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Abstract
Objectives: Neutrophil apoptosis is mandatory for resolving inflammation and is regulated by expression of pro- and anti-apoptotic genes. We studied neutrophils isolated from patients with granulomatosis with polyangiitis (GPA) to investigate apoptosis alterations and to identify transcriptional and circulating factors affecting this process.
Method: We enrolled 36 patients (18 in active stage, 18 in remission) and 18 healthy controls. Circulating levels of tumour necrosis factor-α (TNF-α), granulocyte–macrophage colony-stimulating factor (GM-CSF), macrophage migration inhibitory factor, plasminogen activator inhibitor-1, interferon-γ, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, soluble Fas (sFas), sFas ligand, survivin, and pentraxin-3 (PTX3) were evaluated by enzyme-linked immunosorbent assay/Luminex; circulating apoptotic neutrophils by flow cytometry; and apoptosis-related gene transcripts by real-time polymerase chain reaction.
Results: Patients had decreased fractions of circulating apoptotic neutrophils and delayed neutrophil apoptosis was present in vitro. Circulating levels of TNF-α, GM-CSF, sFas, and PTX3 were higher in GPA. Delayed neutrophil apoptosis was accompanied by decreased mRNA of pro-apoptotic genes and transcription factors (DIABLO, PMAIP1, BAX, CASP3, CASP7, RUNX3, E2F1, TP53) and increased anti-apoptotic CFLAR and BCL2A1 mRNA. TNF-α and sFas levels correlated with circulating apoptotic neutrophils and expression of apoptosis genes. Stimulation with TNF-α of neutrophils from controls significantly down-regulated E2F1 and CASP3 expression.
Conclusions: Circulating neutrophils in GPA have anti-apoptotic phenotype involving both intrinsic and extrinsic pathways of apoptosis. This is accompanied by increased levels of circulating pro-survival factors (GM-CSF, TNF-α, sFas), independent of disease activity. Anti-apoptotic phenotype of neutrophils in GPA is reproduced by exposure to low concentrations of TNF-α.
Acknowledgement
This work was supported by the National Centre of Science in Poland [2011/03/N/NZ6/01578, 2015/17/B/NZ6/03459].
Disclosure statement
No potential conflict of interest was reported by the authors.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
Supplementary figure S1. Quality tests of isolated neutrophils.
Supplementary figure S2. Gating strategy used in flow cytometry analysis of neutrophil apoptosis.
Supplementary figure S3. Neutrophil expression of 93 apoptosis-related genes in patients with active GPA (n = 6).
Supplementary figure S4. (A) Percentage of circulating apoptotic neutrophils in patients with GPA. (B) mRNA expression of analysed genes in neutrophils of patients with GPA.
Supplementary figure S5. Correlation between percentage of circulating apoptotic neutrophils and: neutrophils CD11b expression (A), DNA-MPO complexes (B), MPO (C) and NE (D) level. (E) Correlation between neutrophil activation markers (CD11b, DNA-MPO complexes, MPO, NE) and expression of analysed genes.
Supplementary figure S6. Correlation between circulating DNA-MPO complexes and plasma levels of PTX3 in patients with GPA and healthy controls.
Supplementary table S1. List of genes (with assay number) analysed in the study.
Supplementary table S2. Neutrophil abundance of mRNA of analysed genes (not shown in main part of article).
Supplementary table S3. Circulating levels of measured proteins.
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