Low Density Granulocytes in ANCA Vasculitis Are Heterogenous and Hypo-Responsive to Anti-Myeloperoxidase Antibodies.

Aisling Ui Mhaonaigh,Joana Cabral,Ursula Fearon,Mark A Little,Rosemary Lucey,Barry Moran,Kiva Brennan,Achilleas Floudas,Sarah L Doyle,Susan Mcgrath,Alice M Coughlan,Katherine Hughes,Aine De Bhailis,Jack Hartnett,Amrita Dwivedi,Gareth Brady,Eleanor J Molloy,Sarah Cormican

doi:10.3389/fimmu.2019.02603

Abstract

Low Density Granulocytes (LDGs), which appear in the peripheral blood mononuclear cell layer of density-separated blood, are seen in cancer, sepsis, autoimmunity, and pregnancy. Their significance in ANCA vasculitis (AAV) is little understood. As these cells bear the autoantigens associated with this condition and have been found to undergo spontaneous NETosis in other diseases, we hypothesized that they were key drivers of vascular inflammation. We found that LDGs comprise a 3-fold higher fraction of total granulocytes in active vs. remission AAV and disease controls. They are heterogeneous, split between cells displaying mature (75%), and immature (25%) phenotypes. Surprisingly, LDGs (unlike normal density granulocytes) are hyporesponsive to anti-myeloperoxidase antibody stimulation, despite expressing myeloperoxidase on their surface. They are characterized by reduced CD16, CD88, and CD10 expression, higher LOX-1 expression and immature nuclear morphology. Reduced CD16 expression is like that observed in the LDG population in umbilical cord blood and in granulocytes of humanized mice treated with G-CSF. LDGs in AAV are thus a mixed population of mature and immature neutrophils. Their poor response to anti-MPO stimulation suggests that, rather than being a primary driver of AAV pathogenesis, LDGs display characteristics consistent with generic emergency granulopoiesis responders in the context of acute inflammation.

Highlights

Neutrophils have conventionally been considered a uniform, short-lived, and functionallyrestricted population of immune cells [1]
To determine whether Low density granulocytes (LDGs) were elevated in acute Anti-neutrophil cytoplasm autoantibody (ANCA) vasculitis (AAV), PBMC were isolated from peripheral blood of AAV patients and healthy controls (HC) by density gradient centrifugation
As acute AAV is characterized by neutrophil leucocytosis, we assessed whether the observed LDG expansion was merely in proportion to the overall granulocyte expansion

Summary

Introduction

Neutrophils have conventionally been considered a uniform, short-lived, and functionallyrestricted population of immune cells [1]. Anti-neutrophil cytoplasm autoantibody (ANCA) vasculitis (AAV) is a systemic autoimmune disease in which. Low-Density Granulocytes in ANCA-Vasculitis neutrophils play a pivotal role [4]. It is characterized by autoantibodies directed against neutrophil proteins myeloperoxidase (MPO) and proteinase-3 (PR3) and is associated clinically with rapidly progressive glomerulonephritis and inflammatory necrosis of small blood vessels in lungs, skin, and other organs [5,6,7]. Neutrophils obtained from patients with active AAV aberrantly transcribe the autoantigens MPO and PR3, a feature that correlates with subsequent clinical outcome [8]. Most studies in autoimmune diseases suggest that LDGs are pro-inflammatory, relatively longlived and undergo NETosis more readily than NDGs [3, 23]. It has been postulated that these cells are a key pathogenic force of autoimmunity [25]

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