Background Low density granulocyte (LDG), a proinflammatory population of neutrophils, was first described in systemic lupus erythematosus (SLE) and has been shown to contribute to lupus pathogenesis. It has been suggested that LDGs have a pathogenic role in ANCA associated vasculitis (AAV) based on the data of gene expression signature in AAV and the ability of excessive neutrophil extracellular traps (NETs) formation by LDGs in AAV. However, more detailed analysis of LDG in AAV patients has not been reported.

Objectives In this study we investigated the characteristics of LDG in AAV patients using flow cytometry and proteomics approach and examined the correlations with disease activity.

Methods We examined the presence of LDGs in peripheral blood of 10 AAV patients before treatment and followed them for 4 months with immunosuppressive therapy. Normal-density granulocytes (NDGs) were isolated by dextran sedimentation and PBMCs were isolated by Ficoll gradient. LDGs were assessed using cell surface expression of CD14 and CD15 by flow cytometry and isolated by magnetic bead procedure from PBMCs. We performed comparative proteomic analysis among LDGs and autologous NDGs and healthy controls (HC)-NDGs.

Results LDG frequencies were 9.8-fold higher in AAV patients before treatment relative to HC. There were two distinct populations of LDGs showing different cell surface expressions of CD10, CD14, and CD15 in AAV patients. One population of LDGs was mainly CD10 positive and another one was CD10 negative. Although the frequency of CD10 positive-LDG decreased along with decrease of disease activity, the frequency of CD10 negative-LDG increased in 4 months (17.9-fold higher than before treatment). Comparative proteomic analysis revealed these two populations of LDGs were distinct from each other and had common differences from autologous NDGs and HC-NDGs.

LDG frequencies were 9.8-fold higher in AAV patients before treatment relative to HC. There were two distinct populations of LDGs showing different cell surface expressions of CD10, CD14, and CD15 in AAV patients. One population of LDGs was mainly CD10 positive and another one was CD10 negative. Although the frequency of CD10 positive-LDG decreased along with decrease of disease activity, the frequency of CD10 negative-LDG increased in 4 months (17.9-fold higher than before treatment). Comparative proteomic analysis revealed these two populations of LDGs were distinct from each other and had common differences from autologous NDGs and HC-NDGs.

Conclusions We identified two distinct subsets of LDGs in AAV. It is possible that they play different roles in the pathogenesis of AAV.

References [1] Denny MF, et al. A distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type I Interferons. J Immunol2010;184(6):3284–3297.

[2] Grayson, et al. Neutrophil-Related Gene Expression And Low-Density Granulocytes Associated with Disease Activity and Response to Treatment in ANCA-Associated Vasculitis. Arthritis Rheumatol2015;67:1922–1932.

Disclosure of Interest None declared