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Published ahead of print on July 18, 2007
J Am Soc Nephrol 18: 2330-2339, 2007
© 2007 American Society of Nephrology
doi: 10.1681/ASN.2006121309
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BASIC RESEARCH

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

Soumeya Brachemi*, Agnès Mambole*, Fadi Fakhouri{dagger}, Luc Mouthon{ddagger}, Loïc Guillevin{ddagger}, Philippe Lesavre*,{dagger} and Lise Halbwachs-Mecarelli*

* INSERM U845 and Université Paris V René Descartes, {dagger} Necker Hospital Nephrology Department, and {ddagger} Internal Medicine Department, Cochin Hospital and Unité Propre de Recherche de l'Enseignement Supérieur (UPRES) EA 4058, Paris, France

Correspondence: Dr. Lise Halbwachs-Mecarelli, INSERM U845, Hôpital Necker, 161 rue de Sèvres, 75015 Paris, France. Phone: +33-144-49-52-32; Fax: +33-145-66-51-33; E-mail: mecarelli{at}necker.fr

Received for publication December 3, 2006. Accepted for publication April 27, 2007.


   Abstract
Top
 Abstract
Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 Disclosures
 References
 
We investigated membrane proteinase 3 (mPR3) expression during TNF-{alpha}–induced adhesion of neutrophils 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 on the mPR3+ neutrophil subset after stepwise cell activation. TNF-{alpha} activation without adhesion, TNF-{alpha}–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 neutrophils, whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neutrophil adhesion to activated human umbilical vein endothelial cells. It involved beta2 integrins and Fc{gamma} 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-myeloperoxidase or anti–HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhesion, were mobilized from secretory vesicles (CD35+) and secondary granules (CD11b+). The adhesion- and antibody-dependent upregulations of mPR3 expression occurred with little azurophilic granule degranulation, 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.


   Introduction
Top
 Abstract
 Introduction
RESULTS
 DISCUSSION
 CONCISE METHODS
 Disclosures
 References
 
Wegener granulomatosis, microscopic polyangiitis, pauci-immune necrotizing glomerulonephritis, and Churg-Strauss syndrome constitute a group of small vessel vasculitis associated with anti-neutrophil cytoplasmic autoantibodies (ANCA). The pathogenesis of these vasculitides is closely linked to increased ANCA levels and polymorphonuclear neutrophils (PMN) found in the glomeruli and interstitium.1–4

ANCA are directed against proteinase 3 (PR3) or myeloperoxidase (MPO), both proteinases mainly stored in intracellular granules. Membrane PR3 is present on a variable proportion of resting PMN (mPR3+), as opposed to membrane MPO, which is found only after extensive cell activation.5 A high proportion of mPR3+ neutrophils is a risk factor for the development of vasculitis, and elevated mPR3 levels are associated with a higher relapse rate in patients with Wegener granulomatosis.6,7 In vitro ANCA-induced responses are higher in mPR3+ 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 release of proteolytic enzymes and reactive oxygen species.9–13 They modulate neutrophil adhesion and migration14,15 and induce 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 {alpha}1 anti-trypsin (A1AT) binding to PR3.21 The association between A1AT-deficient phenotypes and anti-PR3–positive vasculitis suggests that A1AT controls ANCA access to its antigen.22

Adherent neutrophils are surrounded by an area where protease inhibitors, such as A1AT, are inactivated by neutrophil-derived metalloproteases.23–25 We hypothesized that mPR3 becomes 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 adhesion induced by TNF-{alpha} in the presence of anti-PR3 antibodies, as seen in the microvascular environment of vasculitis patients.


   RESULTS
Top
 Abstract
 Introduction
 RESULTS
DISCUSSION
 CONCISE METHODS
 Disclosures
 References
 
Upregulation of Membrane PR3 Expression during TNF-{alpha}–Induced Adhesion
TNF-{alpha} is known to trigger adhesion-independent and -dependent neutrophil responses. The addition of EDTA distinguishes the effects that are caused by TNF-{alpha} per se from those that result from TNF-induced integrin engagement and "outside-in" signaling.26 We analyzed mPR3 expression on TNF-activated PMN from individuals with varying proportions of PMN expressing PR3 (mPR3+; Figure 1A).


Figure 1
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  		Figure 1. Membrane proteinase 3 (mPR3) expression after TNF-{alpha}–induced PMN activation and adhesion. (A) Representative flow cytometry histograms obtained with polymorphonuclear neutrophils (PMN) from three healthy donors with 80% (left), 40% (middle), and 0% (right) of mPR3+ PMN. PMN (2 x 106/ml in HBSS-EDTA in BSA-coated tubes) were incubated at 37°C for 45 min (Resting PMN) or, after 15 min at 37°C, for 30 min with TNF-{alpha} (20 ng/ml; TNF-EDTA). PMN (2 x 106/ml in HBSS2+-BSA) were allowed to settle for 15 min at 37°C in 1% gelatin–coated wells and incubated with TNF-{alpha} (20 ng/ml) for 30 min at 37°C without (TNF + Adhesion) and with 2 µg/ml anti-PR3 mAb (TNF adhesion + anti-PR3). The anti-PR3 mAb was previously centrifuged for 20 min at 12,000 x g to remove Ig aggregates. After washing off nonadherent cells, adherent cells were released by a 20-min incubation with ice-cold PBS, 1% BSA, 10 mM EDTA, and 0.1% sodium azide. Resting and TNF-EDTA cells were similarly incubated in 0.1% sodium azide before neutrophil labeling with anti-PR3 mAb (solid line) or control IgG1 (dotted line) and PE-labeled secondary antibody. (B) Synthesis of 22 experiments performed as described in A (means ± SD of mPR3+ subset mean fluorescence intensity [MFI]). ***P < 0.001.

 
TNF-{alpha} activation without cell adhesion, in HBSS-EDTA, doubled the mPR3 level of the mPR3+ neutrophil subset (Figure 1). Similar results were obtained with TNF-{alpha} activation in HBSS–, with or without shaking (data not shown). When neutrophils were allowed to adhere to gelatin-coated plates, upon activation by TNF-{alpha} in HBSS2+, the mPR3 level was further enhanced, reaching 7 ± 3-fold the resting cells level (Figure 1).

Anti-PR3 Antibody-Dependent Upregulation of Membrane PR3 Expression
We analyzed TNF-{alpha}–activated neutrophils in the presence of anti-PR3 mAb to mimic further the situation occurring in ANCA-positive vasculitis. As expected, TNF-{alpha} induced 58 ± 23% PMN adhesion, which was increased to 65 ± 21% in the presence of 2 µg/ml anti-PR3 mAb (data not shown).14,15 Surprisingly, the presence of anti-PR3 mAb throughout adhesion resulted in a stronger mPR3 upregulation, reaching 24 ± 15 times the level of resting mPR3+ PMN (Figure 1).

We excluded an in vitro artifact related to cell incubation with mAb at 37°C, allowing a more rapid antibody binding than during the labeling performed at 4°C. The antibody concentration was not limiting, because the mPR3 upregulation was similar with 60 ng/ml anti-PR3 mAb (n = 3) instead of 2 µg/ml. Moreover, incubation of TNF-activated adherent PMN for up to 2 h at 4°C with the mAb, to improve PR3 labeling, resulted in <10% 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 mPR3+ neutrophil subset. TNF-{alpha} activation had no effect on the mPR3+ neutrophil percentage, and the mPR3– subset remained mostly negative during neutrophil activation and adhesion. It is worth noting that some mPR3 was detected on the mPR3– subpopulation as a result of TNF-induced adhesion in the presence of anti-PR3 mAb. However, this mPR3 expression did not reach 3% of the level expressed by mPR3+ cells in the same conditions.

Human Anti-PR3 but not Anti-MPO ANCA Enhance mPR3 Expression during PMN Adhesion
The Fc domain of ANCA or control IgG binds indistinctly to mPR3+ and mPR3– PMN subsets. As described previously,27 it leads to a high background that obscures the specific binding of anti–PR3-ANCA IgG (Figure 2A, left). The little shift of the ANCA IgG peak, as compared with normal IgG, was NS and was not observed with all normal IgG. Specific binding of anti-PR3 ANCA to mPR3+ neutrophils occurs, however (Figure 2A, right), and F(ab')2 fragments of anti–PR3-ANCA labeled the same proportion of PMN as the anti-PR3 mAb.


Figure 2
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  		Figure 2. mPR3 expression upregulation by human anti-PR3 ANCA. (A) Binding of anti-PR3 ANCA IgG and F(ab')2 to PMN from a donor bimodal in mPR3 expression. PMN activated by TNF-{alpha} in EDTA, as described in Figure 1, were labeled at 4°C with IgG (100 µg/ml) purified from a normal control (plain line) or from an anti-PR3 anti-neutrophil cytoplasmic autoantibodies (ANCA)-positive vasculitis patient (bold line, left) or with anti-PR3 ANCA F(ab')2 (500 µg/ml; bold line, right), followed by a secondary FITC anti-human IgG antibody. The dotted line represents nonspecific binding of the secondary antibody and is superimposed, in the right diagram, with the peak obtained with control IgG F(ab')2. ANCA and control human IgG bind nonspecifically to PMN, via their Fc domain, with no distinction of the mPR3+ and mPR3– subsets. Conversely, F(ab')2 ANCA anti-PR3 specifically labels the mPR3+ subset. (B) Effect of ANCA anti-PR3 on mPR3 expression during PMN adhesion. PMN were activated as in Figure 1, in the presence or absence of human IgG, ANCA, or control (50 µg/ml). mPR3 expression was measured after labeling with biotin-labeled anti-PR3 mAb and PE-streptavidin. mPR3 flow cytometry histograms of TNF-{alpha}–activated PMN in EDTA (dotted line), adherent PMN (plain line), or PMN adherent in the presence of ANCA or control IgG (bold line) are shown.

 
We took advantage of the fact that anti–PR3-ANCA do not hinder anti-PR3 mAb CLB 12.8 binding: The anti-PR3 mAb binding on mPR3+ PMN was not modified when 200 µg/ml anti-PR3+ patient IgG were added during labeling (data not shown). We thus used the biotinylated anti-PR3 mAb to quantify mPR3 after PMN adhesion with ANCA-IgG. A significant 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, with no effect observed with IgG from five of five control subjects and four of four anti–MPO-ANCA–positive patients (data not shown).

Specificity of Antibody-Induced Upregulation of mPR3 Expression and Role of Fc{gamma} Receptors
We first looked for antibody-induced upregulations of neutrophil MPO or elastase. As shown in Figure 3, TNF-induced adhesion did not promote significant MPO or elastase membrane expressions. The presence of anti-MPO or anti-elastase mAb throughout adhesion resulted in a small, statistically significant membrane expression of MPO or elastase, respectively. However, such expressions, homogeneous within the neutrophil pool, represented <10% of the mPR3+ expression seen after PMN adhesion with anti-PR3 mAb (Figure 3B).


Figure 3
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  		Figure 3. Membrane myeloperoxidase (MPO), elastase (HNE), and HLA labeling after PMN adhesion. (A) Increase of membrane MPO and HNE expression during PMN adhesion (performed as described in Figure 1) in the presence of anti-MPO mAb and anti-HNE mAb (2 µg/ml), respectively (replacing anti-PR3 mAb described in Figure 1). The figure shows nonspecific binding of isotype-matched control antibody (dark dotted line) and binding of anti-MPO or anti-HNE mAb on resting PMN (light solid line), on TNF-{alpha}–activated PMN in EDTA (light dotted line), on TNF-{alpha}–activated adherent PMN (plain line), and on adherent PMN in the presence of either anti-MPO mAb or anti-HNE mAb (bold line; n = 4). No difference in membrane HLA was noted between TNF-{alpha}–activated adherent PMN (plain line) and those adherent in the presence of anti-HLA mAb (bold line; n = 3). (B) Membrane MPO (mMPO) levels, measured after TNF-{alpha}–induced PMN adhesion (performed as described in Figure 1) with anti-MPO, are compared with mPR3 levels (mPR3+ subset) of adherent PMN with anti-PR3 mAb. No modulation of nonspecific binding of IgG1 was observed during PMN adhesion with IgG1. Results are expressed as means ± SD of anti-MPO, anti-PR3, or IgG1 MFI, using PE-labeled secondary anti-mouse IgG antibody (n = 8). **P < 0.01. ***P < 0.001.

 
No change of HLA A/B expression and no binding of IgG1 were observed when PMN adhered in the presence of anti-HLA mAb (Figure 3A) or IgG1 (Figure 3B), respectively. Then, neutrophils were allowed to adhere in the presence of various IgG1 mAb before analysis of mPR3 expression of the mPR3+ subset. This was enhanced only by anti-PR3 mAb and was not modified by isotypic IgG1 control, anti-MPO, or irrelevant anti-neutrophil mAb such as anti-CD43 or anti-HLA A/B, despite the high levels of membrane CD43 or HLA antigens reacting with these mAb (Table 1).


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  		Table 1. Antibody-induced mPR3 upregulation during PMN adhesion is specific of anti-PR3 and involves the antibody Fc portiona

 
Incubation of TNF-{alpha}–activated PMN with F(ab')2 anti-PR3 mAb during adhesion did not enhance mPR3 expression, emphasizing the role of Fc{gamma} receptors (Fc{gamma}R) in mPR3 upregulation (Table 1). However, nonspecific Fc{gamma}R activation by 1 mg/ml heat-aggregated IgG during PMN adhesion was not sufficient to increase the mPR3 expression (Table 1).

Role of Adhesion and beta2 Integrin Engagement
The presence of anti-PR3 antibodies during TNF-induced adhesion to gelatin (Figure 1), fibrinogen, or type I collagen (data not shown) upregulated mPR3 expression of the mPR3+ subset. Pretreating PMN with anti-CD18 blocking mAb (IB4), which resulted in 86% inhibition of cell adhesion (data not shown), significantly prevented the antibody-induced mPR3 upregulation (Figure 4A). TNF activation with this anti-CD18 mAb resulted, however, in a slight significant increase of PR3 expression similar to that observed with EDTA.


Figure 4
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  		Figure 4. Role of beta2 integrin engagement in mPR3 upregulation. (A) PMN were pretreated with 10 µg/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 TNF-{alpha}, by the addition of 1 mM Mn2+. (C) PMN adhesion was induced, in the absence of TNF-{alpha}, by anti-CD18 activation KIM 185 mAb. RestingHBSS–, PMN in HBSS with no Ca/Mg, incubated at 37°C for 45 min in BSA-coated tubes; IgG1 HBSS++, PMN incubated in HBSS++ at 37°C for 45 min with control IgG1 (30 µg/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 µg/ml) or TNF-{alpha} (20 ng/ml). {blacksquare}, same protocols performed in the presence of 2 µg/ml anti-PR3 mAb. Adhesion was performed as described in Figure 1. Nonadherent cells were removed by washing with HBSS++, and adherent cells were released by a 20-min incubation with ice-cold PBS-BSA-EDTA-azide. Suspended PMN (resting, TNF-EDTA, anti-CD18 blocking [A] and IgG1/HBSS++[C]) were also incubated in this buffer before labeling. The mPR3 MFI of the mPR3+ subset was obtained by flow cytometry after labeling with biotinylated anti-PR3 mAb and PE-streptavidin. Results are expressed as means ± SD of MFI (n = 4 [A], 3 [B], and 5 [C]). *P < 0.05.

 
For further analysis if their role of in mPR3 expression, beta2 integrins were switched into an active conformation by manganese (Mn2+) or by an anti-CD18 activating mAb (KIM 185)28 in the absence of TNF-{alpha} signaling. Both resulted in an enhanced mPR3 level on adherent cells, which was further increased by the presence of anti-PR3 throughout adhesion (Figure 4, B and C).

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-{alpha} and with IL-8 or formyl-methionyl-leucyl-phenylalanine (fMLP) (Figure 5). Background cell adhesion, observed in the absence of priming, with or without anti-PR3 mAb, resulted in slight nonsignificant increases of mPR3 expression (data not shown).


Figure 5
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  		Figure 5. Upregulation of mPR3 expression during IL-8–and formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion of PMN and during adhesion to preactivated endothelial cells. PMN were activated as described in Figure 1, where TNF-{alpha} was replaced by 2 µM fMLP (A), 25 ng/ml IL-8, (B) or TNF-preactivated human umbilical vein endothelial cells (HUVEC; see the Concise Methods section; C). Neutrophil adhesion to HUVEC-coated plates was performed in M199 medium. Adherent cells were detached in EDTA, and PMN finally were labeled with biotinylated anti-PR3 mAb and PE-streptavidin. (C) After adhesion to HUVEC, cells were also labeled with anti-CD146 to exclude CD146+ endothelial cells (see the Concise Methods section). Results are expressed as means ± SD of mPR3 MFI from three experiments.

 
Finally, neutrophils were allowed to adhere to TNF-{alpha}–preactivated human umbilical vein endothelial cells (HUVEC) to mimic further the situation occurring in ANCA vasculitis. This caused a 11 ± 4-fold increase of mPR3 expression of the mPR3+ subset, which further doubled when the anti-PR3 mAb was present during adhesion (Figure 5).

Role of Degranulation in the mPR3 Upregulation
We assessed membrane expression of granules and vesicles markers to investigate the newly exposed mPR3 origin (Figure 6). As previously reported.,26 TNF-{alpha} activation of PMN in the absence of adhesion significantly increased membrane CD35 and CD11b, consistent with secretory vesicles and tertiary granules degranulation.29


Figure 6
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  		Figure 6. Mobilization of secretory vesicles and specific and azurophilic granules during PMN activation and adhesion with anti-PR3 mAb and during artificial degranulation. Neutrophils were activated as in Figure 1 and labeled with mAb specific for membrane markers of secretory vesicles (FITC–anti-CD35), of secretory vesicles and specific granules (FITC–anti-CD11b; top), or of azurophilic granules (PE–anti-CD63; middle). mPR3 expression of the mPR3+ subset was measured in parallel. Results are expressed as means ± SD of MFI (n = 5). The table shows mPR3 upregulation and azurophil granule mobilization observed after TNF-induced adhesion with anti-PR3 and after artificial degranulation promoted by cytochalasin B and fMLP or by calcium ionophore. PMN degranulation was performed as follows: 2 x 106/ml neutrophils in HBSS++-BSA were incubated at 37°C with calcium ionophore A23187 (1 µM and Ca2+ 5 mM) for 15 min or with cytochalasin B (10 µg/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 granule exocytosis were CD63 levels, measured by flow cytometry and MPO release in the cell supernatant, measured enzymatically. Results are given as means ± SD (n = 11).

 
TNF-{alpha}–induced PMN adhesion further enhanced CD11b membrane expression, indicating specific granule mobilization. Adhesion in the presence of anti-PR3 mAb again amplified CD11b levels and resulted in a slight expression of CD63, a marker of azurophilic granules. However, CD63 expression did not reach 3% of the levels observed after maximum degranulation by fMLP/cytochalasin or by calcium ionophore A23187 (Figure 6, table). Similarly, the amount of MPO released in supernatants of TNF-{alpha}–activated neutrophils, with or without anti-PR3 mAb, was <10% (detection threshold) of the amount released after total degranulation. Neutrophil adhesion in the presence of anti-PR3 thus resulted in little mobilization of azurophilic granules. Surprising, after PMN extensive degranulation by fMLP/cytochalasin, leading to maximum CD63 expression and MPO release, mPR3 expression did not reach the levels observed after PMN adhesion in the presence of anti-PR3 (Figure 6, table).

Role of Putative Membrane Mechanisms
We assessed putative apoptosis-related membrane flip-flop, resulting from cell adhesion or mechanic recovery of adherent cells, by measuring annexin V binding. Less than 10% of annexin-positive cells were detected after adhesion, with or without anti-PR3 mAb (data not shown).

A close association between CD177 and mPR3 was recently reported, suggesting that CD177 itself mediates PR3 membrane expression.30,31 We therefore analyzed CD177 during mPR3 upregulation. As expected, there was a bimodal distribution of CD177 on PMN (Figure 7A), and we confirmed, by double labeling, that the CD177+ and mPR3+ subsets are identical (data not shown).


Figure 7
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  		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) bimodal expression pattern, showing 60% mPR3+ and CD177+ PMN. (B) Modulation mPR3 and mCD177 expression (positive PMN subsets) during PMN activation by TNF-{alpha} and adhesion. Conditions of TNF-{alpha} activation and adhesion are those described in Figure 1. PMN were labeled with FITC–anti-CD177 ({square}) or anti-PR3 ({square}). Results are expressed as means ± SD of MFI of five experiments. *P < 0.05.

 
CD177 expression of the mPR3/CD177+ subset nearly doubled after TNF-{alpha} activation in EDTA. However, contrasting with mPR3, CD177 levels were not further upregulated by adhesion, regardless of whether anti-PR3 mAb was present (Figure 7B).

mPR3 Accessibility to Anti-PR3 Antibodies in Plasma
As already mentioned, plasma factors such as A1AT seem to inhibit the binding of anti-PR3 antibodies to mPR3+ PMN: When neutrophil incubations and labeling were performed in undiluted autologous plasma, no mPR3 was detected on resting PMN or upon TNF-activation in EDTA (Figure 8). However, TNF-induced adhesion allowed anti-PR3 mAb binding to mPR3+ PMN in plasma. Increased binding was obtained when the anti-PR3 mAb, diluted in plasma, was present throughout adhesion.


Figure 8
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  		Figure 8. The increase of mPR3 expression during PMN adhesion overcomes the inhibition of anti-PR3 mAb access to mPR3 by plasma. PMN in plasma (heparinized plasma from the same donor as neutrophils) or in HBSS-BSA were activated and analyzed for mPR3 expression as in Figure 1. The presence of plasma prevented the binding of anti-PR3 mAb to suspended PMN (resting or TNF-EDTA) but allowed it after cell adhesion. The numbers refer to the mPR3+ MFI of the mPR3+ neutrophil subset.

 
Similar experiments were performed with and without 100 µg/ml ANCA-PR3+ human IgG diluted in plasma. As previously mentioned, the binding of ANCA antibodies could not be assessed directly because of the nonspecific binding of IgG (Figure 2). The effect of ANCA was therefore measured using the anti-PR3 mAb. We observed a 2.4-fold increase of mPR3 expression when ANCA antibodies, diluted in plasma, were present throughout adhesion, as compared with PMN adherent in plasma without ANCA (data not shown). This clearly demonstrates the ANCA–anti-PR3 interaction with adherent PMN in plasma.


   DISCUSSION
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
CONCISE METHODS
 Disclosures
 References
 
We show that neutrophil activation by inflammatory stimuli, cell adhesion, and anti-PR3 mAb leads to step-wise increases of mPR3 available to ANCA. PMN priming is required for ANCA-induced PMN activation.9,32 Priming by TNF-{alpha}, platelet-activating factor (PAF), or LPS induces a PR3 translocation to the cell surface, enhanced upon further activation by IL-8 or fMLP.33,34 As shown here, the adhesion process and the presence of anti-PR3 antibodies amplify the mPR3 upregulations induced by TNF-{alpha}, IL-8, fMLP, or activated HUVEC.

Of interest, mPR3 expression is restricted to a constant neutrophil subset in all of these conditions.5 The following discussion refers to the mPR3+ subpopulation and does not address the question of the fundamental differences between mPR3+ and mPR3– subsets.

Fc{gamma}R and beta2 integrins are known to be involved in ANCA-induced PMN activation.27,34–37 The originality of our work is to show that these effects can be explained, at least in part, by the striking upregulation of ANCA antigen expression promoted by beta2 integrin-dependent adhesion and amplified by ANCA. The role of beta2 integrins is demonstrated here, because anti-CD18 blocking mAb prevent the adhesion-dependent mPR3 upregulation. Conversely, anti-CD18 activating mAb enhance the mPR3 expression by promoting adhesion in the absence of TNF priming. The lack of effect of F(ab')2 anti-PR3 antibodies on neutrophil mPR3 expression emphasizes the role of Fc{gamma}R. We demonstrated for the first time an amplifying loop in PMN activation: ANCA are involved in membrane expression of their own specific antigens.

The mechanism of the described mPR3 upregulation is complex and involves a synergy among (1) TNF-{alpha} inside-out signaling, analyzed in the absence of Ca/Mg or with integrin-blocking antibodies to prevent adhesion, which is not TNF-specific but also is observed with IL-8 or fMLP; (2) integrin outside-in signaling, resulting from the adhesion process; and (3) Fc{gamma}R signaling promoted by the Fc portion of antibodies. Moreover, these three signaling pathways alone are not sufficient to account for the striking mPR3 levels observed on adherent neutrophils reacting with anti-PR3 mAb. Indeed, these levels could not be reached when TNF-activated and adherent PMN were incubated with 1 mg/ml heat-aggregated IgG to trigger Fc{gamma}R pathways. Similarly, mAb reacting with highly expressed irrelevant membrane antigens had no effect on mPR3 expression. This supports the proposed model of ANCA-mediated effects occurring via both the binding of their Fc regions to Fc{gamma}R and the binding of their Fab regions to membrane antigens.3 PMN activation by ANCA F(ab')2 has indeed been reported.27,38,39 It suggests a specific effect of mPR3, possibly through cis-interactions with transmembrane signaling molecules, which could be cross-linked by anti-PR3 antibodies and/or be clustered in close contact with Fc{gamma}R. In that respect, the previously described localization of PR3 in membrane rafts, together with beta2 integrins and Fc{gamma}R, is worth noting.40,41

Recent data have suggested that PR3 membrane expression would result from mPR3 close association with CD177.30,31 We confirm that CD177, exclusively present on the mPR3+ neutrophil subset, is upregulated as mPR3 by TNF-{alpha} activation without adhesion. However, unlike mPR3, CD177 level is not further enhanced by cell adhesion and by anti-PR3 antibodies. This suggests the existence of two mPR3 species: mPR3 molecules that are present on resting PMN and recruited after limited activation and degranulation would form a complex with CD177, as previously suggested.31 New mPR3 molecules that are translocated to the membrane upon adhesion, especially in the presence of anti-PR3, may not be associated with CD177. We cannot exclude, however, that each CD177 can bind several mPR3 molecules and that CD177 molecules that are expressed on resting PMN are sufficient to anchor newly recruited mPR3 after cell adhesion with anti-PR3 antibodies.

We previously showed that intracellular storage pools of PR3 are located not only in azurophilic but also in specific granules and secretory vesicles.42 TNF activation is sufficient to mobilize secretory vesicles and possibly tertiary granules.26,29 This results in a first increase of mPR3 expression, together with exposure of the secretory vesicle marker CD35 and with CD11b upregulation. TNF-induced adhesion further mobilizes specific granules, enhancing CD11b and mPR3 expression at the same time. Membranes of the fused vesicles or granules seem to be richer in CD11b than in mPR3, because CD11b expression increases twice more than mPR3 during cell adhesion. The opposite situation is observed when anti-PR3 antibodies are present during adhesion, when mPR3 levels increase four times more than CD11b. The origin of these newly appearing mPR3 molecules therefore is not limited to secretory vesicles and specific granules. Some may result from an azurophilic granule exocytosis, suggested by the slight shift of CD63 labeling and the decrease, possibly elastase-dependent, of CD35 expression.43 However, azurophilic granules are unlikely the only source of the impressive mPR3 upregulation occurring during adhesion with anti-PR3 antibodies—first because their exocytosis is very limited and second because maximum degranulation of azurophilic granules and all other vesicles, by fMLP/cytochalasin or Ca ionophore, was not sufficient to reach similar mPR3 levels. Apart from secretory vesicles and granules, an intracellular localization of PR3 at the inner face of the plasma membrane has been proposed to explain the increase of mPR3 expression on apoptotic cells.44 In our experimental settings, mPR3 upregulation occurred without apoptosis. However, transient phospholipid flip-flop also occurred during PMN activation,45 and constant recycling of membrane PR3 was recently described.35 Whether TNF-induced adhesion results in a similar phenomenon deserves further studies. It might promote a transient PR3 molecule transfer from the inner to the outer plasma membrane leaflet. We hypothesize that anti-PR3 antibody binding would then prevent this PR3 to be re-internalized.

As previously reported,46 TNF activation increased mPR3 but did not result in MPO membrane expression. Although we were able to detect some membrane MPO or elastase on adherent PMN with specific anti-MPO or -elastase mAb, these expressions were far less important (<10%) than mPR3 levels. They could result from the nonspecific binding of fluid phase MPO and elastase, released during TNF-induced adhesion, and likely bind neutrophil membranes as a result of their positive charge. The same mechanism could explain the low mPR3 levels detected on the mPR3– subset in response to TNF-induced adhesion in the presence of anti-PR3 antibodies. Finally, confirming our initial working hypothesis, our results show that PMN adhesion favors the access of anti-PR3 mAb or ANCA to their antigens in whole blood.

We have shown that cytokine activation, cell adhesion, and the presence of anti-PR3 antibodies are three conditions required to allow a maximal mPR3 expression and an efficient binding of anti-PR3 antibodies to PMN in plasma. This would explain, at least in part, that vascular lesions observed in ANCA-associated vasculitis are restricted to capillaries and postcapillary venules, where small vessel diameters and low blood flow allow PMN adhesion to the endothelium.


   CONCISE METHODS
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
Disclosures
 References
 
Antibodies and Reagents
Anti-PR3 (CLB-12.8) and anti-MPO (7.17) mAb were from Sanquin (Amsterdam, Netherlands), anti-elastase mAb was from Biogenesis (Poole, UK), anti-CD43 was from BD Pharmingen (San Diego, CA), FITC-conjugated F(ab')2 anti-human IgG was from Jackson ImmunoResearch (West Grove, PA), FITC–anti-CD177 mAb (MEM-166) was from Biolegend (San Diego, CA), and FITC–anti-CD146 was from Serotec (Oxford, UK). Anti-CD18 clone IB4 was from Ancell (Bayport, MN), and KIM-185 was provided by M. Robinson (CellTech, Cambridge, UK). Other antibodies were from Beckman Coulter (Roissy, France). TNF-{alpha} was from PeproTech (Rocky Hill, NJ), IL-8 was from R&D Systems (Abingdon Oxon, UK), fibrinogen was from Diagnostica Stago (Genevilliers, France), and collagen I was from Invitrogen (Palo Alto, CA). Other reagents were from Sigma (St. Louis, MO).

Neutrophil Isolation and Activation
PMN from healthy donors, prepared as described previously,47 were suspended (2 x 106/ml) in BSA 0.1% HBSS (Life Technologies, Paisley, UK) without (HBSS–) or with (HBSS2+) 1 mM Ca2+/Mg2+ or with 10 mM EDTA (HBSS-EDTA). Protocols used for PMN activation and adhesion are given in corresponding figure legends.

Endothelial Cells (HUVEC)
Endothelial cells were isolated from human umbilical cord veins as described previously48 and cultured in M199 medium (Life Technologies) with 20% FCS. They were subcultured in 48-well culture plates coated with 0.5% gelatin and pretreated for 4 h with 10 ng/ml TNF-{alpha}. After rinsing three times with medium, they were used as substrate for PMN adhesion, as described in Figure 5.

IgG Purification, Biotinylation, and F(ab')2 Preparations
Blood was obtained from three PR3-ANCA+ and four MPO-ANCA+ vasculitis patients with high ANCA titers. Human IgG were isolated from patient and healthy donor sera by Sepharose–protein G chromatography (Amersham Bioscience AB, Uppsala, Sweden) and, when indicated, biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL), according to manufacturers’ instructions. When mentioned, IgG (10 mg/ml) were heat-aggregated by a 20-min incubation at 60°C. F(ab')2 fragments of IgG preparations were made by pepsin digestion, as described previously.36

Flow Cytometry
After stimulation, PMN were washed in ice-cold PBS, 1% BSA, and 0.1% sodium azide and incubated at 4°C with unlabeled or biotinylated primary antibody, followed by PE-labeled secondary antibody or streptavidin. When anti-PR3 antibodies were present during PMN activation, similar results were obtained whether labeling was performed directly with the secondary antibody or a new sample of anti-PR3 mAb was added after adhesion.

Cell suspensions recovered after PMN adhesion to HUVEC were labeled with anti-PR3 mAb and with FITC–anti-CD146. mPR3 expression was analyzed after exclusion of CD146+ HUVEC.

Cells were analyzed by flow cytometry on a Becton Dickinson FACSCalibur (Mountainview, CA). Results are given as mean fluorescence intensity.

MPO Enzymatic Activity Dosage
The amount of active MPO released in neutrophil supernatant was measured enzymatically by colorimetry.49

Apoptosis Evaluation
Annexin V and 7AAD binding were used to analyze the apoptotic and necrotic state of neutrophils by flow cytometry, as described previously.50

Statistical Analyses
Mean fluorescence intensities were compared using a paired t test analysis.


   Disclosures
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 Disclosures
References
 
None.


   Acknowledgments
 
S.B. and A.M. were supported by grants from the Fondation Nationale d'Aide aux Insuffisants Renaux and from the Association pour la Recherche sur la Polyarthrite Rhumatoïde, respectively. We acknowledge support from Baxter, Amgen, and AURA.

Part of our results were previously reported as abstracts at the annual meetings of the American Society of Nephrology; November 8 through 13, 2005, Philadelphia, PA; and November 14 through 19, 2006; San Diego, CA.

We thank Martyn Robinson (CellTech, Cambridge, UK) for the invaluable gift of the anti-CD18 activating KIM185 mAb.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 Disclosures
 References
 

  1. Jayne DR, Gaskin G, Pusey CD, Lockwood CM: ANCA and predicting relapse in systemic vasculitis. QJM 88 : 127 –133, 1995[Abstract/Free Full Text]
  2. Tervaert JW, van der Woude FJ, Fauci AS, Ambrus JL, Velosa J, Keane WF, Meijer S, van der Giessen M, van der Hem GK, The TH, et al.: Association between active Wegener's granulomatosis and anticytoplasmic antibodies. Arch Intern Med 149 : 2461 –2465, 1989[Abstract]
  3. Reumaux D, Duthilleul P, Roos D: Pathogenesis of diseases associated with antineutrophil cytoplasm autoantibodies. Hum Immunol 65 : 1 –12, 2004[CrossRef][Medline]
  4. Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L: Neutrophils: Molecules, functions and pathophysiological aspects. Lab Invest 80 : 617 –653, 2000[Medline]
  5. Halbwachs-Mecarelli L, Bessou G, Lesavre P, Lopez S, Witko-Sarsat V: Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool. FEBS Lett 374 : 29 –33, 1995[CrossRef][Medline]
  6. Witko-Sarsat V, Lesavre P, Lopez S, Bessou G, Hieblot C, Prum B, Noel LH, Guillevin L, Ravaud P, Sermet-Gaudelus I, Timsit J, Grunfeld JP, Halbwachs-Mecarelli L: A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis. J Am Soc Nephrol 10 : 1224 –1233, 1999[Abstract/Free Full Text]
  7. Rarok AA, Stegeman CA, Limburg PC, Kallenberg CG: Neutrophil membrane expression of proteinase 3 (PR3) is related to relapse in PR3-ANCA-associated vasculitis. J Am Soc Nephrol 13 : 2232 –2238, 2002[Abstract/Free Full Text]
  8. Schreiber A, Luft FC, Kettritz R: Membrane proteinase 3 expression and ANCA-induced neutrophil activation. Kidney Int 65 : 2172 –2183, 2004[CrossRef][Medline]
  9. Charles LA, Caldas ML, Falk RJ, Terrell RS, Jennette JC: Antibodies against granule proteins activate neutrophils in vitro. J Leukoc Biol 50 : 539 –546, 1991[Abstract]
  10. Falk RJ, Terrell RS, Charles LA, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci U S A 87 : 4115 –4119, 1990[Abstract/Free Full Text]
  11. Keogan MT, Esnault VL, Green AJ, Lockwood CM, Brown DL: Activation of normal neutrophils by anti-neutrophil cytoplasm antibodies. Clin Exp Immunol 90 : 228 –234, 1992[Medline]
  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 neutrophils. Arthritis Rheum 54 : 1619 –1628, 2006[CrossRef][Medline]
  13. Savage CO, Pottinger BE, Gaskin G, Pusey CD, Pearson JD: Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial cells. Am J Pathol 141 : 335 –342, 1992[Abstract]
  14. Mayet WJ, Meyer zum Buschenfelde KH: Antibodies to proteinase 3 increase adhesion of neutrophils to human endothelial cells. Clin Exp Immunol 94 : 440 –446, 1993[Medline]
  15. Radford DJ, Luu NT, Hewins P, Nash GB, Savage CO: Antineutrophil cytoplasmic antibodies stabilize adhesion and promote migration of flowing neutrophils on endothelial cells. Arthritis Rheum 44 : 2851 –2861, 2001[CrossRef][Medline]
  16. Buttrum SM, Drost EM, MacNee W, Goffin E, Lockwood CM, Hatton R, Nash GB: Rheological response of neutrophils to different types of stimulation. J Appl Physiol 77 : 1801 –1810, 1994[Abstract/Free Full Text]
  17. Tse WY, Nash GB, Hewins P, Savage CO, Adu D: ANCA-induced neutrophil F-actin polymerization: implications for microvascular inflammation. Kidney Int 67 : 130 –139, 2005[CrossRef][Medline]
  18. Abdel-Salam B, Iking-Konert C, Schneider M, Andrassy K, Hansch GM: Autoantibodies to neutrophil cytoplasmic antigens (ANCA) do not bind to polymorphonuclear neutrophils in blood. Kidney Int 66 : 1009 –1017, 2004[CrossRef][Medline]
  19. Ambrose LR, Little MA, Nourshargh S, Pusey CD: Anti-proteinase 3 antibody binding to neutrophils as demonstrated by confocal microscopy. Kidney Int 68 : 2912 –2913, 2005[Medline]
  20. Van Rossum AP, van der Geld YM, Limburg PC, Kallenberg CG: Human anti-neutrophil cytoplasm autoantibodies to proteinase 3 (PR3-ANCA) bind to neutrophils. Kidney Int 68 : 537 –541, 2005[CrossRef][Medline]
  21. Rooney CP, Taggart C, Coakley R, McElvaney NG, O'Neill SJ: Anti-proteinase 3 antibody activation of neutrophils can be inhibited by alpha1-antitrypsin. Am J Respir Cell Mol Biol 24 : 747 –754, 2001[Abstract/Free Full Text]
  22. Esnault VL, Testa A, Audrain M, Roge C, Hamidou M, Barrier JH, Sesboue R, Martin JP, Lesavre P: Alpha 1-antitrypsin genetic polymorphism in ANCA-positive systemic vasculitis. Kidney Int 43 : 1329 –1332, 1993[Medline]
  23. Campbell EJ, Campbell MA: Pericellular proteolysis by neutrophils in the presence of proteinase inhibitors: Effects of substrate opsonization. J Cell Biol 106 : 667 –676, 1988[Abstract/Free Full Text]
  24. Vissers MC, George PM, Bathurst IC, Brennan SO, Winterbourn CC: Cleavage and inactivation of alpha 1-antitrypsin by metalloproteinases released from neutrophils. J Clin Invest 82 : 706 –711, 1988[Medline]
  25. Nie J, Pei D: Rapid inactivation of alpha-1-proteinase inhibitor by neutrophil specific leukolysin/membrane-type matrix metalloproteinase 6. Exp Cell Res 296 : 145 –150, 2004[CrossRef][Medline]
  26. Bouaouina M, Blouin E, Halbwachs-Mecarelli L, Lesavre P, Rieu P: TNF-induced beta2 integrin activation involves Src kinases and a redox-regulated activation of p38 MAPK. J Immunol 173 : 1313 –1320, 2004[Abstract/Free Full Text]
  27. Kettritz R, Jennette JC, Falk RJ: Crosslinking of ANCA-antigens stimulates superoxide release by human neutrophils. J Am Soc Nephrol 8 : 386 –394, 1997[Abstract]
  28. Andrew D, Shock A, Ball E, Ortlepp S, Bell J, Robinson M: KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA-1- and CR3-dependent adhesion. Eur J Immunol 23 : 2217 –2222, 1993[Medline]
  29. Chakrabarti S, Zee JM, Patel KD: Regulation of matrix metalloproteinase-9 (MMP-9) in TNF-stimulated neutrophils: novel pathways for tertiary granule release. J Leukoc Biol 79 : 214 –222, 2006[Abstract/Free Full Text]
  30. Bauer S, Abdgawad M, Gunnarsson L, Segelmark M, Tapper H, Hellmark T: Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils. J Leukoc Biol 81 : 458 –464, 2007[Abstract/Free Full Text]
  31. von Vietinghoff S, Tunnemann G, Eulenberg C, Wellner M, Cardoso MC, Luft FC, Kettritz R: NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils. Blood 109 : 4487 –4493, 2007[Abstract/Free Full Text]
  32. Falk RJ, Jennette JC: Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N Engl J Med 318 : 1651 –1657, 1988[Abstract]
  33. Campbell EJ, Campbell MA, Owen CA: Bioactive proteinase 3 on the cell surface of human neutrophils: Quantification, catalytic activity, and susceptibility to inhibition. J Immunol 165 : 3366 –3374, 2000[Abstract/Free Full Text]
  34. Porges AJ, Redecha PB, Kimberly WT, Csernok E, Gross WL, Kimberly RP: Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa. J Immunol 153 : 1271 –1280, 1994[Abstract]
  35. Mulder AH, Heeringa P, Brouwer E, Limburg PC, Kallenberg CG: Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA): A Fc gamma RII-dependent process. Clin Exp Immunol 98 : 270 –278, 1994[Medline]
  36. Reumaux D, Vossebeld PJ, Roos D, Verhoeven AJ: Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: Relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies. Blood 86 : 3189 –3195, 1995[Abstract/Free Full Text]
  37. Hewins P, Williams JM, Wakelam MJ, Savage CO: Activation of Syk in neutrophils by antineutrophil cytoplasm antibodies occurs via Fcgamma receptors and CD18. J Am Soc Nephrol 15 : 796 –808, 2004[Abstract/Free Full Text]
  38. Yang JJ, Preston GA, Alcorta DA, Waga I, Munger WE, Hogan SL, Sekura SB, Phillips BD, Thomas RP, Jennette JC, Falk RJ: Expression profile of leukocyte genes activated by anti-neutrophil cytoplasmic autoantibodies (ANCA). Kidney Int 62 : 1638 –1649, 2002[CrossRef][Medline]
  39. Williams JM, Ben-Smith A, Hewins P, Dove SK, Hughes P, McEwan R, Wakelam MJ, Savage CO: Activation of the G(i) heterotrimeric G protein by ANCA IgG F(ab')2 fragments is necessary but not sufficient to stimulate the recruitment of those downstream mediators used by intact ANCA IgG. J Am Soc Nephrol 14 : 661 –669, 2003[Abstract/Free Full Text]
  40. David A, Fridlich R, Aviram I: The presence of membrane proteinase 3 in neutrophil lipid rafts and its colocalization with FcgammaRIIIb and cytochrome b558. Exp Cell Res 308 : 156 –165, 2005[CrossRef][Medline]
  41. Fridlich R, David A, Aviram I: Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes. J Cell Biochem 99 : 117 –125, 2006[CrossRef][Medline]
  42. Witko-Sarsat V, Cramer EM, Hieblot C, Guichard J, Nusbaum P, Lopez S, Lesavre P, Halbwachs-Mecarelli L: Presence of proteinase 3 in secretory vesicles: Evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules. Blood 94 : 2487 –2496, 1999[Abstract/Free Full Text]
  43. Tosi MF, Zakem H, Berger M: Neutrophil elastase cleaves C3bi on opsonized pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch. J Clin Invest 86 : 300 –308, 1990[Medline]
  44. Durant S, Pederzoli M, Lepelletier Y, Canteloup S, Nusbaum P, Lesavre P, Witko-Sarsat V: Apoptosis-induced proteinase 3 membrane expression is independent from degranulation. J Leukoc Biol 75 : 87 –98, 2004[Abstract/Free Full Text]
  45. Frasch SC, Henson PM, Nagaosa K, Fessler MB, Borregaard N, Bratton DL: Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils. J Biol Chem 279 : 17625 –17633, 2004[Abstract/Free Full Text]
  46. Reumaux D, Hordijk PL, Duthilleul P Roos D: Priming by tumor necrosis factor-alpha of human neutrophil NADPH-oxidase activity induced by antiproteinase-3 or antimyeloperoxidase antibodies. J Leukoc Biol 80 : 1424 –1433, 2006[Abstract/Free Full Text]
  47. Seveau S, Lopez S, Lesavre P, Guichard J, Cramer EM, Halbwachs-Mecarelli L: Leukosialin (CD43, sialophorin) redistribution in uropods of polarized neutrophils is induced by CD43 cross-linking by antibodies, by colchicine or by chemotactic peptides. J Cell Sci 110 : 1465 –1475, 1997[Abstract]
  48. Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52 : 2745 –2756, 1973[Medline]
  49. Capeillere-Blandin C, Gausson V, Nguyen AT, Descamps-Latscha B, Drueke T, Witko-Sarsat V: Respective role of uraemic toxins and myeloperoxidase in the uraemic state. Nephrol Dial Transplant 21 : 1555 –1563, 2006[Abstract/Free Full Text]
  50. Nusbaum P, Laine C, Bouaouina M, Seveau S, Cramer EM, Masse JM, Lesavre P, Halbwachs-Mecarelli L: Distinct signaling pathways are involved in leukosialin (CD43) down-regulation, membrane blebbing, and phospholipid scrambling during neutrophil apoptosis. J Biol Chem 280 : 5843 –5853, 2005[Abstract/Free Full Text]

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