

doi: 10.1371/journal.pone.0088742

Figure Lengend Snippet: (A, B) PBMCs from kidney graft recipients were recovered before transplantation (D0), and 3 hours (H3) and day 3 (POD3) after transplantation. They were membrane-labelled with anti-CD3-FITC, anti-iNKT-PE 6B11 clonotype, and anti-CD69-PerCP/Cy5.5. CD69 analysis was performed by flow cytometry gating on CD3(+)6B11(+) cells, defined as iNKT cells: (A) Flow cytometry plot showing expression profiles of surface marker CD69 on iNKT cells ex vivo from one representative patient at D0 (filled histogram), H3 (bold line) and POD3 (dotted line). Numbers indicate MFI of CD69 expression on iNKT lymphocytes. (B) Mean Fluorescence Intensity (MFI) of CD69 expression on iNKT lymphocytes from the patient cohort (n = 16 at D0, H3, and POD3). (C, D) PBMCs from healthy adult donors (n = 6) were cultured with (black columns) or without (white columns) HMGB1(C) or IL-33 (D) for 3, 6 or 24 hours of culture. MFI of CD69 expression on iNKT lymphocytes was analysed by Flow cytometry as described in (A, B). Data are expressed as means ± SEM. **p<0.01, ***p<0.001 by Wilcoxon test.

Article Snippet: Membrane antibodies FITC mouse anti-human CD3, PE mouse anti-human iNKT cell (clone 6B11) and PerCP/Cy5.5 mouse anti-human CD69 were provided by BD Biosciences (San Jose, CA).

Techniques: Transplantation Assay, Flow Cytometry, Expressing, Marker, Ex Vivo, Fluorescence, Cell Culture

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Panel (a) of this figure depicts the expression of the T cell activation marker GITR on CD25+, CD25 bright and CD25 verybright cells from healthy controls (white bars, n = 24) and SSc patients having limited cutaneous SSc (light gray bars, n = 18), late diffuse SSc (dark gray bars, n = 22) and early diffuse SSc (black bars, n = 22) patients. In panel (b) the expression of CD62L on Tregs is investigated. CD25+ and CD25 bright cells from SSc and healthy controls express similar levels of CD62L, whereas CD25 verybright from SSc patient subsets exhibit lower levels of CD62L compared to those from healthy controls. Panel (c) reflects the expression of CD69 on Tregs from healthy donors and SSc patients. CD25+, CD25 bright and CD25 verybright cells from SSc patients express significant lower levels of CD69 than those from healthy donors. CD69 expression on CD25 bright and CD25 verybright cells from edSSc patients was significantly lower then that from ldSSc patients, and ldSSc expressed CD69 significantly lower than those from lSSc. In panel (d) the expression on CD3+ cells is shown for all investigated groups. In contrast with that observed on Tregs from SSc patients, CD69 expression on CD4+ cells was significantly higher in all SSc patient groups. Panel (e) reflects the potential association between CD69 expression on Tregs and disease duration. CD69 expression on T regs from patients with lSSc correlated with disease duration, whereas this was not the case either with ldSSc nor edSSc. In all figures the white bars represent healthy controls, whereas lSSc, ldSSc and edSSc patients are represented by light gray, dark gray and black bars, respectively.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Expressing, Activation Assay, Marker

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Unsorted CD3+ (MACS bead isolated) were stimulated with PHA (5 µg/ml) and consecutively incubated with CD25 high CD127 - or CD25 low CD127 high cells for 5 days. Thereafter, CD3+ cells were incubated with 3 H -thymidine for 24 more hours after which 3 H -thymidine incorporation was measured. Panel (a) reflects the suppressive capacity of Tregs from healthy donors and SSc patients. Proliferation of CD3+ effector cells was effectively inhibited by T regulatory cells from healthy controls, whereas a clearly diminished suppressive activity was observed in the experiments with Tregs from SSc patients. Suppressive effect of Treg (CD25 high CD127 - ) and non-Tregs (CD25 low CD127 high ) is presented in black and white bars, respectively. Results are the mean and SEM of 6 separate experiments using cells from healthy donors (n = 9), lSSc (n = 7), ldSSc (n = 9) and edSSc (n = 7). Panel (b) represents the correlation of CD69 expression and Treg suppressive capacity in Tregs from the various groups under investigation. The percentage of CD69 positive regulatory T cells (CD25 high CD127 - ) correlates well with the percentage of inhibition of CD3+ cells in healthy controls (triangles), lSSc (diamonds), ldSSc (circles) and edSSc (squares). Panel (c) reflects the expression of intracellular TGFβ in Tregs from healthy controls and SSc patients as measured using intracellular flow cytometry. CD25 high CD127 - cells from all SSc patients express lower TGFβ levels compared to controls. Left panel reflects an representative individual from each group whereas the right panel displays the mean of each group comprising 6 individuals (per group) coming forth from 4 independent experiments.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Isolation, Incubation, Activity Assay, Expressing, Inhibition, Flow Cytometry

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: (a) During the co-cultures of unsorted CD3+ cells with either Tregs (CD25 high CD127 - ) or non-Tregs (CD25 low CD127 high ) 10 or 25% plasma from an edSSc patient or healthy control was added to the culture. The graph represents data from 3 independent experiments using 3 healthy control cells, and plasma derived from two edSSc patients and two control individuals. (b) The effect of SSc plasma was evaluated by adding 10% to CD3+ cells for 24 hrs stimulated with PHA or unstimulated. As a control, CD69 expression was measured on CD3+ cells stimulated with PHA only. CD4 and CD25 high /FoxP3 high cells were gated based on the expression of these markers using flow cytometry. (c) CD69 expression and induction upon PHA mediated stimulation of CD4+ and CD25 high /FoxP3 high obtained from healthy donors, lSSc, ldSSc and edSSc patients was investigated using flow cytometry. One representative patient from each group is shown.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Clinical Proteomics, Control, Derivative Assay, 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: 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