Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Increased interleukin-21 (IL-21) in sera and CD4+ T cells of patients with systemic lupus erythematosus (SLE). (a) The concentrations of IL-21 in sera isolated from 22 SLE patients and 16 healthy controls were analysed by ELISA. (b) Peripheral blood mononuclear cells (PBMCs) and CD4+ T cells were isolated from heparinized peripheral blood obtained from three patients and three healthy controls. The mRNA expression levels of IL-21 and IL-21 receptor were determined by RT-PCR. Results are expressed as means ± standard deviation (SD). (**P < 0·001).

Article Snippet: The wells were treated with blocking solution (PBS containing 1% BSA and 0·05% Tween-20), samples and the standard recombinant IL-21 (R&D Systems) were added to the 96-well plate, and the plate was incubated.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Oestrogen treatment increased interleukin-21 (IL-21) expression in CD4+ T cells of systemic lupus erythematosus (SLE) patients in a dose- and time-dependent manner. (a, b) Isolated CD4+ T cells were treated with 10, 100 and 1000 nm 17β-oestradiol for 48 hr. The mRNA expression of IL-21 was measured by RT-PCR (a). The concentration of IL-21 in the supernatant was determined by ELISA (b). (c) CD4+ T cells were cultured with 1000 nm 17β-oestradiol for 24, 48 and 72 hr. The concentration of IL-21 in the supernatant was determined by ELISA. Data are expressed as means ± SD of three independent experiments using cells obtained from three patients and three healthy controls. (*P < 0·05, **P < 0·01, versus untreated cells of SLE patients).

Article Snippet: The wells were treated with blocking solution (PBS containing 1% BSA and 0·05% Tween-20), samples and the standard recombinant IL-21 (R&D Systems) were added to the 96-well plate, and the plate was incubated.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Synergistic effect of T-cell receptor (TCR) stimulation on interleukin-21 (IL-21) production by oestrogen-pretreated CD4+ T cells in systemic lupus erythematosus (SLE) patients. Isolated CD4+ T cells from SLE patients were cultured with 10, 100 and 1000 nm 17β-oestradiol for 48 hr in the presence of 0·5 μg/ml CD3 antibody. The concentration of IL-21 in the supernatant was determined by ELISA. Data are expressed as means ± SD of three independent experiments using cells obtained from three different patients. (*P < 0·05, versus cells treated with 1000 nm 17β-oestradiol only).

Article Snippet: The wells were treated with blocking solution (PBS containing 1% BSA and 0·05% Tween-20), samples and the standard recombinant IL-21 (R&D Systems) were added to the 96-well plate, and the plate was incubated.

Techniques: Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Signalling pathways involved in oestrogen-induced interleukin 21 (IL-21) production. (a) Isolated CD4+ T cells from systemic lupus erythematosus (SLE) patients were pre-treated with 20 μm PD98509, 10 μm SB203850, 1 μm SP600125, 20 μm LY294002, 50 μm AG490, 50 μm BAY 11, or 10 μm curcumin for 2 hr, and then cultured with 1000 nm of 17β-oestradiol for 48 hr. The concentration of IL-21 in the supernatant was measured by ELISA. Data are expressed as means ± SD of three independent experiments using cells obtained from three different patients. (b) CD4+ T cells from SLE patients were treated with 1000 nm 17β-oestradiol for 20 min. Phosphorylated forms of mitogen-activated protein kinases were measured by Western blot analysis. The representative Western blots of three independent experiments using cells from three different patients are shown. (*P < 0·05, **P < 0·01).

Article Snippet: The wells were treated with blocking solution (PBS containing 1% BSA and 0·05% Tween-20), samples and the standard recombinant IL-21 (R&D Systems) were added to the 96-well plate, and the plate was incubated.

Techniques: Isolation, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Immunology

Article Title: Oestrogen up-regulates interleukin-21 production by CD4 + T lymphocytes in patients with systemic lupus erythematosus

doi: 10.1111/imm.12263

Figure Lengend Snippet: Increased antibody secretion by B cells co-cultured with oestrogen-stimulated CD4+ T cells. (a, b) CD4+ T cells from systemic lupus erythematosus (SLE) patients were treated with 1000 nm 17β-oestradiol for 48 hr. CD4+ T cells and culture supernatants were then separated by centrifugation. B cells isolated from the peripheral blood of healthy controls were cultured for 96 hr with oestrogen-pretreated T cells (a) and the culture supernatant (b), respectively, in the absence or presence of interleukin-21 (IL-21) blocking antibody. The concentrations of secreted IgG, IgG1 and IgG2a by B cells were measured by ELISA. Data are expressed as means ± SD of three independent experiments using cells from three patients and three healthy controls. (*P < 0·05, **P < 0·01, versus untreated cells, #P < 0·05, ##P < 0·01, versus 1000 nm 17β-oestradiol-treated cells).

Article Snippet: The wells were treated with blocking solution (PBS containing 1% BSA and 0·05% Tween-20), samples and the standard recombinant IL-21 (R&D Systems) were added to the 96-well plate, and the plate was incubated.

Techniques: Cell Culture, Centrifugation, Isolation, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: IL‐21 and IL‐21 receptors are elevated during the acute phase of MIRI and CD4+ T‐cells are the major source of IL‐21 in the ischaemic myocardium. The time course of changes in the (A) mRNA and (B) protein expression of IL‐21 in the myocardium following MIRI were measured by quantitative real‐time PCR and western blotting, respectively (n = 6 per group). The time course of changes in the (C) mRNA and (D) protein expression of IL‐21 receptors (IL‐21R) in the myocardium following MIRI were measured via quantitative real‐time PCR and western blotting, respectively (n = 6 per group). (E) The infiltrated IL‐21+ leukocytes in myocardial I/R mice after 6 h of reperfusion were analysed by flow cytometry. CD45+ cells were isolated and restimulated. The IL‐21+ CD45+ cells were further analysed for CD3, CD4 and γδTCR expression to detect the cellular source of IL‐21. The proportion of different IL‐21‐secreting cells in the IL‐21+CD45+ cells were quantitatively analysed (n = 5 per group). *P < 0.05 versus sham.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Isolation

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: IL‐21 neutralization attenuates, whereas exogenous IL‐21 aggravates, myocardial injury. (A) Representative images of left ventricular slices from different groups 1 day after reperfusion. The non‐ischaemic area is indicated in blue, the area at risk (AAR) in red and the infarct area (I) in white. (B) Quantification of infarct size of myocardial tissues 1 day after reperfusion. (C) Serum cTnT was measured 1 day after reperfusion. (D) Representative M‐mode echocardiographic images of the left ventricular 1 day after reperfusion. (E) Left ventricular EF and FS were measured via echocardiography 1 day after reperfusion. *P < 0.05 versus isotype; # P < 0.05 versus vehicle. Isotype, n = 9; anti‐IL‐21, n = 9; vehicle, n = 11; IL‐21, n = 10.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Neutralization

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: IL‐21 increases the number of neutrophils infiltrating the myocardium and the myocardial expression of KC and MIP‐2 following MIRI, while anti‐IL‐21 mAb reduced cardiac infiltration of neutrophils and the expression of KC and MIP‐2. (A–D) The number, percentage and representative contour plots of CD11b+Gr‐1+ neutrophils infiltrating the myocardium after 30 min of ischaemia and 3 h of reperfusion were analysed by flow cytometry (n = 5 per group). (E, F) The mRNA expression of KC, MIP‐2 and LIX in the myocardium after 30 min of ischaemia followed by 30 min or 3 h of reperfusion was analysed via real‐time PCR (n = 5 per group). *P < 0.05 versus Sham; #P < 0.05 versus vehicle or isotype.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: IL‐21 induces neutrophil migration and the expression of KC and MIP‐2 in CMs and CFs. (A) Neutrophil migration in the presence of conditioned supernatants from stimulated CMs (upper) and CFs (lower) was measured by transwell assay. The values were normalized relative to those of the medium from unstimulated CMs or CFs (control). (B) The mRNA expression of KC and MIP‐2 in response to 1 h of stimulation with IL‐21(100 ng·mL−1) in CMs (left) and CFs (right). (C) ELISA results for KC and MIP‐2 levels in the supernatants of CMs (left) and CFs (right) stimulated by IL‐21(100 ng·mL−1) for 24 h. H2O2 (100 μM) or TNF‐α (5 ng·mL−1) served as positive controls for CMs and CFs respectively. IL‐21‐alone control media was from unstimulated cells ‘spiked’ with IL‐21. Data are representative of five independent experiments. *P < 0.05 versus control.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Expressing, Transwell Assay, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: Activation of the ERK, p38 MAPK, Akt, NF‐κB, STAT1 and STAT3 signalling pathways in the myocardium after the administration of exogenous IL‐21. (A) Representative western blots showing the activation of different signalling pathways 10 min after reperfusion in the myocardium after IL‐21 administration. (B) Quantitative analysis of the levels of phospho‐/total‐ERK, p38 MAPK, Akt, NF‐κB, STAT1 and STAT3 signalling pathways 10 min after reperfusion in different groups (n = 6 per group). (C) Representative western blots showing the activation of different signalling pathways 30 min after reperfusion in the myocardium after IL‐21 administration. (D) Quantitative analysis of the levels of phospho‐/total‐ERK, p38 MAPK, Akt, NF‐κB, STAT1 and STAT3 signalling pathways 30 min after reperfusion in different groups (n = 6 per group). *P < 0.05 versus sham; # P < 0.05 versus vehicle.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Western Blot

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: Direct effects of IL‐21 on the activation of Akt, p38 MAPK and NF‐κB signalling in isolated CMs and CFs. (A, B) Representative western blots showing the phospho‐/total‐Akt, p38 MAPK and NF‐κB p65 levels in CMs and CFs unstimulated (control) or stimulated with IL‐21 (100 ng·mL−1) for indicated durations. (C, D) Quantitative analysis of the phospho‐/total‐Akt, p38 MAPK and NF‐κB p65 levels in CMs and CFs. Data are representative of five independent experiments. *P < 0.05 versus control.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Isolation, Western Blot

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: IL‐21‐induced chemokine expression is Akt/NF‐κB or p38 MAPK/NF‐κB dependent. (A) CMs were pretreated for 1 h with an Akt inhibitor (LY294002, 10 μM) or an NF‐κB inhibitor (BAY11‐7082, 10 μM), followed by IL‐21 (100 ng·mL−1) stimulation for 1 h. The expression of KC and MIP‐2 was measured via real‐time PCR. (B) CMs were pretreated for 1 h with the indicated inhibitors, followed by IL‐21 stimulation for 24 h. Concentrations of KC and MIP‐2 in the culture media were measured by ELISA. (C) CFs were pretreated for 1 h with a p38 MAPK inhibitor (SB203580, 10 μM) or an NF‐κB inhibitor (BAY11‐7082, 10 μM), followed by IL‐21 (100 ng·mL−1) stimulation for 1 h. The expression of KC and MIP‐2 was measured by real‐time PCR. (D) CFs were pretreated for 1 h with the indicated inhibitors, followed by IL‐21 stimulation for 24 h. Concentrations of KC and MIP‐2 in the culture media were measured by ELISA. (E) CMs were pretreated with LY294002, and (F) CFs were pretreated with SB203580 and then cells were stimulated with IL‐21 or vehicle for 1 h. NF‐κB p65 phosphorylation was analysed by western blotting. Representative western blot images (left) and quantitative analyses (right) are shown. Data are representative of five independent experiments. *P < 0.05 versus control; # P < 0.05 versus IL‐21.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: British Journal of Pharmacology

Article Title: IL‐21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration

doi: 10.1111/bph.13781

Figure Lengend Snippet: Schematic illustration of the IL‐21‐mediated effects on neutrophil recruitment in MIRI. MIRI induces the up‐regulation of IL‐21 in the myocardium, which directly acts on CMs and CFs to promote the mRNA expression and production of KC and MIP‐2 via the activation of Akt/NF‐κB and p38 MAPK/NF‐κB signalling in CMs and CFs respectively. KC and MIP‐2 are potent neutrophil chemoattractants, which recruit neutrophils into the injured myocardium.

Article Snippet: Recombinant mouse‐IL‐21 and primary antibodies to mouse IL‐21 and IL‐21 receptor were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Activation Assay

Journal: Mucosal immunology

Article Title: Interleukin (IL)-21 promotes intestinal IgA response to microbiota

doi: 10.1038/mi.2014.134

Figure Lengend Snippet: ( A ) Fecal pellets were collected from age-matched wild-type, IL-21 −/− , and IL-21R −/− B6 mice. IgA levels were quantified through ELISA and normalized to total protein. N = 2 mice per group, data is reflective of 2 independent experiments. *p<0.05 compared to wild-type mice. ( B ) Naïve IgD + B cells were activated with anti-µ (5µg/ml) and anti-CD40 (5µg/ml) in the presence of TGFβ1 (5ng/ml), IL-17 (50ng/ml), IL-21 (20ng/ml), or retinoic acid (1µM), or a combination of TGFβ1, IL-17, IL-21, and/or retinoic acid. On day 5, IgA was measured from the supernatant by ELISA. Data is reflective of 6 independent experiments. *p<0.05, ***p<0.001 compared between samples. + p<0.05, ++ p<0.01 compared to untreated cells.

Article Snippet: The following antibodies were used for flow cytometry: PE-IL-17A (TC11-18H10.1), FITC-B220 (RA3-6B2), PE-α 4 β 7 (DATK32), FITC-CD4 (RM4-5), PE/Cy7-CD38 (90), APC-CD138 (281-2), and PE/Cy7-PD-1 (29F.1A12) were purchased from Biolegend; APC-RORγt (B2D), APC-GL-7, and eFluor450-Streptavidin were from eBioscience; Brilliant Violet 421-CD95 (Jo2) and biotinylated CXCR5 (2G8) were from BD Biosciences; recombinant mouse IL-21R human FC chimera was from R&D Systems; AlexaFluor 647 antihuman IgG FCγ (polyclonal) was from Jackson ImmunoResearch; PE-IgA (polyclonal) was from Southern Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Interleukin (IL)-21 promotes intestinal IgA response to microbiota

doi: 10.1038/mi.2014.134

Figure Lengend Snippet: 10 6 CBir1 Th17 cells were i.v. transferred into TCR®x™ −/− mice. ( A ) On day 44 post-Th17 cell transfer, expression of surface IgA on splenic, intestinal lamina propria, or Peyer’s patch B cells of CBir1 Th17 cell recipients, Th17 cell recipients receiving neutralizing IL-21r (Th17+αIL-21r), or control TCRβxδ −/− mice was determined by flow cytometry. FACS plots are representative of 4 mice per group. ( B ) IgA + plasma cells and mature B cells in the spleens and intestines. Bar charts reflect mean +/− SEM. Data reflects 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 between compared samples. LPL = intestinal lamina propria lymphocytes; PP = Peyer’s patch; PC = plasma cell

Article Snippet: The following antibodies were used for flow cytometry: PE-IL-17A (TC11-18H10.1), FITC-B220 (RA3-6B2), PE-α 4 β 7 (DATK32), FITC-CD4 (RM4-5), PE/Cy7-CD38 (90), APC-CD138 (281-2), and PE/Cy7-PD-1 (29F.1A12) were purchased from Biolegend; APC-RORγt (B2D), APC-GL-7, and eFluor450-Streptavidin were from eBioscience; Brilliant Violet 421-CD95 (Jo2) and biotinylated CXCR5 (2G8) were from BD Biosciences; recombinant mouse IL-21R human FC chimera was from R&D Systems; AlexaFluor 647 antihuman IgG FCγ (polyclonal) was from Jackson ImmunoResearch; PE-IgA (polyclonal) was from Southern Biotechnology.

Techniques: Expressing, Control, Flow Cytometry, Clinical Proteomics

Journal: Mucosal immunology

Article Title: Interleukin (IL)-21 promotes intestinal IgA response to microbiota

doi: 10.1038/mi.2014.134

Figure Lengend Snippet: ( A ) On day 44 post-Th17 cell transfer, IgG and IgA in the serum was measured by ELISA. *p<0.05, **p<0.01, ***p<0.001 ( B ) Fecal pellets were collected from Th17 cell recipients during the course of the experiment. IgA levels were quantified through ELISA and normalized to total protein. Changes in expression over time are expressed as a fold change from individuals pre-transfer. N = 4 mice per group, from 3 independent experiments. *p<0.05 as compared to anti-IL-21r group. ( C ) Intracellular IgA production was measured from total bone marrow cells of CBir1 Th17 cell recipients, Th17 cell recipients receiving neutralizing IL-21r (Th17+αIL-21r), or control TCRβxδ −/− mice by flow cytometry. FACS plots are representative of 4 mice per group.

Article Snippet: The following antibodies were used for flow cytometry: PE-IL-17A (TC11-18H10.1), FITC-B220 (RA3-6B2), PE-α 4 β 7 (DATK32), FITC-CD4 (RM4-5), PE/Cy7-CD38 (90), APC-CD138 (281-2), and PE/Cy7-PD-1 (29F.1A12) were purchased from Biolegend; APC-RORγt (B2D), APC-GL-7, and eFluor450-Streptavidin were from eBioscience; Brilliant Violet 421-CD95 (Jo2) and biotinylated CXCR5 (2G8) were from BD Biosciences; recombinant mouse IL-21R human FC chimera was from R&D Systems; AlexaFluor 647 antihuman IgG FCγ (polyclonal) was from Jackson ImmunoResearch; PE-IgA (polyclonal) was from Southern Biotechnology.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Control, Flow Cytometry

Journal: Mucosal immunology

Article Title: Interleukin (IL)-21 promotes intestinal IgA response to microbiota

doi: 10.1038/mi.2014.134

Figure Lengend Snippet: 10 6 CBir1 Th17 cells were i.v. transferred into TCR®x™ −/− mice. ( A ) On day 44 post-Th17 cell transfer, expression of germinal center markers GL-7 and CD95 on splenic, mesenteric lymph node, intestinal lamina propria, or Peyer’s patch B220 + cells of CBir1 Th17 cell recipients, Th17 cell recipients receiving neutralizing IL-21r (Th17+αIL-21r), or control TCRβxδ −/− mice was determined by flow cytometry. FACS plots are representative of 4 mice per group. ( B ) IF image of Peyer’s patch from control TCRβxδ −/− mice or CBir1 Th17 cell recipients with 20× objective. Germinal center formation was analyzed by staining for GL-7-AlexaFluor 594 and B220-FITC. ( C ) RNA was collected from ileal tissue on day 44 post-Th17 cell transfer from CBir1 Th17 cell recipients or control TCRβxδ −/− mice and Aicda mRNA was analyzed by RT-PCR. Data is reflective of 4 mice per group, from 3 independent experiments.

Article Snippet: The following antibodies were used for flow cytometry: PE-IL-17A (TC11-18H10.1), FITC-B220 (RA3-6B2), PE-α 4 β 7 (DATK32), FITC-CD4 (RM4-5), PE/Cy7-CD38 (90), APC-CD138 (281-2), and PE/Cy7-PD-1 (29F.1A12) were purchased from Biolegend; APC-RORγt (B2D), APC-GL-7, and eFluor450-Streptavidin were from eBioscience; Brilliant Violet 421-CD95 (Jo2) and biotinylated CXCR5 (2G8) were from BD Biosciences; recombinant mouse IL-21R human FC chimera was from R&D Systems; AlexaFluor 647 antihuman IgG FCγ (polyclonal) was from Jackson ImmunoResearch; PE-IgA (polyclonal) was from Southern Biotechnology.

Techniques: Expressing, Control, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction