BASIC RESEARCH www.jasn.org

Increased Membrane Expression of during Adhesion in the Presence of Anti– Proteinase 3 Antibodies

Soumeya Brachemi,* Agne`s Mambole,* Fadi Fakhouri,† Luc Mouthon,‡ Loı¨c Guillevin,‡ Philippe Lesavre,*† and Lise Halbwachs-Mecarelli*

*INSERM U845 and Universite´ Paris V Rene´ Descartes, †Necker Hospital Nephrology Department, and ‡Internal Medicine Department, Cochin Hospital and Unite´ Propre de Recherche de l’Enseignement Supe´rieur (UPRES) EA 4058, Paris, France

ABSTRACT We investigated membrane proteinase 3 (mPR3) expression during TNF-␣–induced adhesion of neutro- phils in the presence of anti-PR3 antibodies, a situation occurring during anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Three increasing levels of mPR3 expression were observed ϩ on the mPR3 neutrophil subset after stepwise cell activation. TNF-␣ activation without adhesion, TNF-␣–induced adhesion, and adhesion in the presence of anti-PR3 mAb or human anti-PR3 ANCA resulted, respectively, in a two-, seven-, and 24-fold increase of mPR3 levels. In plasma, anti-PR3 antibodies poorly recognized suspended , whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neu- trophil adhesion to activated human umbilical vein endothelial cells. It involved ␤2 integrins and Fc␥ Ј receptor, because it was prevented by anti-CD18 antibodies and was not observed with anti-PR3 F(ab )2. Furthermore, it was specific to anti-PR3 mAb, and no mPR3 upregulation was observed with anti- or anti–HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhe- ϩ ϩ sion, were mobilized from secretory vesicles (CD35 ) and secondary granules (CD11b ). The adhesion- and antibody-dependent upregulations of mPR3 expression occurred with little azurophilic , no sign of apoptosis, and no further CD177 upregulation. In conclusion, this study describes an amplifying loop in polymorphonuclear neutrophil activation process, whereby ANCA are involved in the membrane expression of their own antigen during cell adhesion. This could explain the restriction of ANCA-associated vasculitis to small vessels, the main site of neutrophil adhesion.

J Am Soc Nephrol 18: 2330–2339, 2007. doi: 10.1681/ASN.2006121309

Wegener granulomatosis, microscopic polyangiitis, found only after extensive cell activation.5 A high pauci-immune necrotizing glomerulonephritis, proportion of mPR3ϩ neutrophils is a risk factor and Churg-Strauss syndrome constitute a group of for the development of vasculitis, and elevated small vessel vasculitis associated with anti-neutro- mPR3 levels are associated with a higher relapse rate phil cytoplasmic autoantibodies (ANCA). The in patients with Wegener granulomatosis.6,7 In vitro pathogenesis of these vasculitides is closely linked to increased ANCA levels and polymorphonuclear neutrophils (PMN) found in the glomeruli and in- Received December 3, 2006. Accepted April 27, 2007. terstitium.1–4 Published online ahead of print. Publication date available at www.jasn.org. ANCA are directed against proteinase 3 (PR3) or myeloperoxidase (MPO), both proteinases mainly Correspondence: Dr. Lise Halbwachs-Mecarelli, INSERM U845, Hoˆpital Necker, 161 rue de Se`vres, 75015 Paris, France. Phone: stored in intracellular granules. Membrane PR3 is ϩ33-144-49-52-32; Fax: ϩ33-145-66-51-33; E-mail: mecarelli@ present on a variable proportion of resting PMN necker.fr ϩ (mPR3 ), as opposed to membrane MPO, which is Copyright © 2007 by the American Society of Nephrology

2330 ISSN : 1046-6673/1808-2330 J Am Soc Nephrol 18: 2330–2339, 2007 www.jasn.org BASIC RESEARCH

ANCA-induced responses are higher in mPR3ϩ PMN than in the negative subset.8 The binding of ANCA to mPR3 may thus be an important factor in the pathogenesis of ANCA vasculitis. ANCA trigger neutrophil respiratory burst and degranulation, with the re- lease of proteolytic and reactive oxygen species.9–13 They modulate neutrophil adhesion and migration14,15 and in- duce F-actin polymerization and cell stiffening, ultimately leading to neutrophil retention in glomerular capillaries.15–17 Although the role of ANCA in vasculitis pathogenesis has been established, the accessibility of ANCA to their antigens in blood is still debated.18–20 The absence of ANCA detection on neutrophils in whole blood has been proposed to result from plasma ␣1 anti- (A1AT) binding to PR3.21 The associ- ation between A1AT-deficient phenotypes and anti-PR3–pos- itive vasculitis suggests that A1AT controls ANCA access to its antigen.22 Adherent neutrophils are surrounded by an area where pro- tease inhibitors, such as A1AT, are inactivated by neutrophil- derived metalloproteases.23–25 We hypothesized that mPR3 be- comes accessible to ANCA in the presence of plasma during neutrophil adhesion to endothelial cells, as found in vasculitis. We thus analyzed PR3 membrane expression during PMN ad- hesion induced by TNF-␣ in the presence of anti-PR3 antibod- ies, as seen in the microvascular environment of vasculitis pa- tients.

RESULTS

Upregulation of Membrane PR3 Expression during ␣ TNF- –Induced Adhesion Figure 1. Membrane proteinase 3 (mPR3) expression after TNF- ␣ TNF- is known to trigger adhesion-independent and -depen- ␣–induced PMN activation and adhesion. (A) Representative flow dent neutrophil responses. The addition of EDTA distin- cytometry histograms obtained with polymorphonuclear neutro- guishes the effects that are caused by TNF-␣ per se from those phils (PMN) from three healthy donors with 80% (left), 40% (mid- ϩ that result from TNF-induced integrin engagement and “out- dle), and 0% (right) of mPR3 PMN. PMN (2 ϫ 106/ml in HBSS- side-in” signaling.26 We analyzed mPR3 expression on TNF- EDTA in BSA-coated tubes) were incubated at 37°C for 45 min activated PMN from individuals with varying proportions of (Resting PMN) or, after 15 min at 37°C, for 30 min with TNF-␣ (20 ϩ ϫ 6 2ϩ PMN expressing PR3 (mPR3 ; Figure 1A). ng/ml; TNF-EDTA). PMN (2 10 /ml in HBSS -BSA) were al- ␣ lowed to settle for 15 min at 37°C in 1% gelatin–coated wells and TNF- activation without cell adhesion, in HBSS-EDTA, ␣ doubled the mPR3 level of the mPR3ϩ neutrophil subset (Fig- incubated with TNF- (20 ng/ml) for 30 min at 37°C without (TNF ϩ Adhesion) and with 2 ␮g/ml anti-PR3 mAb (TNF adhesion ϩ ure 1). Similar results were obtained with TNF-␣ activation in Ϫ anti-PR3). The anti-PR3 mAb was previously centrifuged for 20 HBSS , with or without shaking (data not shown). When neu- min at 12,000 ϫ g to remove Ig aggregates. After washing off trophils were allowed to adhere to gelatin-coated plates, upon nonadherent cells, adherent cells were released by a 20-min 2ϩ activation by TNF-␣ in HBSS , the mPR3 level was further incubation with ice-cold PBS, 1% BSA, 10 mM EDTA, and 0.1% enhanced, reaching 7 Ϯ 3-fold the resting cells level (Figure 1). sodium azide. Resting and TNF-EDTA cells were similarly incu- bated in 0.1% sodium azide before neutrophil labeling with anti- Anti-PR3 Antibody-Dependent Upregulation of PR3 mAb (solid line) or control IgG1 (dotted line) and PE-labeled Membrane PR3 Expression secondary antibody. (B) Synthesis of 22 experiments performed Ϯ ϩ We analyzed TNF-␣–activated neutrophils in the presence of as described in A (means SD of mPR3 subset mean fluores- Ͻ anti-PR3 mAb to mimic further the situation occurring in cence intensity [MFI]). ***P 0.001. ANCA-positive vasculitis. As expected, TNF-␣ induced 58 Ϯ 23% PMN adhesion, which was increased to 65 Ϯ 21% in the resulted in a stronger mPR3 upregulation, reaching 24 Ϯ 15 ϩ presence of 2 ␮g/ml anti-PR3 mAb (data not shown).14,15 Sur- times the level of resting mPR3 PMN (Figure 1). prisingly, the presence of anti-PR3 mAb throughout adhesion We excluded an in vitro artifact related to cell incubation

J Am Soc Nephrol 18: 2330–2339, 2007 Neutrophil Membrane PR3 Upregulation 2331 BASIC RESEARCH www.jasn.org with mAb at 37°C, allowing a more rapid antibody binding than during the labeling performed at 4°C. The antibody con- centration was not limiting, because the mPR3 upregulation was similar with 60 ng/ml anti-PR3 mAb (n ϭ 3) instead of 2 ␮g/ml. Moreover, incubation of TNF-activated adherent PMN forupto2hat4°Cwith the mAb, to improve PR3 labeling, resulted in Ͻ10% increase of anti-PR3 binding (data not shown). This could not explain the 2.5-fold increase of mPR3 expression observed when the antibody was present during adhesion at 37°C. The mPR3 levels summarized in Figure 1B refer only to the mPR3ϩ neutrophil subset. TNF-␣ activation had no effect on the mPR3ϩ neutrophil percentage, and the mPR3Ϫ subset re- mained mostly negative during neutrophil activation and ad- hesion. It is worth noting that some mPR3 was detected on the mPR3Ϫ subpopulation as a result of TNF-induced adhesion in the presence of anti-PR3 mAb. However, this mPR3 expres- ϩ sion did not reach 3% of the level expressed by mPR3 cells in Figure 2. mPR3 expression upregulation by human anti-PR3 Ј the same conditions. ANCA. (A) Binding of anti-PR3 ANCA IgG and F(ab )2 to PMN from a donor bimodal in mPR3 expression. PMN activated by Human Anti-PR3 but not Anti-MPO ANCA Enhance TNF-␣ in EDTA, as described in Figure 1, were labeled at 4°C with mPR3 Expression during PMN Adhesion IgG (100 ␮g/ml) purified from a normal control (plain line) or from The Fc domain of ANCA or control IgG binds indistinctly to an anti-PR3 anti-neutrophil cytoplasmic autoantibodies (ANCA)- ϩ Ϫ mPR3 and mPR3 PMN subsets. As described previously,27 positive vasculitis patient (bold line, left) or with anti-PR3 ANCA Ј ␮ it leads to a high background that obscures the specific binding F(ab )2 (500 g/ml; bold line, right), followed by a secondary FITC of anti–PR3-ANCA IgG (Figure 2A, left). The little shift of the anti-human IgG antibody. The dotted line represents nonspecific binding of the secondary antibody and is superimposed, in the ANCA IgG peak, as compared with normal IgG, was NS and right diagram, with the peak obtained with control IgG F(abЈ) . was not observed with all normal IgG. Specific binding of anti- 2 ϩ ANCA and control human IgG bind nonspecifically to PMN, via ϩ Ϫ PR3 ANCA to mPR3 neutrophils occurs, however (Figure their Fc domain, with no distinction of the mPR3 and mPR3 Ј Ј 2A, right), and F(ab )2 fragments of anti–PR3-ANCA labeled subsets. Conversely, F(ab )2 ANCA anti-PR3 specifically labels the ϩ the same proportion of PMN as the anti-PR3 mAb. mPR3 subset. (B) Effect of ANCA anti-PR3 on mPR3 expression We took advantage of the fact that anti–PR3-ANCA do not during PMN adhesion. PMN were activated as in Figure 1, in the hinder anti-PR3 mAb CLB 12.8 binding: The anti-PR3 mAb presence or absence of human IgG, ANCA, or control (50 ␮g/ml). binding on mPR3ϩ PMN was not modified when 200 ␮g/ml mPR3 expression was measured after labeling with biotin-labeled anti-PR3ϩ patient IgG were added during labeling (data not anti-PR3 mAb and PE-streptavidin. mPR3 flow cytometry histo- ␣ shown). We thus used the biotinylated anti-PR3 mAb to quan- grams of TNF- –activated PMN in EDTA (dotted line), adherent tify mPR3 after PMN adhesion with ANCA-IgG. A significant PMN (plain line), or PMN adherent in the presence of ANCA or control IgG (bold line) are shown. mPR3 upregulation was observed on adherent PMN treated with anti-PR3 ANCA IgG (Figure 2B). This was true of IgG from two of three patients with high anti–PR3-ANCA titers, No change of HLA A/B expression and no binding of IgG1 with no effect observed with IgG from five of five control sub- were observed when PMN adhered in the presence of anti- jects and four of four anti–MPO-ANCA–positive patients HLA mAb (Figure 3A) or IgG1 (Figure 3B), respectively. Then, (data not shown). neutrophils were allowed to adhere in the presence of various IgG1 mAb before analysis of mPR3 expression of the mPR3ϩ Specificity of Antibody-Induced Upregulation of mPR3 subset. This was enhanced only by anti-PR3 mAb and was not Expression and Role of Fc␥ Receptors modified by isotypic IgG1 control, anti-MPO, or irrelevant We first looked for antibody-induced upregulations of neutro- anti-neutrophil mAb such as anti-CD43 or anti-HLA A/B, de- phil MPO or . As shown in Figure 3, TNF-induced ad- spite the high levels of membrane CD43 or HLA antigens re- hesion did not promote significant MPO or elastase membrane acting with these mAb (Table 1). ␣ Ј expressions. The presence of anti-MPO or anti-elastase mAb Incubation of TNF- –activated PMN with F(ab )2 anti- throughout adhesion resulted in a small, statistically signifi- PR3 mAb during adhesion did not enhance mPR3 expression, cant membrane expression of MPO or elastase, respectively. emphasizing the role of Fc␥ receptors (Fc␥R) in mPR3 upregu- However, such expressions, homogeneous within the neutro- lation (Table 1). However, nonspecific Fc␥R activation by 1 phil pool, represented Ͻ10% of the mPR3ϩ expression seen mg/ml heat-aggregated IgG during PMN adhesion was not after PMN adhesion with anti-PR3 mAb (Figure 3B). sufficient to increase the mPR3 expression (Table 1).

2332 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2330–2339, 2007 www.jasn.org BASIC RESEARCH

mPR3 Upregulation Promoted by IL-8, fMLP, or Adhesion to Endothelial Cells Similar adhesion-dependent and anti–PR3-dependent mPR3 upregulations were observed with 20 ng/ml (Figure 1) or 2 ng/ml TNF-␣ and with IL-8 or formyl-methionyl-leucyl-phe- nylalanine (fMLP) (Figure 5). Background cell adhesion, ob- served in the absence of priming, with or without anti-PR3 mAb, resulted in slight nonsignificant increases of mPR3 ex- pression (data not shown). Finally, neutrophils were allowed to adhere to TNF-␣–pre- activated human umbilical vein endothelial cells (HUVEC) to mimic further the situation occurring in ANCA vasculitis. This caused a 11 Ϯ 4-fold increase of mPR3 expression of the mPR3ϩ subset, which further doubled when the anti-PR3 mAb was present during adhesion (Figure 5).

Figure 3. Membrane myeloperoxidase (MPO), elastase (HNE), Role of Degranulation in the mPR3 Upregulation and HLA labeling after PMN adhesion. (A) Increase of membrane MPO and HNE expression during PMN adhesion (performed as We assessed membrane expression of granules and vesicles described in Figure 1) in the presence of anti-MPO mAb and markers to investigate the newly exposed mPR3 origin (Figure anti-HNE mAb (2 ␮g/ml), respectively (replacing anti-PR3 mAb 6). As previously reported.,26 TNF-␣ activation of PMN in the described in Figure 1). The figure shows nonspecific binding of absence of adhesion significantly increased membrane CD35 isotype-matched control antibody (dark dotted line) and binding and CD11b, consistent with secretory vesicles and tertiary of anti-MPO or anti-HNE mAb on resting PMN ( solid line), on granules degranulation.29 TNF-␣–activated PMN in EDTA (light dotted line), on TNF-␣– TNF-␣–induced PMN adhesion further enhanced CD11b activated adherent PMN (plain line), and on adherent PMN in the membrane expression, indicating mobiliza- presence of either anti-MPO mAb or anti-HNE mAb (bold line; tion. Adhesion in the presence of anti-PR3 mAb again ampli- n ϭ 4). No difference in membrane HLA was noted between fied CD11b levels and resulted in a slight expression of CD63, a TNF-␣–activated adherent PMN (plain line) and those adherent in the presence of anti-HLA mAb (bold line; n ϭ 3). (B) Membrane marker of azurophilic granules. However, CD63 expression MPO (mMPO) levels, measured after TNF-␣–induced PMN adhe- did not reach 3% of the levels observed after maximum de- sion (performed as described in Figure 1) with anti-MPO, are granulation by fMLP/cytochalasin or by calcium ionophore ϩ compared with mPR3 levels (mPR3 subset) of adherent PMN A23187 (Figure 6, table). Similarly, the amount of MPO re- with anti-PR3 mAb. No modulation of nonspecific binding of IgG1 leased in supernatants of TNF-␣–activated neutrophils, with was observed during PMN adhesion with IgG1. Results are ex- or without anti-PR3 mAb, was Ͻ10% (detection threshold) of pressed as means Ϯ SD of anti-MPO, anti-PR3, or IgG1 MFI, using the amount released after total degranulation. Neutrophil ad- PE-labeled secondary anti-mouse IgG antibody (n ϭ 8). **P Ͻ hesion in the presence of anti-PR3 thus resulted in little mobi- Ͻ 0.01. ***P 0.001. lization of azurophilic granules. Surprising, after PMN exten- sive degranulation by fMLP/cytochalasin, leading to Role of Adhesion and ␤2 Integrin Engagement maximum CD63 expression and MPO release, mPR3 expres- The presence of anti-PR3 antibodies during TNF-induced ad- sion did not reach the levels observed after PMN adhesion in the presence of anti-PR3 (Figure 6, table). hesion to gelatin (Figure 1), fibrinogen, or type I collagen (data not shown) upregulated mPR3 expression of the mPR3ϩ sub- Role of Putative Membrane Mechanisms set. Pretreating PMN with anti-CD18 blocking mAb (IB4), We assessed putative apoptosis-related membrane flip-flop, which resulted in 86% inhibition of cell adhesion (data not resulting from cell adhesion or mechanic recovery of adherent shown), significantly prevented the antibody-induced mPR3 cells, by measuring annexin V binding. Less than 10% of an- upregulation (Figure 4A). TNF activation with this anti-CD18 nexin-positive cells were detected after adhesion, with or with- mAb resulted, however, in a slight significant increase of PR3 out anti-PR3 mAb (data not shown). expression similar to that observed with EDTA. A close association between CD177 and mPR3 was recently ␤ For further analysis if their role of in mPR3 expression, 2 reported, suggesting that CD177 itself mediates PR3 mem- integrins were switched into an active conformation by man- brane expression.30,31 We therefore analyzed CD177 during 2ϩ ganese (Mn ) or by an anti-CD18 activating mAb (KIM mPR3 upregulation. As expected, there was a bimodal distri- 185)28 in the absence of TNF-␣ signaling. Both resulted in an bution of CD177 on PMN (Figure 7A), and we confirmed, by enhanced mPR3 level on adherent cells, which was further in- double labeling, that the CD177ϩ and mPR3ϩ subsets are creased by the presence of anti-PR3 throughout adhesion (Fig- identical (data not shown). ure 4, B and C). CD177 expression of the mPR3/CD177ϩ subset nearly dou-

J Am Soc Nephrol 18: 2330–2339, 2007 Neutrophil Membrane PR3 Upregulation 2333 BASIC RESEARCH www.jasn.org

Table 1. Antibody-induced mPR3 upregulation during PMN adhesion is specific of anti-PR3 and involves the antibody Fc portiona IgG Present during Heat- Control Anti- Control Anti- Anti-CD43b Anti- TNF-Induced 0b Anti-PR3b Aggregated 0c IgG1 PR3 IgG1b MPOb (Leukosialin) HLAb b ؅ c ؅ c Adhesion IgG (Fab )2 (Fab )2 mPR3 MFI 181 Ϯ 183 202 Ϯ 176 585 Ϯ 508d 220 Ϯ 262 131 Ϯ 133 190 Ϯ 89 200 Ϯ 87 31 Ϯ 24 26 Ϯ 21 39 Ϯ 19 (n ϭ 6to7)b aTNF-induced adhesion of polymorphonuclear neutrophils (PMN) was performed as described in Figure 1, in the presence or absence of different anti- neutrophil mAb (2 ␮g/ml), heat-aggregated human or goat IgG (1 mg/ml) as compared with 2 ␮g/ml nonaggregated anti–proteinase 3 (anti-PR3) mAb, and Ј Ј ␮ F(ab )2 fragments of anti-PR3 mAb or of F(ab )2 control IgG1 (40 g/ml). MFI, mean fluorescence intensity; MPO, myeloperoxidase. bBiotinylated anti-PR3 mAb was used for incubation with anti-PR3 and for mPR3 labeling of all samples. c Ј Biotinylated F(ab )2 were used for incubations and for mPR3 labeling of all samples. dP Ͻ 0.01.

Figure 4. Role of ␤2 integrin engagement in mPR3 upregulation. (A) PMN were pretreated with 10 ␮g/ml of a blocking anti-CD18 clone IB4 mAb (or control IgG1) for 20 min at room temperature before the adhesion experiment was performed as described in Figure 1. Conditions of resting and TNF-EDTA–activated PMN are also described in Figure 1. (B) PMN adhesion was induced, in the absence of ϩ TNF-␣, by the addition of 1 mM Mn2 . (C) PMN adhesion was induced, in the absence of TNF-␣, by anti-CD18 activation KIM 185 mAb. Ϫ ϩϩ RestingHBSS , PMN in HBSS with no Ca/Mg, incubated at 37°C for 45 min in BSA-coated tubes; IgG1 HBSS , PMN incubated in ϩϩ ϩϩ HBSS at 37°C for 45 min with control IgG1 (30 ␮g/ml) in BSA-coated tubes; Adhesion, PMN in HBSS , incubated for 45 min at 37°C in 1% gelatin–coated wells in the presence of either KIM 185 mAb (30 ␮g/ml) or TNF-␣ (20 ng/ml). f, same protocols performed in the presence of 2 ␮g/ml anti-PR3 mAb. Adhesion was performed as described in Figure 1. Nonadherent cells were removed by washing ϩϩ with HBSS , and adherent cells were released by a 20-min incubation with ice-cold PBS-BSA-EDTA-azide. Suspended PMN (resting, ϩϩ TNF-EDTA, anti-CD18 blocking [A] and IgG1/HBSS [C]) were also incubated in this buffer before labeling. The mPR3 MFI of the ϩ mPR3 subset was obtained by flow cytometry after labeling with biotinylated anti-PR3 mAb and PE-streptavidin. Results are expressed as means Ϯ SD of MFI (n ϭ 4 [A], 3 [B], and 5 [C]). *P Ͻ 0.05. bled after TNF-␣ activation in EDTA. However, contrasting (Figure 2). The effect of ANCA was therefore measured using with mPR3, CD177 levels were not further upregulated by ad- the anti-PR3 mAb. We observed a 2.4-fold increase of mPR3 hesion, regardless of whether anti-PR3 mAb was present (Fig- expression when ANCA antibodies, diluted in plasma, were ure 7B). present throughout adhesion, as compared with PMN adher- ent in plasma without ANCA (data not shown). This clearly mPR3 Accessibility to Anti-PR3 Antibodies in Plasma demonstrates the ANCA–anti-PR3 interaction with adherent As already mentioned, plasma factors such as A1AT seem to PMN in plasma. inhibit the binding of anti-PR3 antibodies to mPR3ϩ PMN: When neutrophil incubations and labeling were performed in DISCUSSION undiluted autologous plasma, no mPR3 was detected on rest- ing PMN or upon TNF-activation in EDTA (Figure 8). How- We show that neutrophil activation by inflammatory stimuli, ever, TNF-induced adhesion allowed anti-PR3 mAb binding cell adhesion, and anti-PR3 mAb leads to step-wise increases of to mPR3ϩ PMN in plasma. Increased binding was obtained mPR3 available to ANCA. PMN priming is required for when the anti-PR3 mAb, diluted in plasma, was present ANCA-induced PMN activation.9,32 Priming by TNF-␣, plate- throughout adhesion. let-activating factor (PAF), or LPS induces a PR3 translocation Similar experiments were performed with and without 100 to the cell surface, enhanced upon further activation by IL-8 or ϩ ␮g/ml ANCA-PR3 human IgG diluted in plasma. As previ- fMLP.33,34 As shown here, the adhesion process and the pres- ously mentioned, the binding of ANCA antibodies could not ence of anti-PR3 antibodies amplify the mPR3 upregulations be assessed directly because of the nonspecific binding of IgG induced by TNF-␣, IL-8, fMLP, or activated HUVEC.

2334 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2330–2339, 2007 www.jasn.org BASIC RESEARCH

Figure 6. Mobilization of secretory vesicles and specific and azurophilic granules during PMN activation and adhesion with Figure 5. Upregulation of mPR3 expression during IL-8– and anti-PR3 mAb and during artificial degranulation. Neutrophils formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion were activated as in Figure 1 and labeled with mAb specific for of PMN and during adhesion to preactivated endothelial cells. membrane markers of secretory vesicles (FITC–anti-CD35), of se- PMN were activated as described in Figure 1, where TNF-␣ was cretory vesicles and specific granules (FITC–anti-CD11b; top), or replaced by 2 ␮M fMLP (A), 25 ng/ml IL-8, (B) or TNF-preactivated of azurophilic granules (PE–anti-CD63; middle). mPR3 expression ϩ human umbilical vein endothelial cells (HUVEC; see the Concise of the mPR3 subset was measured in parallel. Results are ex- Methods section; C). Neutrophil adhesion to HUVEC-coated pressed as means Ϯ SD of MFI (n ϭ 5). The table shows mPR3 plates was performed in M199 medium. Adherent cells were upregulation and azurophil granule mobilization observed after detached in EDTA, and PMN finally were labeled with biotinyl- TNF-induced adhesion with anti-PR3 and after artificial degranu- ated anti-PR3 mAb and PE-streptavidin. (C) After adhesion to lation promoted by cytochalasin B and fMLP or by calcium iono- HUVEC, cells were also labeled with anti-CD146 to exclude phore. PMN degranulation was performed as follows: 2 ϫ 106/ml ϩ ϩϩ CD146 endothelial cells (see the Concise Methods section). neutrophils in HBSS -BSA were incubated at 37°C with calcium ϩ Results are expressed as means Ϯ SD of mPR3 MFI from three ionophore A23187 (1 ␮M and Ca2 5 mM) for 15 min or with Ϫ experiments. cytochalasin B (10 ␮g/ml) for 10 min then fMLP 10 6 M for 5 min. TNF-induced adhesion with or without 2 mg/ml anti-PR3 mAb was performed as described in Figure 1. Markers of azurophilic Of interest, mPR3 expression is restricted to a constant neu- granule exocytosis were CD63 levels, measured by flow cytom- trophil subset in all of these conditions.5 The following discus- ϩ etry and MPO release in the cell supernatant, measured enzymat- sion refers to the mPR3 subpopulation and does not address ically. Results are given as means Ϯ SD (n ϭ 11). the question of the fundamental differences between mPR3ϩ and mPR3Ϫ subsets. ANCA. The role of ␤2 integrins is demonstrated here, because Fc␥R and ␤2 integrins are known to be involved in ANCA- anti-CD18 blocking mAb prevent the adhesion-dependent induced PMN activation.27,34–37 The originality of our work is mPR3 upregulation. Conversely, anti-CD18 activating mAb to show that these effects can be explained, at least in part, by enhance the mPR3 expression by promoting adhesion in the Ј the striking upregulation of ANCA antigen expression pro- absence of TNF priming. The lack of effect of F(ab )2 anti-PR3 moted by ␤2 integrin-dependent adhesion and amplified by antibodies on neutrophil mPR3 expression emphasizes the

J Am Soc Nephrol 18: 2330–2339, 2007 Neutrophil Membrane PR3 Upregulation 2335 BASIC RESEARCH www.jasn.org

Figure 7. Comparison of mPR3 and CD177 expression of PMN. (A) Representative flow cytometry histograms of PMN from a healthy donor with the same mCD177 (left) and mPR3 (right) ϩ ϩ bimodal expression pattern, showing 60% mPR3 and CD177 PMN. (B) Modulation mPR3 and mCD177 expression (positive PMN subsets) during PMN activation by TNF-␣ and adhesion. Conditions of TNF-␣ activation and adhesion are those described u in Figure 1. PMN were labeled with FITC–anti-CD177 ( )or Figure 8. The increase of mPR3 expression during PMN adhesion Ⅺ Ϯ anti-PR3 ( ). Results are expressed as means SD of MFI of five overcomes the inhibition of anti-PR3 mAb access to mPR3 by Ͻ experiments. *P 0.05. plasma. PMN in plasma (heparinized plasma from the same donor as neutrophils) or in HBSS-BSA were activated and analyzed for role of Fc␥R. We demonstrated for the first time an amplifying mPR3 expression as in Figure 1. The presence of plasma pre- loop in PMN activation: ANCA are involved in membrane vented the binding of anti-PR3 mAb to suspended PMN (resting or TNF-EDTA) but allowed it after cell adhesion. The numbers expression of their own specific antigens. ϩ ϩ refer to the mPR3 MFI of the mPR3 neutrophil subset. The mechanism of the described mPR3 upregulation is complex and involves a synergy among (1) TNF-␣ inside-out signaling, analyzed in the absence of Ca/Mg or with integrin- signaling molecules, which could be cross-linked by anti-PR3 blocking antibodies to prevent adhesion, which is not TNF- antibodies and/or be clustered in close contact with Fc␥R. In specific but also is observed with IL-8 or fMLP; (2) integrin that respect, the previously described localization of PR3 in outside-in signaling, resulting from the adhesion process; and membrane rafts, together with ␤2 integrins and Fc␥R, is worth (3)Fc␥R signaling promoted by the Fc portion of antibodies. noting.40,41 Moreover, these three signaling pathways alone are not suffi- Recent data have suggested that PR3 membrane expression cient to account for the striking mPR3 levels observed on ad- would result from mPR3 close association with CD177.30,31 We herent neutrophils reacting with anti-PR3 mAb. Indeed, these confirm that CD177, exclusively present on the mPR3ϩ neu- levels could not be reached when TNF-activated and adherent trophil subset, is upregulated as mPR3 by TNF-␣ activation PMN were incubated with 1 mg/ml heat-aggregated IgG to without adhesion. However, unlike mPR3, CD177 level is not trigger Fc␥R pathways. Similarly, mAb reacting with highly further enhanced by cell adhesion and by anti-PR3 antibodies. expressed irrelevant membrane antigens had no effect on This suggests the existence of two mPR3 species: mPR3 mole- mPR3 expression. This supports the proposed model of cules that are present on resting PMN and recruited after lim- ANCA-mediated effects occurring via both the binding of their ited activation and degranulation would form a complex with Fc regions to Fc␥R and the binding of their Fab regions to CD177, as previously suggested.31 New mPR3 molecules that 3 Ј membrane antigens. PMN activation by ANCA F(ab )2 has are translocated to the membrane upon adhesion, especially in indeed been reported.27,38,39 It suggests a specific effect of the presence of anti-PR3, may not be associated with CD177. mPR3, possibly through cis-interactions with transmembrane We cannot exclude, however, that each CD177 can bind several

2336 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2330–2339, 2007 www.jasn.org BASIC RESEARCH

mPR3 molecules and that CD177 molecules that are expressed We have shown that cytokine activation, cell adhesion, and on resting PMN are sufficient to anchor newly recruited mPR3 the presence of anti-PR3 antibodies are three conditions re- after cell adhesion with anti-PR3 antibodies. quired to allow a maximal mPR3 expression and an efficient We previously showed that intracellular storage pools of binding of anti-PR3 antibodies to PMN in plasma. This would PR3 are located not only in azurophilic but also in specific explain, at least in part, that vascular lesions observed in granules and secretory vesicles.42 TNF activation is sufficient to ANCA-associated vasculitis are restricted to capillaries and mobilize secretory vesicles and possibly tertiary granules.26,29 postcapillary venules, where small vessel diameters and low This results in a first increase of mPR3 expression, together blood flow allow PMN adhesion to the endothelium. with exposure of the secretory vesicle marker CD35 and with CD11b upregulation. TNF-induced adhesion further mobi- lizes specific granules, enhancing CD11b and mPR3 expression CONCISE METHODS at the same time. Membranes of the fused vesicles or granules seem to be richer in CD11b than in mPR3, because CD11b Antibodies and Reagents expression increases twice more than mPR3 during cell adhe- Anti-PR3 (CLB-12.8) and anti-MPO (7.17) mAb were from Sanquin sion. The opposite situation is observed when anti-PR3 anti- (Amsterdam, Netherlands), anti-elastase mAb was from Biogenesis bodies are present during adhesion, when mPR3 levels increase (Poole, UK), anti-CD43 was from BD Pharmingen (San Diego, CA), Ј four times more than CD11b. The origin of these newly ap- FITC-conjugated F(ab )2 anti-human IgG was from Jackson Immu- pearing mPR3 molecules therefore is not limited to secretory noResearch (West Grove, PA), FITC–anti-CD177 mAb (MEM-166) vesicles and specific granules. Some may result from an azuro- was from Biolegend (San Diego, CA), and FITC–anti-CD146 was philic granule exocytosis, suggested by the slight shift of CD63 from Serotec (Oxford, UK). Anti-CD18 clone IB4 was from Ancell labeling and the decrease, possibly elastase-dependent, of (Bayport, MN), and KIM-185 was provided by M. Robinson (Cell- CD35 expression.43 However, azurophilic granules are un- Tech, Cambridge, UK). Other antibodies were from Beckman Coulter likely the only source of the impressive mPR3 upregulation (Roissy, France). TNF-␣ was from PeproTech (Rocky Hill, NJ), IL-8 occurring during adhesion with anti-PR3 antibodies—first be- was from R&D Systems (Abingdon Oxon, UK), fibrinogen was from cause their exocytosis is very limited and second because max- Diagnostica Stago (Genevilliers, France), and collagen I was from In- imum degranulation of azurophilic granules and all other ves- vitrogen (Palo Alto, CA). Other reagents were from Sigma (St. Louis, icles, by fMLP/cytochalasin or Ca ionophore, was not MO). sufficient to reach similar mPR3 levels. Apart from secretory vesicles and granules, an intracellular localization of PR3 at the Neutrophil Isolation and Activation inner face of the plasma membrane has been proposed to ex- PMN from healthy donors, prepared as described previously,47 were plain the increase of mPR3 expression on apoptotic cells.44 In suspended (2 ϫ 106/ml) in BSA 0.1% HBSS (Life Technologies, Pais- our experimental settings, mPR3 upregulation occurred with- ley, UK) without (HBSSϪ) or with (HBSS2ϩ)1mMCa2ϩ/Mg2ϩ or out apoptosis. However, transient phospholipid flip-flop also with 10 mM EDTA (HBSS-EDTA). Protocols used for PMN activa- occurred during PMN activation,45 and constant recycling of tion and adhesion are given in corresponding figure legends. membrane PR3 was recently described.35 Whether TNF-in- duced adhesion results in a similar phenomenon deserves fur- Endothelial Cells (HUVEC) ther studies. It might promote a transient PR3 molecule trans- Endothelial cells were isolated from human umbilical cord veins as fer from the inner to the outer plasma membrane leaflet. We described previously48 and cultured in M199 medium (Life Technol- hypothesize that anti-PR3 antibody binding would then pre- ogies) with 20% FCS. They were subcultured in 48-well culture plates vent this PR3 to be re-internalized. coated with 0.5% gelatin and pretreated for 4 h with 10 ng/ml TNF-␣. As previously reported,46 TNF activation increased mPR3 After rinsing three times with medium, they were used as substrate for but did not result in MPO membrane expression. Although we PMN adhesion, as described in Figure 5. were able to detect some membrane MPO or elastase on ad- ؅ herent PMN with specific anti-MPO or -elastase mAb, these IgG Purification, Biotinylation, and F(ab )2 expressions were far less important (Ͻ10%) than mPR3 levels. Preparations They could result from the nonspecific binding of fluid phase Blood was obtained from three PR3-ANCAϩ and four MPO-ANCAϩ MPO and elastase, released during TNF-induced adhesion, vasculitis patients with high ANCA titers. Human IgG were isolated and likely bind neutrophil membranes as a result of their pos- from patient and healthy donor sera by Sepharose– G chro- itive charge. The same mechanism could explain the low mPR3 matography (Amersham Bioscience AB, Uppsala, Sweden) and, when Ϫ levels detected on the mPR3 subset in response to TNF-in- indicated, biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce, duced adhesion in the presence of anti-PR3 antibodies. Finally, Rockford, IL), according to manufacturers’ instructions. When men- confirming our initial working hypothesis, our results show tioned, IgG (10 mg/ml) were heat-aggregated by a 20-min incubation Ј that PMN adhesion favors the access of anti-PR3 mAb or at 60°C. F(ab )2 fragments of IgG preparations were made by pepsin ANCA to their antigens in whole blood. digestion, as described previously.36

J Am Soc Nephrol 18: 2330–2339, 2007 Neutrophil Membrane PR3 Upregulation 2337 BASIC RESEARCH www.jasn.org

Flow Cytometry 3. Reumaux D, Duthilleul P, Roos D: Pathogenesis of diseases associated After stimulation, PMN were washed in ice-cold PBS, 1% BSA, and with antineutrophil cytoplasm autoantibodies. Hum Immunol 65: 1–12, 2004 0.1% sodium azide and incubated at 4°C with unlabeled or biotinyl- 4. Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs- ated primary antibody, followed by PE-labeled secondary antibody or Mecarelli L: Neutrophils: Molecules, functions and pathophysiological streptavidin. When anti-PR3 antibodies were present during PMN aspects. Lab Invest 80: 617–653, 2000 activation, similar results were obtained whether labeling was per- 5. Halbwachs-Mecarelli L, Bessou G, Lesavre P, Lopez S, Witko-Sarsat V: formed directly with the secondary antibody or a new sample of anti- Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil PR3 mAb was added after adhesion. pool. FEBS Lett 374: 29–33, 1995 Cell suspensions recovered after PMN adhesion to HUVEC were 6. Witko-Sarsat V, Lesavre P, Lopez S, Bessou G, Hieblot C, Prum B, Noel labeled with anti-PR3 mAb and with FITC–anti-CD146. mPR3 ex- LH, Guillevin L, Ravaud P, Sermet-Gaudelus I, Timsit J, Grunfeld JP, pression was analyzed after exclusion of CD146ϩ HUVEC. Halbwachs-Mecarelli L: A large subset of neutrophils expressing mem- Cells were analyzed by flow cytometry on a Becton Dickinson brane proteinase 3 is a risk factor for vasculitis and rheumatoid arthri- tis. J Am Soc Nephrol 10: 1224–1233, 1999 FACSCalibur (Mountainview, CA). Results are given as mean fluo- 7. Rarok AA, Stegeman CA, Limburg PC, Kallenberg CG: Neutrophil rescence intensity. membrane expression of proteinase 3 (PR3) is related to relapse in PR3-ANCA-associated vasculitis. J Am Soc Nephrol 13: 2232–2238, MPO Enzymatic Activity Dosage 2002 The amount of active MPO released in neutrophil supernatant was 8. Schreiber A, Luft FC, Kettritz R: Membrane proteinase 3 expression and ANCA-induced neutrophil activation. Kidney Int 65: 2172–2183, 49 measured enzymatically by colorimetry. 2004 9. Charles LA, Caldas ML, Falk RJ, Terrell RS, Jennette JC: Antibodies Apoptosis Evaluation against granule activate neutrophils in vitro. J Leukoc Biol 50: Annexin V and 7AAD binding were used to analyze the apoptotic and 539–546, 1991 necrotic state of neutrophils by flow cytometry, as described previous- 10. Falk RJ, Terrell RS, Charles LA, Jennette JC: Anti-neutrophil cytoplas- mic autoantibodies induce neutrophils to degranulate and produce 50 ly. oxygen radicals in vitro. Proc Natl Acad Sci U S A 87: 4115–4119, 1990 Statistical Analyses 11. Keogan MT, Esnault VL, Green AJ, Lockwood CM, Brown DL: Activa- Mean fluorescence intensities were compared using a paired t test tion of normal neutrophils by anti-neutrophil cytoplasm antibodies. analysis. Clin Exp Immunol 90: 228–234, 1992 12. Lu X, Garfield A, Rainger GE, Savage CO, Nash GB: Mediation of endothelial cell damage by serine proteases, but not superoxide, released from antineutrophil cytoplasmic antibody-stimulated neutro- ACKNOWLEDGMENTS phils. Arthritis Rheum 54: 1619–1628, 2006 13. Savage CO, Pottinger BE, Gaskin G, Pusey CD, Pearson JD: Autoan- tibodies developing to myeloperoxidase and proteinase 3 in systemic S.B. and A.M. were supported by grants from the Fondation Nation- vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial ale d’Aide aux Insuffisants Renaux and from the Association pour la cells. Am J Pathol 141: 335–342, 1992 Recherche sur la Polyarthrite Rhumatoı¨de, respectively. We acknowl- 14. Mayet WJ, Meyer zum Buschenfelde KH: Antibodies to proteinase 3 edge support from Baxter, Amgen, and AURA. increase adhesion of neutrophils to human endothelial cells. Clin Exp Immunol 94: 440–446, 1993 Part of our results were previously reported as abstracts at the 15. Radford DJ, Luu NT, Hewins P, Nash GB, Savage CO: Antineutrophil annual meetings of the American Society of Nephrology; November 8 cytoplasmic antibodies stabilize adhesion and promote migration of through 13, 2005, Philadelphia, PA; and November 14 through 19, flowing neutrophils on endothelial cells. Arthritis Rheum 44: 2851– 2006; San Diego, CA. 2861, 2001 We thank Martyn Robinson (CellTech, Cambridge, UK) for the 16. Buttrum SM, Drost EM, MacNee W, Goffin E, Lockwood CM, Hatton R, Nash GB: Rheological response of neutrophils to different types of invaluable gift of the anti-CD18 activating KIM185 mAb. stimulation. J Appl Physiol 77: 1801–1810, 1994 17. Tse WY, Nash GB, Hewins P, Savage CO, Adu D: ANCA-induced neutrophil F-actin polymerization: implications for microvascular in- DISCLOSURES flammation. Kidney Int 67: 130–139, 2005 18. Abdel-Salam B, Iking-Konert C, Schneider M, Andrassy K, Hansch GM: None. Autoantibodies to neutrophil cytoplasmic antigens (ANCA) do not bind to polymorphonuclear neutrophils in blood. Kidney Int 66: 1009– 1017, 2004 19. Ambrose LR, Little MA, Nourshargh S, Pusey CD: Anti-proteinase 3 REFERENCES antibody binding to neutrophils as demonstrated by confocal micros- copy. Kidney Int 68: 2912–2913, 2005 1. Jayne DR, Gaskin G, Pusey CD, Lockwood CM: ANCA and predicting 20. Van Rossum AP, van der Geld YM, Limburg PC, Kallenberg CG: relapse in systemic vasculitis. QJM 88: 127–133, 1995 Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 2. Tervaert JW, van der Woude FJ, Fauci AS, Ambrus JL, Velosa J, Keane (PR3-ANCA) bind to neutrophils. Kidney Int 68: 537–541, 2005 WF, Meijer S, van der Giessen M, van der Hem GK, The TH, et al.: 21. Rooney CP, Taggart C, Coakley R, McElvaney NG, O’Neill SJ: Anti- Association between active Wegener’s granulomatosis and anticyto- proteinase 3 antibody activation of neutrophils can be inhibited by plasmic antibodies. Arch Intern Med 149: 2461–2465, 1989 alpha1-antitrypsin. Am J Respir Cell Mol Biol 24: 747–754, 2001

2338 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 2330–2339, 2007 www.jasn.org BASIC RESEARCH

22. Esnault VL, Testa A, Audrain M, Roge C, Hamidou M, Barrier JH, neutrophils by antineutrophil cytoplasm antibodies occurs via Fc- Sesboue R, Martin JP, Lesavre P: Alpha 1-antitrypsin genetic polymor- gamma receptors and CD18. J Am Soc Nephrol 15: 796–808, 2004 phism in ANCA-positive systemic vasculitis. Kidney Int 43: 1329–1332, 38. Yang JJ, Preston GA, Alcorta DA, Waga I, Munger WE, Hogan SL, 1993 Sekura SB, Phillips BD, Thomas RP, Jennette JC, Falk RJ: Expression 23. Campbell EJ, Campbell MA: Pericellular proteolysis by neutrophils in profile of leukocyte activated by anti-neutrophil cytoplasmic the presence of proteinase inhibitors: Effects of substrate opsoniza- autoantibodies (ANCA). Kidney Int 62: 1638–1649, 2002 tion. J Cell Biol 106: 667–676, 1988 39. Williams JM, Ben-Smith A, Hewins P, Dove SK, Hughes P, McEwan R, 24. Vissers MC, George PM, Bathurst IC, Brennan SO, Winterbourn CC: Wakelam MJ, Savage CO: Activation of the G(i) heterotrimeric G Cleavage and inactivation of alpha 1-antitrypsin by metalloproteinases protein by ANCA IgG F(abЈ)2 fragments is necessary but not sufficient released from neutrophils. J Clin Invest 82: 706–711, 1988 to stimulate the recruitment of those downstream mediators used by 25. Nie J, Pei D: Rapid inactivation of alpha-1-proteinase inhibitor by intact ANCA IgG. J Am Soc Nephrol 14: 661–669, 2003 neutrophil specific leukolysin/membrane-type matrix metalloprotein- 40. David A, Fridlich R, Aviram I: The presence of membrane proteinase 3 ase 6. Exp Cell Res 296: 145–150, 2004 in neutrophil lipid rafts and its colocalization with FcgammaRIIIb and 26. Bouaouina M, Blouin E, Halbwachs-Mecarelli L, Lesavre P, Rieu P: cytochrome b558. Exp Cell Res 308: 156–165, 2005 TNF-induced beta2 integrin activation involves Src kinases and a 41. Fridlich R, David A, Aviram I: Membrane proteinase 3 and its interac- redox-regulated activation of p38 MAPK. J Immunol 173: 1313–1320, tions within microdomains of neutrophil membranes. J Cell Biochem 2004 99: 117–125, 2006 27. Kettritz R, Jennette JC, Falk RJ: Crosslinking of ANCA-antigens stim- 42. Witko-Sarsat V, Cramer EM, Hieblot C, Guichard J, Nusbaum P, Lopez ulates superoxide release by human neutrophils. J Am Soc Nephrol 8: S, Lesavre P, Halbwachs-Mecarelli L: Presence of proteinase 3 in 386–394, 1997 secretory vesicles: Evidence of a novel, highly mobilizable intracellular 28. Andrew D, Shock A, Ball E, Ortlepp S, Bell J, Robinson M: KIM185, a pool distinct from azurophil granules. Blood 94: 2487–2496, 1999 monoclonal antibody to CD18 which induces a change in the confor- 43. Tosi MF, Zakem H, Berger M: cleaves C3bi on mation of CD18 and promotes both LFA-1- and CR3-dependent ad- opsonized pseudomonas as well as CR1 on neutrophils to create a hesion. Eur J Immunol 23: 2217–2222, 1993 functionally important opsonin receptor mismatch. J Clin Invest 86: 29. Chakrabarti S, Zee JM, Patel KD: Regulation of matrix metalloprotein- 300–308, 1990 ase-9 (MMP-9) in TNF-stimulated neutrophils: novel pathways for ter- 44. Durant S, Pederzoli M, Lepelletier Y, Canteloup S, Nusbaum P, Le- tiary granule release. J Leukoc Biol 79: 214–222, 2006 savre P, Witko-Sarsat V: Apoptosis-induced proteinase 3 membrane 30. Bauer S, Abdgawad M, Gunnarsson L, Segelmark M, Tapper H, Hell- expression is independent from degranulation. J Leukoc Biol 75: 87– mark T: Proteinase 3 and CD177 are expressed on the plasma mem- 98, 2004 brane of the same subset of neutrophils. J Leukoc Biol 81: 458–464, 45. Frasch SC, Henson PM, Nagaosa K, Fessler MB, Borregaard N, Bratton 2007 DL: Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) 31. von Vietinghoff S, Tunnemann G, Eulenberg C, Wellner M, Cardoso co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neu- MC, Luft FC, Kettritz R: NB1 mediates surface expression of the ANCA trophils. J Biol Chem 279: 17625–17633, 2004 antigen proteinase 3 on human neutrophils. Blood 109: 4487–4493, 2007 46. Reumaux D, Hordijk PL, Duthilleul P Roos D: Priming by tumor necro- 32. Falk RJ, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies with sis factor-alpha of human neutrophil NADPH-oxidase activity induced specificity for myeloperoxidase in patients with systemic vasculitis and by antiproteinase-3 or antimyeloperoxidase antibodies. J Leukoc Biol idiopathic necrotizing and crescentic glomerulonephritis. N Engl 80: 1424–1433, 2006 J Med 318: 1651–1657, 1988 47. Seveau S, Lopez S, Lesavre P, Guichard J, Cramer EM, Halbwachs- 33. Campbell EJ, Campbell MA, Owen CA: Bioactive proteinase 3 on the Mecarelli L: Leukosialin (CD43, sialophorin) redistribution in uropods cell surface of human neutrophils: Quantification, catalytic activity, and of polarized neutrophils is induced by CD43 cross-linking by antibod- susceptibility to inhibition. J Immunol 165: 3366–3374, 2000 ies, by colchicine or by chemotactic peptides. J Cell Sci 110: 1465– 34. Porges AJ, Redecha PB, Kimberly WT, Csernok E, Gross WL, Kimberly 1475, 1997 RP: Anti-neutrophil cytoplasmic antibodies engage and activate hu- 48. Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of human man neutrophils via Fc gamma RIIa. J Immunol 153: 1271–1280, 1994 endothelial cells derived from umbilical veins. Identification by mor- 35. Mulder AH, Heeringa P, Brouwer E, Limburg PC, Kallenberg CG: phologic and immunologic criteria. J Clin Invest 52: 2745–2756, 1973 Activation of by anti-neutrophil cytoplasmic antibodies 49. Capeillere-Blandin C, Gausson V, Nguyen AT, Descamps-Latscha B, (ANCA): A Fc gamma RII-dependent process. Clin Exp Immunol 98: Drueke T, Witko-Sarsat V: Respective role of uraemic toxins and 270–278, 1994 myeloperoxidase in the uraemic state. Nephrol Dial Transplant 21: 36. Reumaux D, Vossebeld PJ, Roos D, Verhoeven AJ: Effect of tumor 1555–1563, 2006 necrosis factor-induced integrin activation on Fc gamma receptor 50. Nusbaum P, Laine C, Bouaouina M, Seveau S, Cramer EM, Masse JM, II-mediated signal transduction: Relevance for activation of neutro- Lesavre P, Halbwachs-Mecarelli L: Distinct signaling pathways are phils by anti-proteinase 3 or anti-myeloperoxidase antibodies. Blood involved in leukosialin (CD43) down-regulation, membrane blebbing, 86: 3189–3195, 1995 and phospholipid scrambling during neutrophil apoptosis. J Biol 37. Hewins P, Williams JM, Wakelam MJ, Savage CO: Activation of Syk in Chem 280: 5843–5853, 2005

J Am Soc Nephrol 18: 2330–2339, 2007 Neutrophil Membrane PR3 Upregulation 2339