ARTHRITIS & RHEUMATISMVol. 60, No. 5, May 2009, pp 1548–1557DOI 10.1002/art.24442© 2009, American College of Rheumatology

Coexpression of CD177 and Membrane Proteinase 3on Neutrophils in

Antineutrophil Cytoplasmic Autoantibody–AssociatedSystemic Vasculitis

Anti–Proteinase 3–Mediated Neutrophil Activation Is Independent of the Role ofCD177-Expressing Neutrophils

N. Hu, J. Westra, M. G. Huitema, M. Bijl, E. Brouwer, C. A. Stegeman, P. Heeringa,P. C. Limburg, and C. G. M. Kallenberg

Objective. Wegener’s granulomatosis (WG) isstrongly associated with antineutrophil cytoplasmicautoantibodies (ANCAs) directed against proteinase 3(PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed andcolocalizes with CD177/NB1 on circulating neutrophils.We undertook this study to assess the differentialexpression of CD177 on neutrophils from patients withANCA-associated systemic vasculitis (ASV) in compar-ison with patients with systemic lupus erythematosus(SLE), patients with rheumatoid arthritis (RA), andhealthy individuals, and to investigate whether colocal-ization of mPR3 and CD177 affects anti-PR3–mediatedneutrophil activation.

Methods. Expression of CD177 and mPR3 wasanalyzed by flow cytometry on isolated neutrophils frompatients with ASV (n � 53), those with SLE (n � 30),those with RA (n � 26), and healthy controls (n � 31).Neutrophil activation mediated by anti-PR3 antibodies

was assessed by measuring the oxidative burst with adihydrorhodamine assay.

Results. Percentages of CD177-expressing neutro-phils were significantly higher in patients with ASV andthose with SLE than in healthy controls. In 3 healthydonors, CD177 expression was not detected. After prim-ing with tumor necrosis factor �, neutrophils remainednegative for CD177 while mPR3 expression was induced.Neutrophils from CD177-negative donors or CD177�neutrophils sorted from donors with bimodal expressionwere susceptible to anti-PR3–mediated oxidative burst.Variation in the extent of anti-PR3–mediated neutrophilactivation among different donors occurred indepen-dent of the percentage of CD177-expressing neutrophils.

Conclusion. Membrane expression of CD177 oncirculating neutrophils is increased in patients withASV and in those with SLE, but not in RA patients.However, primed neutrophils from CD177-negative in-dividuals also express mPR3 and are susceptible toanti-PR3–mediated oxidative burst, suggesting that re-cruitment of CD177-independent mPR3 is involved inanti-PR3–induced neutrophil activation.

Antineutrophil cytoplasmic autoantibody(ANCA)–associated systemic vasculitis (ASV) com-prises Wegener’s granulomatosis (WG), microscopicpolyangiitis (MPA), and Churg-Strauss syndrome (CSS),which share a spectrum of clinical manifestations reflect-ing necrotizing damage of small- and medium-sizedvessels (1,2). The pathogenic role of ANCAs in ASV is

Supported by the Jan Kornelis de Cock Foundation.N. Hu, BSc, J. Westra, PhD, M. G. Huitema, BSc, M. Bijl,

MD, PhD, E. Brouwer, MD, PhD, C. A. Stegeman, MD, PhD, P.Heeringa, PhD, P. C. Limburg, PhD, C. G. M. Kallenberg, MD, PhD:University Medical Center Groningen, Groningen, The Netherlands.

Address correspondence and reprint requests to C. G. M.Kallenberg, MD, PhD, Department of Rheumatology and ClinicalImmunology, University Medical Center Groningen, Hanzeplein 1,9713 GZ Groningen, The Netherlands. E-mail: c.g.m.kallenberg@int.umcg.nl.

Submitted for publication September 5, 2008; accepted inrevised form January 19, 2009.

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supported by a large body of in vitro and in vivoevidence, and the presence of ANCAs in the circulationis an important serologic marker for the diagnosis andfollowup of ASV (3,4). Proteinase 3 (PR3) and myelo-peroxidase (MPO), both of which are mainly stored inprimary granules of neutrophils, have been identified asANCA antigens (5–7). Although either specificity canoccur with any ASV phenotype, PR3 ANCAs are themost frequently detected in the sera of WG patients (8).

In resting neutrophils, PR3 is contained intracell-ularly in azurophilic granules. However, in many indi-viduals, a membrane-bound form of PR3 (mPR3) canalso be detected in a subset of neutrophils, making itaccessible for ANCA binding. In the general population,the percentage of mPR3-expressing neutrophils rangesfrom 0 to 100% (9). Within a given individual, thepercentage of high-expression mPR3 (mPR3high) neu-trophils is constant over time and is not affected byneutrophil activation, disease activity, or therapy, sug-gesting involvement of genetic factors in the regulationof mPR3 expression (9–11).

The mechanism of mPR3 expression is still underinvestigation. Colocalization of mPR3 with other mem-brane proteins, such as �2 integrin and Fc� receptor(Fc�R) type IIIb, has been reported previously (12,13).In recent studies, CD177/NB1 has been proposed as themPR3 receptor on the neutrophil surface (14,15).CD177 is a neutrophil-specific, glycoprotein I–anchoredglycoprotein, compartmentalized in secondary (specific)granules (16). Concurrent with mPR3, CD177 alsoshows differential expression on the neutrophil surface,with percentages of CD177� neutrophils ranging from 0to 100% (17). It has also been observed that mPR3colocalizes with CD177 on the neutrophil membrane,and the subpopulation of neutrophils expressing CD177is identical to that expressing mPR3 (14). Interestingly,the intracellular content of PR3 is similar in all neutro-phil subsets regardless of mPR3 expression status,whereas CD177 molecules are only present in lysates of

neutrophils expressing CD177 on their membrane(11,14).

Collectively, these observations suggest thatCD177 may be a determinant of membrane expressionof PR3 and, as such, influences the potential of neutro-phils to be activated by PR3 ANCAs. Indeed, increasedpercentages of mPR3-expressing neutrophils have beenobserved in patients with ASV, and a high percentage ofmPR3-expressing cells is a risk factor for relapse in WG(10,18,19). However, expression levels of CD177 havenot been studied consistently in patient populations, andthe influence of CD177 expression on ANCA-inducedneutrophil activation has not been described.

Therefore, in the present study, we assessed thedifferential expression of CD177 and mPR3 on neutro-phils from patients with ASV in comparison with pa-tients with other autoimmune diseases and healthycontrols, and investigated the role of CD177 expressionin neutrophil activation by PR3 ANCAs.

PATIENTS AND METHODS

Study population. For the assessment of neutrophilCD177 expression, consecutive patients with ASV (n � 53),those with rheumatoid arthritis (RA) (n � 26), and those withsystemic lupus erythematosus (SLE) (n � 30) were recruitedfrom the outpatient clinic in our hospital. Healthy controlsubjects (n � 31) were recruited from among laboratorypersonnel. Individuals who were pregnant, those receivinggranulocyte–colony-stimulating factor (G-CSF) treatment, orthose with bacterial infection were excluded.

Patients with ASV. The diagnosis of WG, CSS, or MPAwas based on the Chapel Hill Consensus Conference defini-tions (20). ANCA specificity for PR3 or MPO was determinedby capture enzyme-linked immunosorbent assay. ANCA titerswere determined by indirect immunofluorescence assay onethanol-fixed neutrophils (21,22).

Patients with RA. The diagnosis of RA was establishedon the basis of the American College of Rheumatology (ACR;formerly, the American Rheumatism Association) criteria fordefinite RA (23).

Table 1. Characteristics of patients and healthy controls included in the analysis of CD177 expression*

Healthycontrols

ASV patientsSLE

patientsRA

patientsPR3 ANCAs MPO ANCAs

No. of subjects 31 32 21 30 26Sex, no.

Male 17 19 12 5 4Female 14 13 9 25 22

Age, mean � SD years 39 � 11 57 � 13 61 � 14 45 � 15 56 � 12

* ASV � antineutrophil cytoplasmic autoantibody–associated systemic vasculitis; PR3 � proteinase 3;MPO � myeloperoxidase; SLE � systemic lupus erythematosus; RA � rheumatoid arthritis.

EXPRESSION OF CD177 AND mPR3 IN ASV 1549

Patients with SLE. The diagnosis of SLE was deter-mined on the basis of the ACR revised criteria for this disease(24).

The demographic characteristics of all patients andcontrols are given in Table 1. All subjects gave their informedconsent, and the study was approved by the hospital medicalethics committee.

Antibodies. PR3 was detected using 3 murine mono-clonal antibodies: PR3G-3 (25), 4A3 (Wieslab, Lund, Sweden),and CLB12.8 (CLB, Amsterdam, The Netherlands). MEM166(BD PharMingen, San Diego, CA) was used for CD177detection. Irrelevant isotype IgG1 antibodies (MCG1; IQProd-ucts, Groningen, The Netherlands) and IgG2a antibodies(MCG2a; IQProducts) were used as a control. Fluoresceinisothiocyanate– and phycoerythrin (PE)–conjugated goat anti-mouse secondary antibodies (Southern Biotechnology Associ-ates, Birmingham, AL) were used for fluorescence-activatedcell sorter (FACS) analysis. Human IgG fractions were iso-lated from PR3 ANCA–positive sera of WG patients, anti–glomerular basement membrane (anti-GBM)–positive sera ofpatients with Goodpasture syndrome, and sera of age-matchedhealthy donors, using a protein G column (MabTrap G II;Pharmacia Biotech, Uppsala, Sweden) in accordance with themanufacturer’s instructions.

Isolation and priming of neutrophils. Neutrophilswere isolated from the peripheral blood of patients andcontrols, and then primed in accordance with routine proce-dures as published previously (19). Briefly, neutrophils wereisolated from heparinized venous blood by centrifugation onLymphoprep (Axis-Shield, Oslo, Norway). Contaminatingerythrocytes were lysed with ice-cold ammonium chloridebuffer. Cells were washed and resuspended in cold Hanks’balanced salt solution (HBSS) without Ca2�/Mg2�

(HBSS�/�; Gibco/Life Technologies, Breda, The Nether-lands). The cells were then resuspended in RPMI 1640 (Cam-brex BioScience, Verviers, Belgium) supplemented with 10%fetal calf serum (FCS; BioWhittaker Europe, Verviers, Bel-gium) and 50 �g/ml of gentamicin (Gibco, Paisley, UK), toobtain 106 cells/ml.

Where indicated, cells were primed with 2 ng/mlrecombinant human tumor necrosis factor � (rHuTNF�;Boehringer Mannheim, Mannheim, Germany) at 37°C for 15minutes, and nonprimed cells were incubated with controlmedium under the same conditions.

Measurement of expression of membrane-bound PR3and CD177. Membrane expression of PR3 and CD177 wasmeasured by flow cytometry. The complete procedure wasperformed on ice to avoid cell activation. After priming, eachsample, containing 1 � 106 isolated neutrophils, was washedwith ice-cold HBSS�/�/1% FCS and centrifuged at 1,200g at4°C for 3 minutes. Cell pellets were incubated with 0.5 mg/mlheat-aggregated goat immunoglobulin (HAGG; Sigma, Zwijn-drecht, The Netherlands) for 15 minutes to block Fc receptorson the cell surface. Cells were then loaded with a saturatingdose of monoclonal antibodies against human PR3 (PR3G-3)or CD177 (MEM166). An irrelevant isotype IgG1 mousemonoclonal antibody (MCG1) was used as a control. Whereindicated, 4A3 and CLB12.8 were also used for PR3 detection.After 30 minutes, unbound antibodies were washed off andcells were resuspended in PE-conjugated secondary antibodiesdiluted with 0.5 mg/ml HAGG, and then incubated in the dark

for another 30 minutes. Finally, cells were washed and resus-pended in washing buffer.

The fluorescence intensity of the cells was measuredimmediately by FACSCalibur (Becton Dickinson Immunocy-tometry Systems, Mountain View, CA) and calibrated usingCellQuest software (Becton Dickinson). The results wereanalyzed using the Win-List software package (Verity SoftwareHouse, Topsham, ME), and data were edited with FlowJoanalysis software (Treestar, Ashland, OR). Expression levels

Figure 1. Effect of tumor necrosis factor � (TNF�) stimulation onCD177�/high-expression membrane-bound proteinase 3 (mPR3high)neutrophils. CD177/mPR3 expression on TNF�-primed and non-primed neutrophils was measured by flow cytometry. A, Representa-tive example of TNF�-induced bimodal pattern of mPR3/CD177expression. Values over bars indicate the percentage of mPR3- orCD177-expressing cells. B, Extent of TNF�-induced up-regulation ofmPR3 and CD177 on CD177�/mPR3high polymorphonuclear neutro-phils (PMNs) from donors with bimodal expression. Bars show themean and SD percentage of the mean fluorescence intensity of primedneutrophils relative to that of nonprimed neutrophils. PE � phyco-erythrin; HC � healthy controls; PR3 ANCA� � PR3-specificantineutrophil cytoplasmic autoantibody (ANCA)–associated systemicvasculitis; MPO ANCA� � myeloperoxidase-specific ANCA-associated systemic vasculitis; SLE � systemic lupus erythematosus;RA � rheumatoid arthritis.

1550 HU ET AL

are presented as the mean fluorescence intensity (MFI) of10,000 counted cells, corrected for nonspecific binding ofisotype control antibodies. Bimodal expression was defined asthe presence of 10–90% of mPR3high cells or the presence of10–90% of CD177� cells, while percentages out of this rangewere defined as monomodal. Definitions for monomodal-mPR3high and monomodal-mPR3low were based on data froma previous study (19).

Detection of intracellular levels of PR3 and CD177.Isolated neutrophils were suspended in HBSS�/� to obtain aconcentration of 1 � 106 cells/ml. The cell suspensions wereincubated with 10 �g/ml cytochalasin B at 37°C for 5 minutesand primed with 2 ng/ml rHuTNF� at 37°C for 15 minutes. Allneutrophils were then fixed and permeabilized with theFix&Perm kit (Caltag Laboratories, Burlingame, CA) follow-ing the manufacturer’s instructions. Next, intracellular levels ofPR3 and CD177 were detected by incubation with anti-PR3(PR3G-3) or anti-CD177 (MEM166) at saturating concentra-tions. MCG1 was used as a negative control. After 30 minutesof incubation at room temperature, unbound antibodies werewashed off and cells were incubated with PE-conjugatedsecondary antibodies diluted with 0.5 mg/ml HAGG in thedark for another 30 minutes. After an additional washing step,cells were resuspended in washing buffer.

The fluorescence intensity of the cells was measuredimmediately by FACSCalibur and calibrated with CellQuestsoftware. Intracellular levels of PR3 and CD177 are presentedas the MFI, corrected for nonspecific binding as detected fromisotype control antibody incubation.

Dihydrorhodamine-123 (DHR-123) oxidation assay.Intracellular hydrogen peroxide produced during neutrophilactivation was measured by the DHR-123 oxidation assay, asdescribed previously (26). Isolated neutrophils from healthydonors were suspended in HBSS with Ca2�/Mg2� to a con-centration of 2.5 � 106 cells/ml and incubated with cytochala-sin B (5 �g/ml; Serva Electrophoresis, Heidelberg, Germany)at 37°C, to enhance oxygen radical production. The cells werethen loaded with 1 �g/ml DHR-123 (Molecular Probes Eu-rope, Leiden, The Netherlands) and kept at 37°C for 15minutes. Sodium azide (2 mM) was added in order to preventintracellular breakdown of H2O2 by catalase. Part of theDHR-123–loaded cells was incubated for 15 minutes in thepresence of a priming concentration of rHuTNF� (2 ng/ml).

Neutrophils were then stimulated for 1 hour withanti-PR3 antibody (PR3G-3; 5 �g/ml) or an irrelevant mouseIgG1 monoclonal antibody (MCG1) at the same concentra-tion. Where indicated, neutrophils were also stimulated with200 �g/ml of PR3 ANCA–positive or irrelevant human IgG

fractions (anti-GBM or normal IgG). Stimulation with 0.2 �Mphorbol myristate acetate (Sigma) for 30 minutes was per-formed as a positive control. The reaction was stopped withice-cold HBSS�/�. Finally, cells were resuspended in washingbuffer. The fluorescence intensity resulting from the intracell-ular oxidation of DHR-123 into the fluorescent rhodamine-123was measured using FACSCalibur and calibrated usingCellQuest software.

Statistical analysis. Results for each group are ex-pressed as the median. Statistical analysis was performed usingthe Mann-Whitney U test, Kruskal-Wallis test, and Spearman’snonparametric correlation analysis. Data were analyzed usingGraphPad Prism, version 4.03 (GraphPad Software, San Di-ego, CA). P values less than 0.05 were considered significant.

RESULTS

Correlation of CD177 expression with mPR3expression on primed neutrophils. Combining the datafrom the healthy controls and the patient groups (n �140), 120 donors were identified as displaying a bimodalexpression of mPR3. Consistent with the results fromother studies (14,15), CD177 expression was stronglycorrelated with mPR3 expression on primed neutrophils(results not shown). However, neutrophil priming was anessential procedure for assessing the correlation be-tween mPR3 expression and CD177 expression, sincepriming with TNF� is necessary for standardized assess-ment of mPR3 levels, as has been shown previously (19).

In 19 of the 31 healthy donors (61%), isolatedneutrophils in the absence of priming showedmonomodal-negative expression of mPR3, whereas afterincubation with TNF� for 15 minutes, the pattern ofmPR3 expression turned bimodal in 12 of these donors,with a low-expression mPR3 subset and a high-expression mPR3 subset (Figure 1A). Percentages ofmPR3high neutrophils ranged from 38% to 85%, andthese levels remained constant in repeated tests within agiven individual (results not shown).

Similarly, the amount of mPR3 on the surfaceof neutrophils, expressed as the MFI of the cells, wasalso remarkably up-regulated by TNF� stimulation

Table 2. Bimodal expression of mPR3 and CD177 induced by tumor necrosis factor � priming*

Healthycontrols

ASV patientsSLE

patientsRA

patientsPR3 ANCAs MPO ANCAs

No. of subjects 31 32 21 30 26No. with expression of mPR3

before/after priming12/24 25/31 18/21 15/27 16/22

No. with expression of CD177before/after priming

28/28 32/32 21/21 30/30 25/25

* mPR3 � membrane-bound proteinase 3 (see Table 1 for other definitions).

EXPRESSION OF CD177 AND mPR3 IN ASV 1551

(Figure 1B). The effect of neutrophil priming on mPR3recruitment was comparable between the patient groups(Table 2 and Figure 1B). Therefore, priming with TNF�revealed the potential of neutrophils to increase expres-sion of mPR3.

In contrast to the effects of priming on mPR3expression, the percentages of CD177� neutrophilswere not modified by neutrophil priming (Table 2). Theamount of membrane-expressed CD177 was only slightlyup-regulated after priming, with a mean � SD increasein the MFI of 21 � 19% relative to the expression levelon nonprimed neutrophils (Figure 1B).

Presence of mPR3 expression on the neutrophilsfrom CD177-negative donors. In 3 healthy donors,CD177 expression was not detected on the membrane ofisolated neutrophils either before or after priming. How-ever, after incubation with TNF�, neutrophils fromthese CD177-negative donors expressed mPR3, whichwas detected using 3 different monoclonal antibodies(Figures 2A and B). Absence of CD177 expression wasconfirmed by intracellular FACS staining. In contrast,PR3 and MPO were uniformly detected in the neutro-phils from CD177-postive and -negative donors (Figure

2C). These results demonstrate that expression of mPR3on the neutrophil surface can be independent of CD177expression.

Percentages of mPR3high and CD177-expressingneutrophils in inflammatory diseases. The percentagesof CD177� and mPR3high neutrophils were comparedamong patients with ASV (n � 53), those with SLE (n �30), and those with RA (n � 26), and healthy donors(n � 31). In this analysis, donors with monomodalexpression were also included. The median percentageof mPR3high neutrophils was significantly higher in pa-tients with ASV (median 67%, range 16–100%) andpatients with SLE (median 80%, range 25–100%), butnot in patients with RA (median 61%, range 0–100%),as compared with that in healthy controls (median 51%,range 0–100%) (Figure 3A). Furthermore, the medianpercentage of CD177-expressing neutrophils was alsosignificantly higher in patients with ASV (median 71%,range 18–97%) and patients with SLE (median 82%,range 33–94%) than in healthy controls (median 62%,range 0–87%) or patients with RA (median 61%, range0–93%) (Figure 3B).

Figure 2. Detection of TNF�-induced mPR3 expression on CD177� neutrophils. Membrane expression of mPR3 and CD177 onneutrophils was measured in parallel by flow cytometry. A, Expression of CD177 and mPR3 on nonprimed and primed neutrophilsfrom a CD177-negative donor. Results are representative of 3 independent experiments with different CD177-negative donors. B,Detection of mPR3 on neutrophils from CD177-negative donors with the use of the anti-PR3 antibodies 4A3, PR3G-3, and CLB12.8and with PR3-ANCA–positive IgG fractions, in comparison with detection of CD177 expression with MEM166 (anti-CD177). Barsshow the mean and SD mean fluorescence intensity (MFI) of mPR3 and CD177 expression on the membrane of neutrophils from 3CD177-negative donors, after correcting for nonspecific binding. C, Histograms showing the intracellular content of CD177 (shadedarea) and PR3 (solid line) in CD177-negative and -positive donors, as measured by flow cytometry. An isotype control IgG1 antibody(Ab) (broken line) and anti-MPO antibody (dotted line) were used as the negative and positive control, respectively. Results arerepresentative of 3 independent experiments with different donors. See Figure 1 for other definitions.

1552 HU ET AL

Within the ASV patient group, the expressionprofile was also analyzed according to ANCA specificityand ASV phenotypes. There were no statistically signif-icant differences in the percentages of CD177� neutro-phils between patients with MPO ANCA–associatedvasculitis (median 77%, range 25–94%) and those withPR3 ANCA–associated vasculitis (median 67%, range18–97%) or between patients with WG (median 67%,range 18–97%) and those with MPA (median 80%,range 25–94%) (Figure 3C).

Immunosuppressive treatment or prednisoloneadministration did not increase the proportion ofCD177� neutrophils in patients with SLE and MPOANCA–associated vasculitis. However, among the pa-tients with PR3 ANCA–associated vasculitis, patientstreated with cyclophosphamide or prednisolone hadhigher percentages of CD177-expressing neutrophilsthan did patients who had not received these treatments(Figure 3D). We did not find any correlation betweenthe percentages of CD177-expressing neutrophils anddisease activity or disease duration in PR3 ANCA–associated vasculitis (results not shown). However, neu-

trophils from patients who had relapsing disease showedsignificantly higher percentages of CD177� neutrophilsthan did those from patients without relapses (Figure3D). Concurrently, 4 of 6 patients treated with cyclo-phosphamide and 10 of 12 patients treated with pred-nisolone had relapsing disease.

Neutrophil activation by PR3 ANCAs. Neutrophilactivation induced by a mouse anti-human PR3 mono-clonal antibody (PR3G-3) or by PR3 ANCA–positiveIgG fractions was evaluated by measuring oxidativeburst using the dihydrorhodamine assay. Consistent withthe results from previous studies, oxidative burst wasdetected only when neutrophils were primed with TNF�(results not shown) (26,27). Neutrophils from CD177-negative donors (n � 3) could be activated by theanti-PR3 antibody and showed comparable levels ofneutrophil activation as were observed in the neutrophilsfrom donors with moderate expression of CD177 (40–70% positive; n � 3) and donors with high expression ofCD177 (�70% positive; n � 3) (Figure 4A). Moreover,no correlation was observed between the levels of neu-trophil oxidative burst induced by the anti-PR3 antibody

Figure 3. Increased percentages of CD177-expressing neutrophils in patients with ANCA-associated sys-temic vasculitis (ASV) and patients with SLE. Membrane expression of CD177 and mPR3 were measured onisolated neutrophils by flow cytometry. Results are presented as the percentages of CD177�/mPR3high

neutrophils in the total neutrophil population. A and B, Percentages of mPR3high neutrophils (A) andCD177� neutrophils (B) were compared among healthy controls (n � 31), patients with ASV (n � 53),patients with SLE (n � 30), and patients with RA (n � 26). C, Percentages of CD177� neutrophils werecompared between MPO ANCA–positive (n � 22) and PR3 ANCA–positive (n � 32) patients with ASV, andbetween Wegener’s granulomatosis (WG; n � 33) and microscopic polyangiitis (MPA; n � 19) ASVphenotypes. D, Comparison of CD177� neutrophils between PR3 ANCA–positive patients with or withouttreatment with cyclophosphamide (CYC) or prednisolone and patients with or without relapsing disease.Horizontal line denotes the median. � � P � 0.05; �� � P � 0.01; ��� � P � 0.001. ns � not significant (seeFigure 1 for other definitions).

EXPRESSION OF CD177 AND mPR3 IN ASV 1553

and the percentages of CD177� neutrophils (results notshown).

Next, CD177� and CD177� neutrophils fromdonors with bimodal expression of CD177 were sortedby flow cytometry. The purity of each sorted sample wasmore than 95% (Figure 4B). These subsets were used forinduction of the oxidative burst by the anti-PR3 anti-body. The results were comparable between theCD177� and CD177� subpopulations in each donor(Figure 4C).

DISCUSSION

In this study, we described the profile of CD177expression in ASV and the contribution of CD177 toPR3 ANCA–mediated neutrophil activation. Consistentwith the results reported by others, mPR3 expressionand CD177 expression were highly correlated on primedneutrophils from donors with a bimodal expressionpattern of both molecules (14,15). Percentages ofmPR3high/CD177� neutrophils were increased in pa-tients with ASV and those with SLE compared withhealthy individuals. However, colocalization of mPR3and CD177 was not crucial for PR3 ANCA–mediated

neutrophil activation, which is considered a central eventin disease pathogenesis leading to vessel wall damage(28). After priming, mPR3 was expressed on CD177�neutrophils, suggesting that there are additional mech-anisms of membrane presentation of PR3 independentof the role of CD177. Anti-PR3 antibody–mediatedneutrophil activation, as analyzed by DHR-123 oxida-tion, could also be induced in CD177� neutrophils, andamong healthy individuals, there was no correlationbetween the level of neutrophil activation and thepercentage of CD177-expressing neutrophils.

Although the pathogenesis of ASV is still un-clear, it has been shown that ANCAs are pathogenic inanimal models and that the antibodies in vitro activatecytokine-primed neutrophils, leading to oxidative burstand neutrophil degranulation (3,4). Membrane-boundPR3, which is accessible for ANCA binding, showsheterogeneous expression on the neutrophil surface (9).The percentage of mPR3high neutrophils is increased inpatients with ASV and is a risk factor for relapse of WG(10,18,29). Results from recent studies have suggestedthat CD177 acts as a receptor of mPR3, by showing thatmPR3 expression is highly correlated with CD177 ex-

Figure 4. Anti-PR3 antibody–mediated neutrophil activation independently of CD177 expression. Oxidative burstwas assessed by dihydrorhodamine-123 oxidation assay. A, Levels of neutrophil activation induced by the PR3.G3antibody or PR3 ANCAs were compared among individuals with CD177� neutrophils (0% CD177�, n � 3),neutrophils with moderate expression of CD177 (40–70% CD177�, n � 3), and high expression of CD177 (�70%CD177�, n � 3). Bars show the mean and SD activation, presented as the mean fluorescence intensity (MFI) ofrhodamine-123. B, Neutrophils from the same donor were sorted by flow cytometry into CD177� and CD177�subsets; the purity of each sorted sample was �95%. Results are representative of 3 independent experiments. C,Levels of neutrophil activation induced by the anti-PR3 antibody were compared between CD177� and CD177�neutrophils from 3 individual donors. See Figure 1 for other definitions.

1554 HU ET AL

pression and mPR3 colocalizes with CD177 on theneutrophil membrane (14,15). It is tempting to speculatethat increased proportions of mPR3-expressing neutro-phils in patients with ASV reflect up-regulated expres-sion of CD177. Furthermore, it is relevant, with regardto the pathogenesis of ASV, whether CD177�/mPR3high

neutrophils act differently from CD177�/mPR3low neu-trophils when encountering PR3 ANCAs in, for in-stance, PR3 ANCA–mediated neutrophil activation.

It has been reported that the percentage ofCD177� neutrophils is constant within a given individ-ual (30). However, enlarged CD177� subsets have beenobserved in some clinical conditions with altered neu-trophil production. The percentage of CD177� cellsincreased to 90% in healthy individuals receiving G-CSFtreatment; this type of treatment can mobilize myeloidprogenitor cells into the peripheral blood and is used forstem cell collection (31,32). Women in early- or late-stage pregnancy have �10% more CD177-expressingneutrophils and increased neutrophil counts as com-pared with healthy female blood donors (33,34). Thecurrent study is the first to show that percentages ofCD177-expressing neutrophils are also increased in pa-tients with ASV and those with SLE compared withhealthy individuals, which could account for the elevatedexpression of mPR3 in both patient groups.

It has been shown that the percentage of mPR3-expressing neutrophils cannot be modified by diseaseactivity or treatment (10,18). In parallel, we did not findany effect of treatment on the proportion of CD177�neutrophils in patients with SLE and MPO ANCA–associated vasculitis. However, in patients with PR3ANCA–associated vasculitis, patients treated with cyclo-phosphamide or prednisolone showed significantlyhigher percentages of CD177-expressing neutrophilsthan did patients who did not receive these treatments(Figure 3D). In our disease control groups, treatmentwith prednisolone did not increase the percentage ofCD177� neutrophils in patients with SLE, and neithercyclophosphamide nor prednisolone induced up-regulation of CD177 in patients with MPO ANCA–positive vasculitis, suggesting that increased CD177 ex-pression is not a consequence of treatment, but isprobably associated with other factors.

In patients with PR3 ANCA–positive vasculitis,we did not find any correlation between the percentagesof CD177� neutrophils and disease activity or diseaseduration. However, we found that patients who hadrelapsing disease showed significantly higher percent-ages of CD177� neutrophils than did patients withoutrelapses. Concurrently, 4 of 6 patients treated with

cyclophosphamide and 10 of 12 patients treated withprednisolone had relapsing disease, which probably ex-plains the differences in CD177 expression betweenpatients receiving cyclophosphamide or prednisoloneand those who did not receive these treatments. There-fore, it appears that treatment with immunosuppressiveagents and corticosteroids has, in itself, no effect onCD177/mPR3 expression. Moreover, the present resultsconfirm our previous observations that a high propor-tion of mPR3-expressing (and CD177-expressing) neu-trophils is associated with relapse in WG (18).

Whereas increased mPR3 expression on neutro-phils in ASV has been linked to PR3 ANCA–inducedneutrophil activation in vitro (29) and associated withrelapsing disease in patients with ASV (18), the patho-genetic significance of increased expression of mPR3and CD177 in SLE is not clear. Because ANCAs in SLEare, in most cases, not directed to PR3 (35–37), a role formPR3 expression on neutrophil activation by ANCAscannot be substantiated. Otherwise, neutrophils do playa role in the pathogenesis of SLE, by interacting withimmune complexes or by inducing or exacerbating auto-immune responses when these cells accumulate in anapoptotic state (38). Indeed, accelerated apoptosis ofneutrophils and decreased uptake of apoptotic neutro-phils has been observed in SLE (39,40). Whether theincreased expression of PR3 on the membrane ofprimed neutrophils in SLE is related to an increasedlevel of activation or even to a preapoptotic stateremains speculative.

CD177 is a heterophilic binding partner of CD31,which is expressed on endothelial cells, suggesting a roleof CD177 in neutrophil migration (41). Apart from thisrole, the biologic function of CD177 is largely unknown.Furthermore, CD177-negative individuals, comprising�3% of Caucasians, are healthy, although detailedstudies on neutrophil function in these individuals arelacking. In contrast, PR3 executes multiple biologicfunctions, such as regulation of granulopoiesis, microbi-cidal activity, degradation of extracellular matrix, andmodulation of inflammatory factors (42,43). It has beenshown, in vivo, that PR3 is essential for immunecomplex–mediated neutrophil activation by cleavingantiinflammatory progranulin into its inactive form, andboth PR3 and elastase deficiency diminish neutrophilinfiltration into the site of inflammation (44). Apparently,enzymatic activity of PR3 is required in most of its impor-tant actions, and it is probably protected from endogenousinhibitors by anchoring at the neutrophil membrane in-stead of being secreted in a soluble form (45,46). There-

EXPRESSION OF CD177 AND mPR3 IN ASV 1555

fore, one may expect that mPR3 expression also occurs inhealthy persons with CD177 deficiency.

In our study, PR3 was detected by 3 differentmonoclonal antibodies on the surface of primed neutro-phils from CD177-negative donors. Membrane-boundPR3 expression on mPR3low neutrophils has also beenobserved in other studies (9,19,47), and we have previ-ously shown that the extent of TNF�-induced mPR3up-regulation on mPR3low neutrophils is comparablewith that of neutrophil elastase (19). These resultssuggest that CD177 is not an exclusive receptor ofmPR3, and the binding sites for CD177-independentmPR3 expression are still open for discussion. It hasbeen shown that mPR3 colocalizes with activated �2integrin and Fc�R in the neutrophil membrane(12,13,48). PR3 can insert into the membrane lipidbilayer via the hydrophobic region (49). Elastase, aproteinase homologous to PR3, binds to chondroitinsulfate– and heparin sulfate–containing proteoglycans inthe neutrophil membrane, and PR3 is competitive forthese binding sites (50). In a recent study, Hajjar et alassumed mPR3 to be a peripheral membrane proteinand predicted an interfacial binding site in the hydro-phobic region, using a model of molecular dynamicssimulation on a membrane (46). All of these datasuggest that mPR3 binds to the neutrophil membranevia its cationic nature and functionally colocalizes withsome receptor molecules, such as integrins, Fc�R, andCD177, for signal transduction.

Although mPR3 is differentially expressed onneutrophils, the oxidative burst mediated by the anti-PR3 antibody has been uniformly induced in neutrophilsfrom donors with bimodal mPR3 expression (26). In thepresent study, we showed that the level of neutrophilactivation induced by the anti-PR3 antibody was notcorrelated with the percentage of CD177�/mPR3high

neutrophils, and that CD177� neutrophils could also beactivated by the anti-PR3 antibody. Additionally, consis-tent with the results from previous studies (26,27),priming with TNF� was required in this process, regard-less of the high levels of CD177-dependent mPR3expression on resting neutrophils. These results suggestthat colocalization of CD177 and mPR3 on the mem-brane is neither sufficient nor necessary for neutrophilactivation mediated by PR3 ANCAs.

Thus, the proportions of CD177-expressing neu-trophils are increased in patients with ASV and thosewith SLE, while CD177� neutrophils also express mPR3on their membrane and are susceptible to anti-PR3antibody–induced oxidative burst. The results indicatethat CD177�/mPR3high and CD177�/mPR3low neutro-

phils may be equally involved in the pathogenesis of PR3ANCA–associated vasculitis, suggesting that there maybe different mechanisms of membrane binding of PR3other than to CD177.

AUTHOR CONTRIBUTIONS

Dr. Kallenberg had full access to all of the data in the studyand takes responsibility for the integrity of the data and the accuracyof the data analysis.Study design. Hu, Westra, Heeringa, Kallenberg.Acquisition of data. Hu, Huitema, Bijl, Brouwer, Stegeman.Analysis and interpretation of data. Hu, Westra, Huitema, Heeringa,Limburg.Manuscript preparation. Hu, Westra, Heeringa, Kallenberg.Statistical analysis. Hu, Westra, Bijl.

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