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

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.

Neutrophils in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus: Same Foe Different M.O.

“…Whilst the main focus of investigation into the phenotype of LDGs has focused on SLE and to a lesser extent RA, their presence in the blood is not exclusive to rheumatic disease. LDGs have been identified in many other disease settings, including asthma, vasculitis, multiple sclerosis and chronic kidney disease (287)(288)(289)(290). Indeed, they are even present in low numbers in healthy controls (291).…”

Ultra-pure isolation of low density neutrophils casts doubt on their exceptionality in health and disease

“…Granulocyte markers, CD66b, CD15 and CD16 (FcγRIII) are frequently used to identify LDNs within the PBMC cell population (see Table 1). In Rheumatoid Arthritis and ANCA positive Vasculitis, CD16 expression is reduced on LDNs [30,39] while in lung adenocarcinoma and HIV infection, CD15 is elevated on LDNs. [3,13] Furthermore LDNs in gastric cancers have high CD66b expression.…”

Neutrophils: Need for Standardized Nomenclature

“…LDNs display either an immature morphology with banded nuclei or myelocyte features, and are likely released in response to emergency granulopoiesis, or have a mature morphology with segmented nuclei (3,20). Uı́Mhaonaigh et al found that CD16 int/-CD10 -LDNs resemble immature neutrophils while CD16 + /CD10 + LDNs share morphological features with NDNs (44). Similar to descriptions of TANs, LDNs exhibit protumorigenic and immunosuppressive functions (20).…”