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Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis.

Pepper RJ, Hamour S, Chavele KM, Todd SK, Rasmussen N, Flint S, Lyons PA, Smith KG, Pusey CD, Cook HT, Salama AD - Kidney Int. (2013)

Bottom Line: By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least.Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation.Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not.

View Article: PubMed Central - PubMed

Affiliation: UCL Centre for Nephrology, Royal Free Hospital, London, UK. r.pepper@ucl.ac.uk

ABSTRACT
Antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) commonly results in glomerulonephritis, in which neutrophils and monocytes have important roles. The heterodimer calprotectin (S100A8/S100A9, mrp8/14) is a Toll-like receptor-4 ligand found in neutrophils and monocytes and is elevated in inflammatory conditions. By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least. Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation. Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not. As assessed by flow cytometry, patients with AAV had higher monocyte and neutrophil cell surface calprotectin expression than healthy controls, but this was not associated with augmented mRNA expression in CD14(+) monocytes or CD16(+) neutrophils. Thus, serum calprotectin is a potential disease biomarker in patients with AAV, and may have a role in disease pathogenesis.

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Flow cytometry to demonstrate the cell surface expression of calprotectin on monocytes and neutrophils in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis. (a) Flow cytometry plot demonstrating the identification of the population of CD14-positive cells, neutrophils, and CD3-positive T lymphocytes. Monocytes were identified by anti-CD14 FITC antibody, neutrophils by forward and side scatter, and T cells by anti-CD3 PE antibody. (b, c) Graph to demonstrate calprotectin surface expression of (b) neutrophils and (c) CD14 cells in two individual patients (compared with isotype control). Shaded plot shows the FITC isotype control antibody, whereas other plots show the increase in calprotectin-positive cells in a patient with active AAV (blue) and an ANCA–associated vasculitis (AAV) remission patient (red). (d) Graph to demonstrate % gated neutrophils expressing extracellular calprotectin (% of positive cells based upon gating of the isotype control) (controls vs. AAV active P<0.001, AAV active vs. AAV inactive P<0.05, one-way ANOVA). (e) Graph showing % gated monocytes (CD14) expressing extracellular calprotectin, which follows a similar but nonsignificant trend to the neutrophils (P=NS) (d, e). Monocytes and neutrophils from active AAV (n=10) patients demonstrate the highest surface expression of calprotectin (controls n=7, inactive AAV n=18). (f) Mean fluorescent intensity (MFI) (calprotectin FITC antibody/isotype control antibody) of cells stained for calprotectin, comparing controls, active AAV, and inactive AAV. There was a significant increase in the MFI on neutrophils from acute patients compared with healthy controls (P<0.001). (g) MFI of calprotectin on monocytes demonstrating a significant increase in MFI between patients with active disease and healthy controls (P<0.01).
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fig3: Flow cytometry to demonstrate the cell surface expression of calprotectin on monocytes and neutrophils in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis. (a) Flow cytometry plot demonstrating the identification of the population of CD14-positive cells, neutrophils, and CD3-positive T lymphocytes. Monocytes were identified by anti-CD14 FITC antibody, neutrophils by forward and side scatter, and T cells by anti-CD3 PE antibody. (b, c) Graph to demonstrate calprotectin surface expression of (b) neutrophils and (c) CD14 cells in two individual patients (compared with isotype control). Shaded plot shows the FITC isotype control antibody, whereas other plots show the increase in calprotectin-positive cells in a patient with active AAV (blue) and an ANCA–associated vasculitis (AAV) remission patient (red). (d) Graph to demonstrate % gated neutrophils expressing extracellular calprotectin (% of positive cells based upon gating of the isotype control) (controls vs. AAV active P<0.001, AAV active vs. AAV inactive P<0.05, one-way ANOVA). (e) Graph showing % gated monocytes (CD14) expressing extracellular calprotectin, which follows a similar but nonsignificant trend to the neutrophils (P=NS) (d, e). Monocytes and neutrophils from active AAV (n=10) patients demonstrate the highest surface expression of calprotectin (controls n=7, inactive AAV n=18). (f) Mean fluorescent intensity (MFI) (calprotectin FITC antibody/isotype control antibody) of cells stained for calprotectin, comparing controls, active AAV, and inactive AAV. There was a significant increase in the MFI on neutrophils from acute patients compared with healthy controls (P<0.001). (g) MFI of calprotectin on monocytes demonstrating a significant increase in MFI between patients with active disease and healthy controls (P<0.01).

Mentions: Surface expression was investigated in a subset of healthy controls (n=7), active AAV (n=10), and remission AAV (n=18) patients. Twenty-three patients had renal disease, whereas five had extrarenal disease with no renal involvement. Fifteen were MPO-ANCA-positive and eight were PR3-ANCA-positive, whereas five were ANCA negative. Monocyte gates were determined on the flow cytometer by counterstaining with CD14, lymphocyte gates were determined by a CD3 counterstain, and neutrophil gates were identified by forward- and side-scatter characteristics (Figure 3). There was a greater proportion (% of gated cells) of neutrophils and monocytes with detectable extracellular expression of calprotectin in AAV patients during active disease and convalescence compared with healthy controls (percentage of neutrophils expressing calprotectin (median (range)): healthy controls, 0% (0–3.6%); acute AAV, 78.9% (14.7–98.1%); convalescence AAV, 6.8% (0–98.8%); active AAV compared with controls, P<0.001; and active AAV compared with convalescence AAV, P<0.05 (one-way ANOVA) (Figure 3d); percentage of monocytes expressing calprotectin (median (range)): healthy controls, 5.4% (0–16.9%); acute AAV, 50.5% (0–91.6%); and convalescence AAV, 5.6% (1–94.6%) (P=NS) (Figure 3e). We were unable to detect any surface calprotectin expression on CD3-positive cells. Among patients, the decrease in the proportion of monocytes expressing cell surface calprotectin between the acute and convalescent samples was large but did not reach statistical significance (Figure 3e). There was a statistically significant difference in calprotectin mean fluorescent intensity between acute patients and healthy controls for both neutrophil (P<0.001) (Figure 3f) and monocyte (P<0.01)(Figure 3g) cell surface expression. However, when we compared the levels of S100A8 and S100A9 mRNA extracted from purified patients' CD16-positive neutrophils and CD14-positive monocytes, from the Addenbrooke's hospital cohort, we found no correlation between serum calprotectin and mRNA expression. Moreover, there were no significant differences in mRNA expression between AAV patients and healthy controls (data not shown).


Leukocyte and serum S100A8/S100A9 expression reflects disease activity in ANCA-associated vasculitis and glomerulonephritis.

Pepper RJ, Hamour S, Chavele KM, Todd SK, Rasmussen N, Flint S, Lyons PA, Smith KG, Pusey CD, Cook HT, Salama AD - Kidney Int. (2013)

Flow cytometry to demonstrate the cell surface expression of calprotectin on monocytes and neutrophils in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis. (a) Flow cytometry plot demonstrating the identification of the population of CD14-positive cells, neutrophils, and CD3-positive T lymphocytes. Monocytes were identified by anti-CD14 FITC antibody, neutrophils by forward and side scatter, and T cells by anti-CD3 PE antibody. (b, c) Graph to demonstrate calprotectin surface expression of (b) neutrophils and (c) CD14 cells in two individual patients (compared with isotype control). Shaded plot shows the FITC isotype control antibody, whereas other plots show the increase in calprotectin-positive cells in a patient with active AAV (blue) and an ANCA–associated vasculitis (AAV) remission patient (red). (d) Graph to demonstrate % gated neutrophils expressing extracellular calprotectin (% of positive cells based upon gating of the isotype control) (controls vs. AAV active P<0.001, AAV active vs. AAV inactive P<0.05, one-way ANOVA). (e) Graph showing % gated monocytes (CD14) expressing extracellular calprotectin, which follows a similar but nonsignificant trend to the neutrophils (P=NS) (d, e). Monocytes and neutrophils from active AAV (n=10) patients demonstrate the highest surface expression of calprotectin (controls n=7, inactive AAV n=18). (f) Mean fluorescent intensity (MFI) (calprotectin FITC antibody/isotype control antibody) of cells stained for calprotectin, comparing controls, active AAV, and inactive AAV. There was a significant increase in the MFI on neutrophils from acute patients compared with healthy controls (P<0.001). (g) MFI of calprotectin on monocytes demonstrating a significant increase in MFI between patients with active disease and healthy controls (P<0.01).
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Show All Figures
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fig3: Flow cytometry to demonstrate the cell surface expression of calprotectin on monocytes and neutrophils in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis. (a) Flow cytometry plot demonstrating the identification of the population of CD14-positive cells, neutrophils, and CD3-positive T lymphocytes. Monocytes were identified by anti-CD14 FITC antibody, neutrophils by forward and side scatter, and T cells by anti-CD3 PE antibody. (b, c) Graph to demonstrate calprotectin surface expression of (b) neutrophils and (c) CD14 cells in two individual patients (compared with isotype control). Shaded plot shows the FITC isotype control antibody, whereas other plots show the increase in calprotectin-positive cells in a patient with active AAV (blue) and an ANCA–associated vasculitis (AAV) remission patient (red). (d) Graph to demonstrate % gated neutrophils expressing extracellular calprotectin (% of positive cells based upon gating of the isotype control) (controls vs. AAV active P<0.001, AAV active vs. AAV inactive P<0.05, one-way ANOVA). (e) Graph showing % gated monocytes (CD14) expressing extracellular calprotectin, which follows a similar but nonsignificant trend to the neutrophils (P=NS) (d, e). Monocytes and neutrophils from active AAV (n=10) patients demonstrate the highest surface expression of calprotectin (controls n=7, inactive AAV n=18). (f) Mean fluorescent intensity (MFI) (calprotectin FITC antibody/isotype control antibody) of cells stained for calprotectin, comparing controls, active AAV, and inactive AAV. There was a significant increase in the MFI on neutrophils from acute patients compared with healthy controls (P<0.001). (g) MFI of calprotectin on monocytes demonstrating a significant increase in MFI between patients with active disease and healthy controls (P<0.01).
Mentions: Surface expression was investigated in a subset of healthy controls (n=7), active AAV (n=10), and remission AAV (n=18) patients. Twenty-three patients had renal disease, whereas five had extrarenal disease with no renal involvement. Fifteen were MPO-ANCA-positive and eight were PR3-ANCA-positive, whereas five were ANCA negative. Monocyte gates were determined on the flow cytometer by counterstaining with CD14, lymphocyte gates were determined by a CD3 counterstain, and neutrophil gates were identified by forward- and side-scatter characteristics (Figure 3). There was a greater proportion (% of gated cells) of neutrophils and monocytes with detectable extracellular expression of calprotectin in AAV patients during active disease and convalescence compared with healthy controls (percentage of neutrophils expressing calprotectin (median (range)): healthy controls, 0% (0–3.6%); acute AAV, 78.9% (14.7–98.1%); convalescence AAV, 6.8% (0–98.8%); active AAV compared with controls, P<0.001; and active AAV compared with convalescence AAV, P<0.05 (one-way ANOVA) (Figure 3d); percentage of monocytes expressing calprotectin (median (range)): healthy controls, 5.4% (0–16.9%); acute AAV, 50.5% (0–91.6%); and convalescence AAV, 5.6% (1–94.6%) (P=NS) (Figure 3e). We were unable to detect any surface calprotectin expression on CD3-positive cells. Among patients, the decrease in the proportion of monocytes expressing cell surface calprotectin between the acute and convalescent samples was large but did not reach statistical significance (Figure 3e). There was a statistically significant difference in calprotectin mean fluorescent intensity between acute patients and healthy controls for both neutrophil (P<0.001) (Figure 3f) and monocyte (P<0.01)(Figure 3g) cell surface expression. However, when we compared the levels of S100A8 and S100A9 mRNA extracted from purified patients' CD16-positive neutrophils and CD14-positive monocytes, from the Addenbrooke's hospital cohort, we found no correlation between serum calprotectin and mRNA expression. Moreover, there were no significant differences in mRNA expression between AAV patients and healthy controls (data not shown).

Bottom Line: By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least.Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation.Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not.

View Article: PubMed Central - PubMed

Affiliation: UCL Centre for Nephrology, Royal Free Hospital, London, UK. r.pepper@ucl.ac.uk

ABSTRACT
Antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) commonly results in glomerulonephritis, in which neutrophils and monocytes have important roles. The heterodimer calprotectin (S100A8/S100A9, mrp8/14) is a Toll-like receptor-4 ligand found in neutrophils and monocytes and is elevated in inflammatory conditions. By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least. Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation. Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not. As assessed by flow cytometry, patients with AAV had higher monocyte and neutrophil cell surface calprotectin expression than healthy controls, but this was not associated with augmented mRNA expression in CD14(+) monocytes or CD16(+) neutrophils. Thus, serum calprotectin is a potential disease biomarker in patients with AAV, and may have a role in disease pathogenesis.

Show MeSH
Related in: MedlinePlus