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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

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 μg/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
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f7: 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 μg/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

Mentions: As we found MPO to be differentially expressed on different monocyte subsets, we next investigated if the production of IL-1β, IL-6 and IL-8 observed following stimulation of total monocytes differed in a subset-specific manner. Classical, intermediate and non-classical cells were sorted from MACS purified monocytes based on CD14 and CD16 expression (Supplemental Fig. S5). Sorted cells were primed with TNF-α and treated with anti-MPO mAb. This stimulation failed to induce secretion of any of the cytokines tested (IL-1β; IL-6; IL-8) from non-classical monocytes (Fig. 7A–C). IL-1β production was found to vary most between monocyte subsets, with intermediate monocytes producing higher quantities than classical monocytes both basally and in response to anti-MPO stimulation (Fig. 7A). IL-6 production was found to be similar in classical and intermediate monocytes and to be increased in both subsets following incubation of cells with anti-MPO (Fig. 7B). Conversely only classical monocytes were found to secrete IL-8 in response to anti MPO mAb (Fig. 7C).


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)

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

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

f7: 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 μg/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
Mentions: As we found MPO to be differentially expressed on different monocyte subsets, we next investigated if the production of IL-1β, IL-6 and IL-8 observed following stimulation of total monocytes differed in a subset-specific manner. Classical, intermediate and non-classical cells were sorted from MACS purified monocytes based on CD14 and CD16 expression (Supplemental Fig. S5). Sorted cells were primed with TNF-α and treated with anti-MPO mAb. This stimulation failed to induce secretion of any of the cytokines tested (IL-1β; IL-6; IL-8) from non-classical monocytes (Fig. 7A–C). IL-1β production was found to vary most between monocyte subsets, with intermediate monocytes producing higher quantities than classical monocytes both basally and in response to anti-MPO stimulation (Fig. 7A). IL-6 production was found to be similar in classical and intermediate monocytes and to be increased in both subsets following incubation of cells with anti-MPO (Fig. 7B). Conversely only classical monocytes were found to secrete IL-8 in response to anti MPO mAb (Fig. 7C).

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