Membrane Proteinase 3 Expression in Patients with Wegener’s Granulomatosis and in Human Hematopoietic Stem Cell–Derived Neutrophils

Abstract

A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3 high ) is a risk for Wegener's granulomatosis (WG).The relationship between mPR3 expression and clinical manifestations was investigated in 81 WG patients and mPR3 expression was studied in CD34 ؉ stem cell-derived human neutrophils. The mPR3 high neutrophil percentage correlated with renal function, anemia, and albumin at the time of presentation. The mPR3 high neutrophil percentage and renal failure severity correlated directly after 5 yr. For elucidating mechanisms that govern mPR3 expression, studies were conducted to determine whether the genetic information that governs mPR3 expression resides within the neutrophils, even without stimuli possibly related to disease. CD34 ؉ hematopoietic stem cells were differentiated to neutrophils, and their mPR3 expression was determined. A two-step amplification/differentiation protocol was used to differentiate human CD34 ؉ hematopoietic stem cells into neutrophils with G-CSF. The cells progressively expressed the neutrophil surface markers CD66b, CD35, and CD11b. The ferricytochrome C assay demonstrated a strong respiratory burst at day 14 in response to PMA but none at day 0. Intracellular PR3 was detectable from day 4 by Western blotting. An increasing percentage of a mPR3-positive neutrophil subset became detectable by flow cytometry, whereas a second subset remained negative, consistent with a bimodal expression. Finally, human PR3-anti-neutrophil cytoplasmic autoantibodies induced a stronger respiratory burst, compared with human control IgG in stem cell-derived neutrophils. Taken together, these studies underscore the clinical importance of the WG mPR3 phenotype. The surface mPR3 on resting cells is probably genetically determined rather than being dictated by external factors.

Trafficking of QD‐Conjugated MPO‐ANCA in Murine Systemic Vasculitis and Glomerulonephritis Model Mice

“…It is noteworthy that some healthy controls show lower MPO translocation on the surface of neutrophils even in unstimulated conditions in our experiments, and the ratio of MPOtranslocated neutrophils also varied among patients and even in healthy controls. Similar features are also reported by PR3 expression levels on the surface of activated neutrophils; PR3 expression levels varied significantly in patients and even in healthy controls (44)(45)(46). These results indicate that resident translocation levels of MPO might be involved in the potential risk for developing MPO-ANCA-associated RPGN.…”

NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils

“…11 Mononuclear cells from heparinanticoagulated cord blood were obtained by centrifugation over a LSM1077 (PAA, Pasching, Austria) gradient (800g, 30 minutes). Cells were washed and stained with direct CD34 ϩ progenitor isolation kit (Miltenyi Biotech) and sorted according to the manufacturer's instructions.…”

“…To study mPR3 and mNB1 expression during neutrophil development, we used a model of in vitro differentiation from umbilical CD34 ϩ hematopoietic stem cells as described earlier. 11 Differentiation was assessed by morphology and neutrophil surface marker expression (Figure 4). Identical mPR3-and mNB1-positive percentages were detected after 7 days of differentiation.…”

“…[6][7][8] The mean percentage of mPR3-positive neutrophils is increased in patients with ANCA-associated vasculitis, and a higher percentage predicts an unfavorable clinical course. [9][10][11][12] In contrast to the importance of mPR3, little is known about the mechanism of its membrane display. Hydrophobic insertion has been proposed in a system of reconstituted lipid bilayers.…”

Endothelium-Neutrophil Interactions in ANCA-Associated Diseases

“…52 -56 Positive correlations are also reported between a high percentage of mPR3 + neutrophils and abnormal renal function parameters in patients with GPA. 57 Priming with TNF-a increases the mPR3 expression in both CD177 2 and CD177 + subpopulations 55 and the low level of mPR3 then present on CD177 2 neutrophils appears to be sufficient for some of the effects promoted by anti-PR3 antibodies, such as actin polymerization and oxidative bursts. 55,58 More recently, anti-PR3 ANCA were shown phox , and PR3 from secretory vesicles to the plasma membrane, resulting in a co-localization of gp91 phox , b2-integrins, and FcgRII.…”