Disease Activity and Tendency to Relapse in ANCA-Associated Vasculitis Are Reflected in Neutrophil and Intermediate Monocyte Frequencies

Abstract

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases with inflammation affecting small blood vessels and includes granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). In this study, we investigated granulocyte and monocyte subsets in a large cohort of AAV patients with emphasis on disease activity and tendency to relapse. A cohort of 105 patients with GPA or MPA and 126 healthy controls (HCs) were included. Clinical and laboratory data were collected for all patients, including disease activity, tendency to relapse, and pharmacological treatment. Using flow cytometry, circulating eosinophils, basophils, neutrophils, and monocytes were assessed. The monocytes were subdivided into classical (CD14⁺⁺CD16^-), intermediate (CD14⁺⁺CD16⁺), and nonclassical (CD14^-CD16⁺) monocytes. Mature (CD16^high) or newly released (CD16^dim) neutrophils were defined, as well as the frequency of CD177⁺ neutrophils. AAV patients displayed increased frequencies of intermediate monocytes, mature and newly released neutrophils, and an expanded population of CD177⁺ neutrophils compared to HC. MPA patients differed from GPA patients in terms of lower frequency of classical monocytes. No differences in cell frequencies regarding ANCA phenotype were observed. Paired data from 23 patients demonstrated that active disease was associated with an increased frequency of mature neutrophils and a decreased frequency of monocytes, in particular intermediate monocytes. Moreover, GPA patients with a tendency to relapse displayed an increased frequency of mature neutrophils with increased expression of CD177⁺. Relapsing MPA patients, on the other hand, showed decreased frequency of intermediate monocytes. Finally, rituximab treatment was associated with increased frequencies of classical and intermediate monocytes. In conclusion, AAV patients exhibit a skewing of different neutrophil and monocyte subpopulations that are associated with disease subtypes, disease activity, rituximab treatment, and propensity to relapse. These changes may contribute to the inflammatory process and could potentially be used as biomarkers for relapse prediction.

Figure 1

Increased frequency of intermediate monocytes in AAV patients compared to healthy controls. The frequencies of (a) classical (CD14⁺⁺CD16⁻), (b) intermediate (CD14⁺⁺CD16⁺), (c) nonclassical (CD14⁻CD16⁺) monocytes, and (d) neutrophils CD177⁺ in AAV patients and HC, analyzed with flow cytometry, as described in Section 2. AAV patients present a higher frequency of intermediate monocytes and neutrophils CD177⁺, but not of classical and nonclassical, compared to HC. Mann–Whitney U test was used to calculate the level of significance. Data are presented with medians.  ^∗∗∗∗Indicates p-value < 0.0001. WBC, white blood cell; AAV, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; HC, healthy controls; ns, not significant.

Figure 2

Decreased frequency of classical monocytes in MPA compared to GPA patients. The frequencies of (a) classical (CD14⁺⁺CD16⁻), (b) intermediate (CD14⁺⁺CD16⁺), and (c) nonclassical (CD14⁻CD16⁺) monocytes in GPA and MPA patients. Analysis of monocytes in MPA patients compared to GPA patients evidenced a lower frequency of classical monocytes. No significant difference was shown between the two groups regarding intermediate and nonclassical monocytes. The Mann–Whitney U test was used to calculate the level of significance. Data are presented with medians.  ^∗Indicates p-value < 0.01. WBC, white blood cell; GPA, granulomatosis with polyangiitis; MPA, microscopic polyangiitis, ns, not significant.

Figure 3

Concentration of (a) mature (CD16^high) neutrophils, and frequencies of (b) total monocytes, and (c) intermediate (CD14⁺⁺CD16⁺) monocytes in 23 patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in active disease and remission. Patients with active disease present higher concentrations of mature neutrophils but lower frequencies of total monocytes and intermediate monocytes compared to patients in remission. Wilcoxon matched-pairs signed rank test was used to calculate the level of significance. ^∗and  ^∗∗∗Indicate p-value < 0.05 and <0.001, respectively. WBC, white blood cell.

Figure 4

Comparison of granulocyte and monocyte frequencies in patients with and without a tendency to relapse (Ttr). Frequencies of (a) eosinophils, (b) mature CD16^high, and (c) CD177⁺ neutrophils in AAV patients with Ttr or No Ttr, where patients with Ttr presented decreased frequency of eosinophils, but increased frequency of mature neutrophils and CD177⁺ neutrophils. Analysis of cell subsets in GPA patients with Ttr and without Ttr demonstrated statistically significant increased frequencies of (d) mature CD16^high and (e) CD177⁺ neutrophils. The investigation of the cell subsets in MPA patients with Ttr and without Ttr identified decreased frequency of (g) intermediate (CD14⁺⁺CD16⁺) monocytes but no statistically significant difference of (f) classical (CD14⁺⁺CD16⁻), and (h) nonclassical (CD14⁻CD16⁺) monocytes. Mann–Whitney U test was used to calculate the level of significance. Data are presented with medians.  ^∗and  ^∗∗Indicate p-value < 0.05 and <0.01, respectively. AAV, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis; Ttr, tendency to relapse; No Ttr, no tendency to relapse; GPA, granulomatosis with polyangiitis; MPA, microscopic polyangiitis; ns, not significant.

Figure 5

Increased monocyte frequencies in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients with rituximab treatment during the past 365 days. The frequencies of (a) total monocytes, (b) classical (CD14⁺⁺CD16⁻), (c) intermediate (CD14⁺⁺CD16⁺), and (d) nonclassical (CD14⁻CD16⁺) monocytes in patients with RTX or No RTX treatment. Total, classical, and intermediate monocytes were presented with higher frequency in patients in RTX treatment. No significant difference was observed in the comparison of nonclassical monocytes between the two groups. Mann–Whitney U test was used to calculate the level of significance. Data are presented with medians.  ^∗Indicates p-value < 0.05. WBC, white blood cell; RTX, rituximab treatment; No RTX, no rituximab treatment; ns, not significant.