Decreased Neutrophil Apoptosis in Quiescent ANCA-Associated Systemic Vasculitis

Background: ANCA-Associated Systemic Vasculitis (AASV) is characterized by leukocytoclasis, accumulation of unscavenged apoptotic and necrotic neutrophils in perivascular tissues. Dysregulation of neutrophil cell death may contribute directly to the pathogenesis of AASV. less thanbrgreater than less thanbrgreater thanMethods: Neutrophils from Healthy Blood Donors (HBD), patients with AASV most in complete remission, Polycythemia Vera (PV), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and renal transplant recipients (TP) were incubated in vitro, and the rate of spontaneous apoptosis was measured by FACS. Plasma levels of cytokines and sFAS were measured with cytometric bead array and ELISA. Expression of pro/anti-apoptotic factors, transcription factors C/EBP-alpha, C/EBP-beta and PU.1 and inhibitors of survival/JAK2-pathway were measured by real-time-PCR. less thanbrgreater than less thanbrgreater thanResults: AASV, PV and RA neutrophils had a significantly lower rate of apoptosis compared to HBD neutrophils (AASV 50 +/- 14% vs. HBD 64 +/- 11%, p andlt; 0.0001). In RA but not in AASV and PV, low apoptosis rate correlated with increased plasma levels of GM-CSF and high mRNA levels of anti-apoptotic factors Bcl-2A1 and Mcl-1. AASV patients had normal levels of G-CSF, GM-CSF and IL-3. Both C/EBP-alpha, C/EBP-beta were significantly higher in neutrophils from AASV patients than HBD. Levels of sFAS were significantly higher in AASV compared to HBD. less thanbrgreater than less thanbrgreater thanConclusion: Neutrophil apoptosis rates in vitro are decreased in AASV, RA and PV but mechanisms seem to differ. Increased mRNA levels of granulopoiesis-associated transcription factors and increased levels of sFAS in plasma were observed in AASV. Additional studies are required to define the mechanisms behind the decreased apoptosis rates, and possible connections with accumulation of dying neutrophils in regions of vascular lesions in AASV patients.Funding Agencies|Swedish Research Council|71X-15152|Crafoord Foundation||</p

Proteinase 3 and Neutrophil Apoptosis in ANCA-Associated Systemic Vasculitis

ANCA-Associated Systemic Vasculitis (AASV) is characterized by leukocytoclasis- dying neutrophils surrounding small blood vessels. Previous studies have shown increased plasma PR3 and increased proportion of neutrophils expressing plasma membrane-associated PR3 (mPR3+). Neutrophils were isolated by polymorphprep and membrane expression was measured by FACS. Plasma PR3, pro-PR3 and cytokines were measured by ELISA. Neutrophil apoptosis was measured by FACS after 20h in vitro culture. Expression of pro/anti-apoptotic factors and transcription factors C/EBP-α, C/EBP-β and PU.1 was measured by real-time PCR. Plasma PR3, pro-PR3, and proportion of mPR3+neutrophils were elevated in AASV. mPR3 and CD177 were co-expressed on a subset of neutrophils both in AASV and controls. mPR3 expression was not correlated directly to circulating PR3 or PR3 gene transcription, but was dependent on transcription of the CD177 gene. AASV neutrophils had a significantly lower rate of apoptosis than neutrophils from healthy blood donors (HBD). Lower apoptosis rate did not correlate with clinical data, plasma PR3, cytokine levels, mPR3 expression or level of pro/anti-apoptotic factors. However, C/EBP-α and C/EBP-β were significantly higher in neutrophils from AASV patients than in HBD neutrophils. These results indicate an altered neutrophil phenotype in AASV and suggest that the rate of granulopoiesis is higher, while the rate of neutrophil apoptosis is lower. This may predispose for the presence of dying neutrophils in regions of inflammation in AASV. Additional studies are required to determine why the rate of neutrophil apoptosis is lower in AASV patients and if dysregulation of neutrophil apoptosis contributes directly to the pathogenesis

Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils.

Proteinase 3 (PR3) is found in granules of all nentrophils but also on the plasma membrane of a subset of nentrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of nentrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where A mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3- and CD177-expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener's granulomatosis and polycythemia vera

Rate of neutrophil survival and apoptosis.

<p>Neutrophils isolated from 60 HBD, 44 AASV patients, 8 PV patients, 18 TP, 21 SLE patients, and 20 RA patients were cultured in vitro in AIM-V medium. The percentage of surviving neutrophils (1a) and apoptotic neutrophils (1b) was measured after 20 hours. % Neutrophil survival = % of annexin-V negative and 7-AAD negative cells after 20 hour culture. % Neutrophil apoptosis = % of annexin-V positive and 7-AAD negative cells after 2o hour culture. HBD = healthy blood donors. AASV = ANCA-Associated Systemic Vasculitis. PV = Polycythemia Vera. TP = renal transplant recipients. SLE = Systemic Lupus Erythematosus. RA = Rheumatoid Arthritis.</p