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

O'Brien EC, Abdulahad WH, Rutgers A, Huitema MG, O'Reilly VP, Coughlan AM, Harrington M, Heeringa P, Little MA, Hickey FB - Sci Rep (2015)

Bottom Line: Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3).Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production.These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

View Article: PubMed Central - PubMed

Affiliation: Trinity Health Kidney Centre, Department of Clinical Medicine, Trinity College Dublin, St. James' Hospital Campus, Dublin 8, Ireland.

ABSTRACT
ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1β in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

No MeSH data available.


Related in: MedlinePlus

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 μg/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.
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f6: 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 μg/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.

Mentions: To investigate activation of monocytes following binding of ANCA to surface antigen, we measured release of pro-inflammatory cytokines IL-1β, IL-6, IL-8 and IL-12p70. Monocytes were purified from peripheral blood mononuclear cells (PBMCs) by positive selection based on CD14 expression, primed with tumour necrosis factor (TNF)-α and stimulated with either monoclonal antibodies (mAb) directed against MPO or PR3 or with immunoglobulin G (IgG) purified from the plasma of patients with AAV. Treatment with anti-MPO mAb resulted in significantly increased IL-1β, IL-6 and IL-8 production (Fig. 6A). This increase in inflammatory cytokine production was also observed when monocytes were stimulated with IgG purified from anti-MPO+ AAV patients (Fig. 6B). Interestingly, this effect was not seen in monocytes stimulated with either mAb directed against PR3 (Fig. 6C) or protein G purified IgG from anti-PR3+ AAV patients (Fig. 6D). Secretion of IL-12p70 was not detected under any of the conditions tested.


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

O'Brien EC, Abdulahad WH, Rutgers A, Huitema MG, O'Reilly VP, Coughlan AM, Harrington M, Heeringa P, Little MA, Hickey FB - Sci Rep (2015)

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 μg/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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493694&req=5

f6: 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 μg/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.
Mentions: To investigate activation of monocytes following binding of ANCA to surface antigen, we measured release of pro-inflammatory cytokines IL-1β, IL-6, IL-8 and IL-12p70. Monocytes were purified from peripheral blood mononuclear cells (PBMCs) by positive selection based on CD14 expression, primed with tumour necrosis factor (TNF)-α and stimulated with either monoclonal antibodies (mAb) directed against MPO or PR3 or with immunoglobulin G (IgG) purified from the plasma of patients with AAV. Treatment with anti-MPO mAb resulted in significantly increased IL-1β, IL-6 and IL-8 production (Fig. 6A). This increase in inflammatory cytokine production was also observed when monocytes were stimulated with IgG purified from anti-MPO+ AAV patients (Fig. 6B). Interestingly, this effect was not seen in monocytes stimulated with either mAb directed against PR3 (Fig. 6C) or protein G purified IgG from anti-PR3+ AAV patients (Fig. 6D). Secretion of IL-12p70 was not detected under any of the conditions tested.

Bottom Line: Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3).Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production.These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

View Article: PubMed Central - PubMed

Affiliation: Trinity Health Kidney Centre, Department of Clinical Medicine, Trinity College Dublin, St. James' Hospital Campus, Dublin 8, Ireland.

ABSTRACT
ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1β in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

No MeSH data available.


Related in: MedlinePlus