JASN Express. Published on May 18, 2005 as doi: 10.1681/ASN.2004070609 Membrane Proteinase 3 Expression in Patients with Wegener’s Granulomatosis and in Human Hematopoietic Stem Cell–Derived Neutrophils Adrian Schreiber,* Bjoern Otto,* Xinsheng Ju,† Martin Zenke,† Ursula Goebel,* Friedrich C. Luft,* and Ralph Kettritz* *HELIOS Klinikum-Berlin, Franz Volhard Clinic and Max Delbru¨ck Center for Molecular Medicine, Medical Faculty of the Charite´, Humboldt University of Berlin, Berlin; and †Helmholtz Institute of Biomedical Engineering, Department of Cell Biology, University Hospital of Aachen, Aachen, Germany A large membrane proteinase 3 (mPR3)-positive neutrophil subset (mPR3high) 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 mPR3high neutrophil percentage correlated with renal function, anemia, and albumin at the time of presentation. The mPR3high 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. J Am Soc Nephrol 16: ???–???, 2005. doi: 10.1681/ASN.2004070609 nti-neutrophil cytoplasmic autoantibodies (ANCA) mPR3high neutrophils is stable during a person’s lifetime and are found in patients with systemic small-vessel vas- does not change appreciably with activation status or the neu- A culitis (1–3). A pathogenic role of ANCA interacting trophil’s age (14–16). A high percentage of mPR3high neutro- with ANCA–antigen-containing neutrophils and monocytes phils was a risk factor for vasculitis, was associated with re- was suggested by numerous in vitro studies (4–10). Further lapse in a Dutch cohort of patients wit WG, and resulted in evidence for a central role of ANCA as a pathogenetic factor of stronger neutrophil activation by PR3 ANCA in vitro in earlier vasculitis was recently shown in an animal model of the disease studies (15–18). Thus, a detailed understanding of mechanisms (11). ANCA directed against proteinase 3 (PR3) are found in that control this dual mPR3 expression pattern is important. We Wegener’s granulomatosis (WG) (12,13). Intracellular PR3 is recently described a strong genetic influence on mPR3 expres- translocated from granules to the cell membrane upon neutro- sion in monozygotic and dizygotic twins, where a high within- phil activation. However, some PR3 is also expressed on cell pair correlation was found only in monozygotic twins (16). membranes of nonactivated resting neutrophils. It is interesting However, whether the genetic information that determines the that the total neutrophil population of a given individual can be percentage of mPR3high neutrophils resides in the cell or in the high divided into a membrane PR3-positive (mPR3 ) and a mem- extracellular host milieu is not known. The aim of this investi- low brane PR3-negative (mPR3 ) (14). Although the amount of gation was to explore the relationship between mPR3 pheno- mPR3 changes with activation, the individual percentage of type and clinical course in our WG patient cohort. In addition, we investigated whether hematopoietic stem cells could be differentiated into neutrophils and whether this differentiation Received July 28, 2004. Accepted March 17, 2005. would provide a feasible approach to study mPR3 expression Published online ahead of print. Publication date available at www.jasn.org. and ANCA-induced neutrophil activation. Finally, we tested the hypothesis that the stem cells were innately equipped with Address correspondence to: Dr. Ralph Kettritz, Franz Volhard Clinic, Wiltberg- low high strasse 50, 13122 Berlin, Germany. Phone: ϩ49-30-9417-2202; Fax: ϩ49-30-9417- the information to generate a mPR3 and mPR3 neutro- 2206; E-mail: [email protected] phil subset. Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1607-0001 2 Journal of the American Society of Nephrology J Am Soc Nephrol 16: ???–???, 2005 Materials and Methods FCS, 2 mM l-glutamine, 0.1 mM 2-mercaptoethanol, 100 U/ml penicil- Patients and Control Subjects lin and streptomycin (Life Technologies-BRL), and 10 ng/ml G-CSF for ϫ 6 We included 81 PR3-ANCA–positive WG patients. At the time of 16d(1 10 cells/ml). Every 2 d, growth factors were added and cells ϫ 6 mPR3 phenotyping, 66 of the 81 patients had a positive ANCA test. The were maintained at 1 10 cells/ml cell density. After differentiation diagnosis was made on the basis of the criteria of the Chapel Hill into neutrophils, duplicate samples were used for all further assays. Consensus Conference (19) and the American College of Rheumatology Stem cell factor and thrombopoietin were from Amgen (Thousand (20). PR3-ANCA was assayed by indirect immunofluorescence on eth- Oaks, CA), and Flt3 ligand and G-CSF was from PeproTech (London, anol-fixed neutrophils and by PR3-specific enzyme-linked immunoad- UK). Hyper–IL-6 was produced in yeast as described previously (21). sorbent assay. Relapse was defined as a rapid rise in creatinine levels accompanied by urinary sediment activity, the detection of active vas- Determination of Surface Antigen Expression by Flow culitis, or glomerulonephritis; pulmonary hemorrhage or expanding Cytometry nodules; the observation of iritis or uveitis; or new mononeuritis mul- FACS was used as described previously to evaluate the expression of tiplex. Relapse occurred in 53% of the patients. A total of 154 healthy surface molecules on neutrophils (18). Briefly, cells were spun down at subjects were assayed for mPR3 expression and served as control. 200 ϫ g for 7 min at 4°C. Pellets were resuspended in HBSS without ϩ ϩ Ca2 /Mg2 before they were incubated with dilutions of the indicated Materials antibodies. In case the primary antibodies were not FITC conjugated, Recombinant TNF-␣ was obtained from Genzyme (Ru¨sselsheim, Ger- we used a secondary FITC-conjugated F(ab)2 fragment of goat anti- many). The monoclonal mouse antibody to PR3 was obtained from CLB mouse IgG. Flow cytometry was performed on the same day using a (Amsterdam, Netherlands), and FITC-conjugated F(ab)-fragment of FACScan (Becton Dickinson, Heidelberg, Germany), and 10,000 events goat anti-mouse IgG was from DAKO (Hamburg, Germany). Dextran per sample were collected. was purchased from Amersham Pharmacia (Amsterdam, Netherlands). HBSS, PBS, and trypan blue were from Seromed (Berlin, Germany). Preparation of Immunoglobulins Histopaque 1083 was obtained from Sigma-Aldrich (Deisenhofen, Ger- Human IgG was prepared from one patient with biopsy-proven WG many). The following antibodies were used: CD11b (FITC-conjugated) (PR3-ANCA) as well as from one healthy donor as described previ- and CD66b (FITC-conjugated) both from Immunotech (Krefeld, Ger- ously (18). Plasma samples were obtained from freshly drawn blood many) and CD35 (FITC-conjugated) from Cymbus Biotech (Hants, UK). and kept at Ϫ20°C. Plasma was filtered through a 0.2-␮m syringe filter Endotoxin-free reagents and plastic disposables were used in all exper- (Gelman Sciences, Ann Arbor, MI) and applied to a HiTrap protein G iments. affinity column (Pharmacia, Uppsala, Sweden). Bound IgG was eluted with 0.1 M glycine-HCl buffer (pH 2.75; elution buffer). After the Isolation of Human Neutrophils antibodies emerged, the pH was immediately adjusted to pH 7.0 using Neutrophils from healthy volunteers were isolated from heparinized 1 M Tris-HCl (pH 9.0). A mouse mAb to myeloperoxidase (MPO; whole blood by red blood cell sedimentation with dextran 1%, followed MPO-7, IgG1) and an isotype-matched control (IgG1) were purchased by Ficoll-Hypaque density gradient centrifugation and hypotonic from Dako (Hamburg, Germany). Before use, IgG preparations were ϫ erythrocyte lyses. Neutrophils were centrifuged (10 min at 1050 rpm) centrifuged at 10,000 g for 5 min to remove aggregates. ϩ and resuspended in HBSS with calcium and magnesium (HBSS2 ). The cell viability was detected by trypan blue exclusion and exceeded 99%. Western Blot Analysis of PR3 The neutrophil percentage in the suspension was Ͼ95% by Wright- Cells were lysed with 20 ␮l of ice-cold lysing buffer (20 mM Tris-HCl Giemsa staining. [pH 8.0] that contained 138 mM NaCl, 1% Triton X-100, 2 mM EDTA, 10% glycerol, 0.2 mM sodium orthovanadate, 1 mM PMSF, 10 ␮g/ml ␮ Differentiation of Human Hematopoietic Stem Cells into aprotinin, 10 g/ml leupeptin, 0.1 mM quercetin, and 5 mM Iodoacet- Neutrophils amide). Samples were kept on ice for 5 min, supernatant was recovered ϩ ϫ Granulocytes were obtained from CD34 stem cells of cord blood by centrifugation at 13,000 g for 5 min at 4°C, and protein concen- from two different donors and from peripheral blood of three different tration was measured by BCA protein assay (Pierce, Munich, Ger- healthy volunteers by using a two-step amplification/differentiation many).