Abstract
ObjectiveDifferential gene expression in CD177+ and CD177− neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV).MethodsNeutrophils were isolated from healthy controls (HC) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177− subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177− subsets in microarray analysis were re-assessed using quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry.ResultsThe proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177− neutrophils resulted in 14 genes with fold change (fc) >3 difference in expression. Interestingly, 10 of these genes have been reported to change significantly in expression during neutrophil maturation, and most of these genes were granule protein (GP) coding genes. mRNA expression levels measured by RT-PCR of a number of these GP, and of PR3 and MPO were higher in the CD177− neutrophil subset in HC, however, particular granule protein amounts were comparable between CD177+ and CD177− neutrophil subsets. AAV patients had higher amounts of CD177+ neutrophils, but contrary to neutrophils from HC expression of GP-genes was increased, possibly due to activation.ConclusionThe neutrophil population can be distinguished by membrane expression of CD177 into subsets that are different in expression of GP mRNA but not in GP protein production. GP gene expression is also elevated in AAV patients, which is not explained by skewed distribution of CD177+ and CD177− subsets but may be associated with neutrophil activation during on-going inflammation.
Highlights
Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) comprise granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), which share a spectrum of clinical manifestations reflecting necrotizing damage to small- and medium-sized vessels [1,2]
We have reported that proportions of both membrane bound form of PR3 (mPR3)- and CD177-expressing neutrophils are increased in AAV patients and a high percentage of mPR3high neutrophils is a risk factor for relapse in GPA [16,17,18]
These observations indicate that two subsets of neutrophils exist based on CD177 expression and that skewed distribution of these two subpopulations may play a role in the pathogenesis of AAV, we showed that CD177+ and CD1772 neutrophils can be activated by Proteinase 3 (PR3)-ANCA [16]
Summary
Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) comprise granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), which share a spectrum of clinical manifestations reflecting necrotizing damage to small- and medium-sized vessels [1,2]. Proteinase 3 (PR3) and myeloperoxidase (MPO), both of which are mainly stored in primary granules of neutrophils, have been identified as ANCA antigens [5,6,7]. Either specificity can occur with any AAV phenotype, PR3-ANCA are most frequently detected in sera of GPA patients [8]. PR3 is mainly contained in azurophilic granules. In many individuals a membrane bound form of PR3 (mPR3) can be detected in a subset of neutrophils making these accessible for ANCA binding. The percentage of mPR3 expressing neutrophils ranges from 0 to 100% and is genetically determined [9,10]. The percentage of mPR3high neutrophils is constant in time and is not affected by neutrophil activation, disease activity or therapy [9,10,11]
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