Journal: Scientific reports

Article Title: Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

doi: 10.1038/srep11888

Figure Lengend Snippet: Figure 2. The ANCA autoantigens MPO and PR3 are preferentially expressed on intermediate monocytes. Peripheral blood was collected from patients with AAV and the percentage of cells expressing cell-surface MPO and PR3 was examined by flow cytometry. The percentage of MPO and PR3 positive cells in each subset is shown for (A–B) anti-MPO+ AAV patients and (C–D) anti-PR3+ AAV patients. Each symbol represents a separate individual. Open circles represent patients in remission and filled triangles patients with active disease. Data are presented as the median and interquartile range. Non-parametric one- way ANOVA (Friedman test) and Dunn’s post-test were used to test for significance (*p < 0.05, **p < 0.01; ****p < 0.0001). Class: classical; Int: intermediate: NC: non-classical.

Article Snippet: The cells were incubated with 2.5 μ g Fc block (BD Biosciences)/1 × 106 cells @ 37 °C for 30 minutes and then stimulated with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO (2C7, Acris Antibodies) or isotype control antibody (IgG1, Acris Antibodies) for 4 hours.

Techniques: Immunopeptidomics, Expressing, Flow Cytometry

Journal: Scientific reports

Article Title: Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

doi: 10.1038/srep11888

Figure Lengend Snippet: Figure 3. Cell-surface expression of MPO and PR3 is not linked on monocytes. Peripheral blood was collected from patients with AAV and the percentage of cells expressing surface MPO and PR3 was examined by flow cytometry. Cells were classified as being MPO+ PR3-, MPO–PR3+ , MPO+ PR3+ . Data are presented for (A) anti–MPO+ patients and (B) anti-PR3+ patients. Each symbol represents an individual patient. Open circles represent patients in remission and filled triangles patients with active disease. Data are presented as the median and interquartile range. Non-parametric one-way ANOVA (Friedman test) and Dunn’s post-test were used to test for significance (**p < 0.01).

Article Snippet: The cells were incubated with 2.5 μ g Fc block (BD Biosciences)/1 × 106 cells @ 37 °C for 30 minutes and then stimulated with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO (2C7, Acris Antibodies) or isotype control antibody (IgG1, Acris Antibodies) for 4 hours.

Techniques: Expressing, Flow Cytometry

Journal: Scientific reports

Article Title: Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

doi: 10.1038/srep11888

Figure Lengend Snippet: Figure 5. MPO and CD16 expression are correlated on intermediate monocytes. Peripheral blood was collected from patients with AAV and analysed by flow cytometry. Following gating on intermediate monocytes the MFI of CD16 was plotted against that of MPO or PR3. Data presented show (A–B) anti-MPO+ AAV patients, (C–D) anti-PR3+ AAV patients. Each symbol represents an individual patient. Open circles represent patients in remission and filled triangles show patients with active disease. Correlation was tested by Spearman Rank Test.

Article Snippet: The cells were incubated with 2.5 μ g Fc block (BD Biosciences)/1 × 106 cells @ 37 °C for 30 minutes and then stimulated with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO (2C7, Acris Antibodies) or isotype control antibody (IgG1, Acris Antibodies) for 4 hours.

Techniques: Expressing, Flow Cytometry

Journal: Scientific reports

Article Title: Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

doi: 10.1038/srep11888

Figure Lengend Snippet: Figure 6. Stimulation of monocytes with anti-MPO mAb or with anti-MPO+ ANCA leads to increased IL-1β, IL-6 and IL-8 production. CD14+ monocytes were isolated from healthy controls PBMCs by MACS separation. The cells were plated and incubated with 5 ng/ml TNF-α @ 37 °C for 30 minutes and then stimulated for 4 hours with 5 μ g/ml of either (A) monoclonal antibody (mAb) directed against MPO, (B) protein G purified IgG from anti-MPO+ patients, (C) mAb against PR3 or (D) IgG purified from anti-PR3+ patients. Supernatants were then removed and levels of IL-1β , IL-6 and IL-8 measured by ELISA. Data are presented as the median and interquartile range of the fold increase over control. Statistical analysis was performed using Wilcoxon signed rank test (*p < 0.05, **p < 0.01, ****p < 0.0001). Iso: isotype control; CTRL: Control.

Article Snippet: The cells were incubated with 2.5 μ g Fc block (BD Biosciences)/1 × 106 cells @ 37 °C for 30 minutes and then stimulated with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO (2C7, Acris Antibodies) or isotype control antibody (IgG1, Acris Antibodies) for 4 hours.

Techniques: Isolation, Incubation, Purification, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific reports

Article Title: Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

doi: 10.1038/srep11888

Figure Lengend Snippet: Figure 7. Intermediate monocytes produce increased amounts of IL-1β and IL-6 both basally and in response to stimulation with anti-MPO mAb. CD14+ monocytes were isolated from PBMCs of healthy control individuals by MACS separation. Monocyte subsets were then sorted from CD14+ cells based on CD14 and CD16 expression. Sorted subsets of cells were incubated with 5 ng/ml TNF-α @ 37 °C for 30 minutes followed by stimulation for 4 hours with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO or an isotype control. Supernatants were then removed and levels of (A) IL-1β , (B) IL-6 and (C) IL-8 measured by ELISA. Data are presented as the median and interquartile range of the fold increase over control. Statistical analysis was performed using Two-way ANOVA and Sidak test to correct for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Class: Classical; Int: Intermediate; NC: Non-Classical

Article Snippet: The cells were incubated with 2.5 μ g Fc block (BD Biosciences)/1 × 106 cells @ 37 °C for 30 minutes and then stimulated with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO (2C7, Acris Antibodies) or isotype control antibody (IgG1, Acris Antibodies) for 4 hours.

Techniques: Isolation, Control, Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

doi: 10.1038/srep11888

Figure Lengend Snippet: Figure 8. Fc receptor binding is not required for anti-MPO-induced IL-1β production by monocytes. CD14+ monocytes were isolated from PBMCs of healthy control individuals by MACS separation. Cells were incubated with 2.5 μ g/1 × 106 cells Fc Block @ 37 °C for 30 minutes followed by stimulation for 4 hours with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO or an isotype control. Supernatants were then removed and levels of IL-1β measured by ELISA. Data are presented as the median and interquartile range. Statistical analysis was performed using Two-way ANOVA and Sidak test to correct for multiple comparisons (**p < 0.01).

Article Snippet: The cells were incubated with 2.5 μ g Fc block (BD Biosciences)/1 × 106 cells @ 37 °C for 30 minutes and then stimulated with 5 μ g/ml of either monoclonal antibody (mAb) directed against MPO (2C7, Acris Antibodies) or isotype control antibody (IgG1, Acris Antibodies) for 4 hours.

Techniques: Binding Assay, Isolation, Control, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay