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Originally published online as doi:10.1189/jlb.0304144 on September 22, 2006

Published online before print September 22, 2006
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(Journal of Leukocyte Biology. 2006;80:1424-1433.)
© 2006 by Society for Leukocyte Biology

Priming by tumor necrosis factor-{alpha} of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies

Dominique Reumaux*,1, Peter L. Hordijk{dagger}, Patrick Duthilleul* and Dirk Roos{dagger}

* Département d’Hématologie-Immunologie-Cytogénétique, Centre Hospitalier de Valenciennes, and Laboratoire d’Hématologie, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Lille-2, Lille, France; and
{dagger} Sanquin Research at Central Laboratory for Blood Transfusion (CLB) and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands

1 Correspondence: Laboratoire d’Hématologie, Faculté des Sciences Pharmaceutiques et Biologiques, Université de Lille-2, 3 rue du Professeur Laguesse, 59006 Lille cedex, France. E-mail: dominique.reumaux{at}libertysurf.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-proteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies are capable of activating human neutrophils primed by TNF-{alpha} in vitro. We described previously the involvement of Fc{gamma}RIIa and β2 integrins in this neutrophil activation. In the literature, the requirement of TNF priming has been attributed to an effect of TNF-{alpha} on the expression of PR3 or MPO on the cell surface. Under our experimental conditions, TNF-{alpha} (2 ng/ml) increased the binding of the antibody against PR3, whereas binding of the antibody against MPO could hardly be detected, not even after TNF-{alpha} treatment. The aim of this study was to consider (an)other(s) role(s) for TNF-{alpha} in facilitating the NADPH-oxidase activation by these antibodies. We demonstrate the early mobilization of the secretory vesicles as a result of TNF-induced increase in intracellular-free calcium ions, the parallel colocalization of gp91phox, the main component of the NADPH oxidase with β2 integrins and Fc{gamma}RIIa on the neutrophil surface, and the Fc{gamma}RIIa clustering upon TNF priming. TNF-{alpha} also induced redistribution of Fc{gamma}RIIa to the cytoskeleton in a dose- and time-dependent manner. Moreover, blocking CD18 MHM23 antibody, cytochalasin B, and D609 (an inhibitor of phosphatidylcholine phospholipase C) inhibited this redistribution and the respiratory burst in TNF-treated neutrophils exposed to anti-PR3 or anti-MPO antibodies. Our results indicate direct effects of TNF-{alpha} in facilitating neutrophil activation by these antibodies and further support the importance of cytoskeletal rearrangements in this priming process.

Key Words: TNF-{alpha} • neutrophil activation • ANCA • vasculitis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-neutrophil cytoplasm autoantibodies (ANCA) have been described in sera from patients with systemic vasculitides, such as Wegener’s granulomatosis [1 ]. Anti-proteinase-3 (anti-PR3) and anti-myeloperoxidase (anti-MPO) antibodies, the two main ANCA specificities [2 , 3 ], are capable of activating human neutrophils primed by TNF-{alpha} in vitro [4 ]. ANCA-IgG bind with their Fab regions to their target antigens expressed on the cell surface and with their Fc regions to Fc{gamma}Rs on the same cell surface (Kurlander phenomenon) [5 ] or on neighboring neutrophils. In our previous studies [6 , 7 ], we described the involvement of Fc{gamma}RIIa and β2 integrins in the dramatic activation of TNF-treated neutrophils induced by anti-PR3 or anti-MPO antibodies. Moreover, we reported the inhibitory effect of the actin-disrupting agent cytochalasin B (Cyto B) on this activation [8 ].

The precise, underlying mechanisms by which low concentrations of TNF-{alpha} prime the NADPH oxidase have not been well-elucidated, despite numerous cellular events implicated in this process [9 ]. Likewise, only little attention has been given in the literature to the requirement of TNF priming for neutrophil activation by anti-PR3 or anti-MPO antibodies. This requirement has been attributed to an effect of TNF-{alpha} on the expression of PR3 or MPO on the cell surface [4 , 10 , 11 ]. We previously demonstrated that TNF-{alpha} (2 ng/ml) increased significantly but weakly the binding of the antibody against PR3, whereas binding of the antibody against MPO could hardly be detected, not even after TNF-{alpha} treatment [6 ].

Alternatively, the expression of ANCA antigens on the neutrophil surface could be a result, in vivo, of neutrophil apoptosis by a mechanism that is independent of priming [12 ]. This provides a novel mechanism by which ANCA may interact with clustered granule constituents on the surface of apoptotic neutrophils and will activate neighboring, viable neutrophils with their Fc regions.

In our opinion, the requirement of TNF priming for neutrophil activation by anti-PR3 or anti-MPO antibodies is probably not only a result of an effect of TNF-{alpha} on the surface expression of PR3 or MPO antigens. In the present study, we consider additional and/or other(s) role(s) for TNF-{alpha} in facilitating neutrophil activation triggered by these antibodies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Diisopropyl fluorophosphate (DFP) was from Fluka Holding (Buchs, Switzerland). Human fibronectin (FN), azide, PMSF, Triton X-100 (TX-100), Tween-20, L-cysteine, N-ethyl maleimide, EDTA, EGTA, papain, protein A, paraformaldehyde, Cyto B, BAPTA-AM, D609 (tricyclodecan-9-yl xanthogenate-K), and PMA were obtained from Sigma-Aldrich (St. Louis, MO). Dihydro-rhodamine-1,2,3 (DHR) and Alexa Fluor 568-goat antimouse (GAM)-IgG (heavy and light chains) were purchased from Molecular Probes (Eugene, OR). R-PE-conjugated F(ab')2 fragment of affinity-isolated GAM-IgG was from Dako (Glostrup, Denmark). Human recombinant (hr)TNF-{alpha} was from Roche Diagnostics (Mannheim, Germany). Vectashield mounting medium was from Vector Laboratories (Burlingame, CA). All other reagents were of analytical grade purity.

Monoclonal antibodies (mAbs) 12.8 against PR3 (mIgG1), mAb 4.15 against MPO (mIgG1), mAb 3G8 (CD16, mIgG1) against Fc{gamma}RIIIb, and mAb IV.3 (CD32, mIgG2b) against Fc{gamma}RIIa were from the Central Laboratory of the Netherlands Blood Transfusion Service (Amsterdam). mAb CD24 (mIgG1) was from Caltag Laboratories (Burlingame, CA). mAb 7D5 (mIgG1) against the extracellular peptide portion of primate gp91phox of the human flavocytochrome b558 was from Dr. Michio Nakamura (Nagasaki, Japan) [13 ]. The β2-integrin-activating CD18 mAb KIM185 was kindly provided by Dr. Nancy Hogg (Leucocyte Adhesion Laboratory, Imperial Cancer Research Fund, London, UK) [14 ]. mAb CD18 MHM23 (LFA-1, β-chain, mIgG1) and mAb mIgG1 (DAK-GO1) were from Dako. mAb IB4 (mIgG2a), which binds to the β2-integrin subunit (CD18), was from Ancell Immunology Research Products (Bayport, MN). Biotinylated CD18 mAb MHM23 was prepared by Interchim (Montluçon, France). MAb IV.3 was biotinylated with biotin-N-hydroxy-succinimide ester (2 mg per mg IgG) for 4 h at room temperature (RT). Fluorescein-conjugated ImmunoPure streptavidin (Pierce, Rockford, IL) was used to cross-link biotinylated antibodies bound to neutrophils.

Fab fragments were made by digestion with 4% (w/w) papain in PBS containing 10 mM cysteine and 5 mM EDTA for 1.5 h at 37°C. The reaction was terminated by addition of 20 mM N-ethyl maleimide. Protein-A affinity chromatography was used to remove Fc fragments and intact antibodies. When Fab fragments were checked on SDS-PAGE, intact antibodies or Fc fragments were not detectable.

A polyclonal rabbit antiserum directed against the cytoplasmic tail of Fc{gamma}RIIa, designated CT10, was generated against a synthetic peptide (CYLTLPPNDHVNSNN) coupled to keyhole limpet hemocyanin and was subsequently purified, as described by Ibarrola et al. [15 ]. A polyclonal rabbit IgG against human MHC Class I {alpha}-chain was a kind gift of Dr. Jacques J. Neefjes (Amsterdam, The Netherlands) [16 ]. A polyclonal goat-IgG against human actin (I-19) was purchased from Santa Cruz Biotechnology (CA).

Isolation of neutrophils
Granulocytes were purified from blood anticoagulated with 0.4% (w/v) trisodium citrate (pH 7.4), as described [17 ]. In short, blood cells were separated by density gradient centrifugation over isotonic Percoll (Amersham Biosciences, Piscataway, NJ) with a specific gravity of 1.076 g/ml. The interphase, containing the mononuclear cells, was removed. The pellet fraction, containing erythrocytes and granulocytes, was treated for 10 min with ice-cold, isotonic NH4Cl solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4) to lyse the erythrocytes. The remaining granulocytes were washed twice in PBS, resuspended in incubation medium containing 132 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 1.2 mM KH2PO4, 20 mM Hepes, 5.5 mM glucose, and 0.5% (w/v) human serum albumin (HSA; pH 7.4), and kept at RT at a final concentration of 2 x 106 cells/ml, unless indicated otherwise. Purity of neutrophils was more than 95% (the contaminating cells were mainly eosinophils), and viability was more than 98%.

Measurement of respiratory burst in DHR-loaded neutrophils
The assay to measure NADPH-oxidase activity in DHR-loaded cells was carried out as described [6 , 7 ]. Cells were analyzed by flow cytometry (Epics XL-MCL, Coulter Corp., Miami, FL). Results are expressed as mean fluorescence intensity (MFI).

Determination of surface-antigen expression on neutrophils
The assay to measure the expression of surface antigens was carried out as described [6 ]. Cells were analyzed by flow cytometry (Epics XL-MCL). Results are expressed as MFI.

Distribution of gp91phox and β2 integrins on the neutrophil surface analyzed by confocal laser scanning microscopy (LSM)
Wells (15.5 mm diameter) of a flat-bottomed, 24-well plate were pretreated for 1 h at 37°C with FN (10 µg/ml) dissolved in PBS and were then washed cautiously once with PBS and once with incubation medium at RT. Neutrophils (2x106/ml) were distributed rapidly at 106 cells/well, and after 10 min at 37°C, TNF-{alpha} (2 ng/ml) was added to some of the wells. Control cells received PBS only. Forty minutes after addition of TNF-{alpha}, the supernatants of the wells, including the nonadherent and the loosely attached cells, were transferred to tubes and immediately fixed with 1% (w/v) paraformaldehyde in incubation medium for 10 min at 4°C. After two extensive washes with PBS containing 0.5% (w/v) BSA and 1 mM CaCl2, the tubes were centrifuged (400 g) for 5 min at 4°C, and the cells were resuspended in 200 µl ice-cold incubation medium with 7D5 mAb against gp91phox (10 µg/ml) for 45 min at 4°C. The cells were washed and resuspended in 200 µl ice-cold incubation medium with Alexa 568-labeled GAM-IgG (10 µg/ml) for 45 min at 4°C. After another washing step, the cells were resuspended in ice-cold incubation medium with biotinylated CD18 mAb MHM23 (10 µg/ml) for 45 min at 4°C, washed, and resuspended in ice-cold incubation medium with fluorescein-conjugated streptavidin (1:100) for 45 min at 4°C. After another washing step, the cells were resuspended in 200 µl ice-cold incubation medium, centrifuged onto glass slides, and mounted with vectashield medium, as an antifading agent. The cells were analyzed by confocal LSM with a Leica TCS-NT (Leica Microsystems, Heidelberg, Germany) using the appropriate filter settings. Overlap of the individual excitation and emission spectra carries the risk of producing incorrect results, termed cross-linking. To allow a separation of the detection of signals, i.e., to avoid possible cross-linking between excitation and emission spectra of a fluorochrome combination, the Acousto-Optic Tunable Filter (AOTF)-based system was used [18 ]. This technology optimatically selects wavelengths and set-up of the intensity of excitation light, which improves discrimination of a wide range of fluorescent dyes. Therefore, the AOTF-based system is relevant for the documentation of spatially, closely neighboring structures.

Distribution of Fc{gamma}RIIa and β2 integrins on the neutrophil surface analyzed by confocal LSM
Neutrophils were treated, fixed, and washed as described above. First, to study the distribution of Fc{gamma}RIIa on the neutrophil surface induced by TNF-{alpha} (2 ng/ml), the cells were resuspended in 200 µl ice-cold incubation medium with IV.3 mAb against Fc{gamma}RIIa (5 µg/ml) for 45 min at 4°C. The cells were washed and incubated with Alexa-568-labeled GAM-IgG (10 µg/ml) for 45 min at 4°C. After another washing step, the cells were resuspended in incubation medium, centrifuged onto glass slides, and mounted with vectashield medium. The cells were analyzed by confocal LSM. Next, to investigate a possible colocalization of Fc{gamma}RIIa and β2 integrins induced by TNF-{alpha} (2 ng/ml), the cells were fixed and washed, resuspended in incubation medium with IV.3 mAb (5 µg/ml), washed again, and incubated with Alexa-568-labeled GAM-IgG (10 µg/ml). After another washing step, the cells were resuspended in incubation medium with biotinylated CD18 mAb MHM23 (10 µg/ml), washed again, and incubated with fluorescein-conjugated streptavidin (1:100). The cells were analyzed by confocal LSM and the AOTF-based system.

Distribution of gp91phox and Fc{gamma}RIIa on the neutrophil surface analyzed by confocal LSM
To investigate a possible colocalization of gp91phox and Fc{gamma}RIIa induced by TNF-{alpha} (2 ng/ml), the cells were fixed and washed, resuspended with 7D5 mAb (10 µg/ml), washed again, and incubated with Alexa-568-labeled GAM-IgG (10 µg/ml). After another washing step, the cells were resuspended with biotinylated IV.3 mAb (5 µg/ml), washed again, and incubated with fluorescein-conjugated streptavidin (1:100). The cells were analyzed by confocal LSM and the AOTF-based system.

Association of Fc{gamma}RIIa with the cytoskeleton by immunoblot analysis
Neutrophils (1x107/ml), resuspended in incubation medium without HSA, were pretreated with a protease inhibitor (5 mM DFP) on ice for 15 min and then washed once. Treatment with DFP proved to be necessary to recover intact Fc{gamma}RIIa from lysed neutrophils (not shown). Afterwards, neutrophils (5x106/ml) were resuspended in incubation medium without HSA and were incubated by shaking in polystyrene tubes in a water-bath at 37°C. After 15 min, hrTNF-{alpha} was added to part of the samples to a final concentration of 2 ng/ml. After 10 min of priming, samples were spun down (12,000 g) for a few seconds at RT, and the supernatant was removed before adding 250 µl lysis buffer (1% TX-100, 0.15 M NaCl, 20 mM Hepes, 100 µM PMSF, 1 mM EGTA, pH 7.4). The cells (20x106/ml) were suspended by vortexing and kept for 30 min at 4°C. Thereafter, TX-100-soluble and -insoluble fractions were prepared by centrifugation (3400 g) for 10 min at 4°C. The TX-100-insoluble fractions were washed once with the lysis buffer and centrifuged again (3400 g) for 10 min at 4°C to remove the supernatant. Then, TX-100-soluble and -insoluble fractions were mixed 1:1 with 4% reduced Laemmli sample buffer and placed in a boiling water-bath for 5 min. Protein concentrations of the TX-100-soluble fractions were determined by a bicinchoninic acid protein assay kit (Pierce).

These samples were subjected to electrophoresis onto a 10% (w/v) polyacrylamide gel under reducing conditions. Thereafter, separated proteins were electrotransferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) in transfer buffer (25 mM Tris base, 0.2 M glycine, 20% methanol, pH 8.5) by means of a semidry blotting apparatus at a constant current of 1.0 mA/cm2 for 1 h. Unoccupied sites on the membrane were blocked with 5% (w/v) skimmed, dry milk in TBST (20 mM Tris base, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at RT, and the blots were probed with CT10 antibody diluted at 1:1000 with 5% (w/v) BSA in TBST overnight at 4°C. Afterwards, the blots were washed extensively in TBST and incubated with a HRP-linked donkey antirabbit-IgG (Santa Cruz Biotechnology) diluted at 1:1000 for 1 h at RT. The blots were washed again thoroughly several times, and bound antibodies were detected by ECL Plus chemiluminescence kit (Amersham Biosciences). Molecular weights were calculated by comparison with recombinant protein molecular weight markers, RPN 800 (Amersham Biosciences).

When necessary, the blots were stripped by incubation in 62.5 mM Tris-HCl (pH 6.8) containing 2% SDS and 100 mM β-mercaptoethanol at 50°C for 30 min prior to washing in TBST and reprobing, e.g., with a polyclonal rabbit-IgG against human MHC Class I {alpha}-chain diluted at 1:1000 for 1 h at RT or with a polyclonal goat-IgG against human actin (I-19) diluted at 1:250 for 2 h at RT. The same procedure as above was then followed but with incubation with a HRP-linked sheep antirabbit-IgG (Amersham Biosciences) diluted at 1:3000 or with a HRP-linked donkey antigoat-IgG (Santa Cruz Biotechnology) diluted at 1:1000, respectively.

Statistical analysis
Results are expressed as mean ± SEM of (n) independent experiments. Statistical significance was evaluated by means of ANOVA to assess a possible significant, overall effect of treatment. When significant effects were detected, individual analyses were performed with the two-sided t-test. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} primes human neutrophils for enhanced NADPH-oxidase activity in response to 3G8 mAb, anti-PR3, or anti-MPO antibodies
TNF-{alpha} (2 ng/ml) primes human neutrophils for enhanced NADPH-oxidase activity in response to anti-PR3 or anti-MPO antibodies [6 , 7 ] but also in response to 3G8 mAb against anti-Fc{gamma}RIIIb, known to activate neutrophils through binding with its Fc region to Fc{gamma}RIIa [19 ] (Fig. 1A ). Similar results were obtained with purified IgG preparations from sera with PR3-ANCA or MPO-ANCA (not shown) [6 ]. NADPH activation of TNF-treated neutrophils exposed to 3G8 mAb and to anti-PR3 or anti-MPO mAb is mediated by Fc{gamma}RIIa- and β2-integrin-dependent pathways, as IV.3 mAb directed against Fc{gamma}RIIa and blocking CD18 mAb MHM23 Fab fragments inhibited this activation (Fig. 1B) [6 ].


Figure 1
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  		Figure 1. Respiratory burst activation in human neutrophils induced by 3G8, anti-PR3, or anti-MPO mAb: (A) Dependence on TNF priming and (B) effect of CD18 mAb MHM23 and IV.3 mAb (anti-Fc{gamma}RIIa). (A) Neutrophils (2x106/ml) were incubated at 37°C with DHR in polystyrene wells coated with FN (10 µg/ml), as described in Materials and Methods, in the presence of TNF-{alpha} (2 ng/ml). Part of the cells was left untreated, as indicated. (B) Neutrophils (2x106/ml) were also incubated in the absence or presence of CD18 mAb MHM23 (10 µg/ml) and IV.3 mAb (anti-Fc{gamma}RIIa; 5 µg/ml) for 5 min prior to the addition of TNF-{alpha} (2 ng/ml). After 10 min of priming, the cells (A and B) were stimulated for 30 min with 3G8, anti-PR3, or anti-MPO mAb at a final concentration of 5 µg/ml. Control cells (–) received PBS only. Samples of adherent cells to FN were processed for flow cytometry to measure rhodamine-1,2,3 fluorescence. Results (MFI) are the mean ± SEM of five independent experiments. With all stimuli, TNF-{alpha} had a significant, enhancing effect in the absence of CD18 mAb MHM23 and IV.3 mAb (P<0.05), and CD18 mAb MHM23 and IV.3 mAb had a significant, inhibiting effect in the presence of TNF-{alpha} (P<0.05).

 
Distribution of gp91phox and β2 integrins on the neutrophil surface
Flavocytochrome b558, the main membrane component of the NADPH oxidase in neutrophils, is a heterodimer composed of gp91phox and p22phox subunits. TNF-{alpha} priming has been described to up-regulate the expression of β2-integrin membrane-activated complex 1 (Mac-1; CD11b/CD18) but also of flavocytochrome b558 on the neutrophil surface [20 , 21 ]. These two last components are located in specific granules and secretory vesicles [22 , 23 ]. We therefore searched by confocal LSM for a possible colocalization of gp91phox and β2 integrins on the neutrophil surface after treatment of cells with TNF-{alpha}. Gp91phox was detected with 7D5 mAb. TNF-{alpha} (2 ng/ml) induced the up-regulation and colocalization of gp91phox and β2 integrins on the neutrophil surface (Fig. 2A ). Spectral curves obtained from TNF-treated "merge" cells (Fig. 2A) are reported in Figure 2B . The intensity of the image points within the selected region of interest is plotted against the wavelength of both fluorochrome emission curves (channels 1 vs. 2). These curves indicate a high degree of colocalization between gp91phox and β2 integrins.


Figure 2
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  		Figure 2. TNF-{alpha} induces colocalization of gp91phox and β2 integrins on the neutrophil surface. (A) Neutrophils (2x106/ml) were incubated at 37°C in polystyrene wells coated with FN (10 µg/ml), as described in Materials and Methods. After 5 min at 37°C, TNF-{alpha} (2 ng/ml) was added to some of the wells. After 40 min, the nonadherent and the loosely attached cells were transferred to tubes and immediately fixed with 1% (w/v) paraformaldehyde in incubation medium and washed. The cells were then resuspended with 7D5 mAb (10 µg/ml) against gp91phox and after another washing step, were incubated with Alexa-568-labeled GAM-IgG. Thereafter, the cells were resuspended with biotinylated CD18 mAb MHM23 (10 µg/ml), washed again, and incubated with fluorescein-conjugated streptavidin. After centrifugation onto glass slides, the cells were analyzed by confocal LSM. Results are representative of three independent experiments. (B) The AOTF-based system applied to merging of gp91phox and β2 integrins on the surface of neutrophils primed by TNF-{alpha}. Spectral curves are related to the fluorochrome emission of 7D5 mAb against gp91phox (10 µg/ml) incubated with Alexa-568-labeled GAM-IgG versus biotinylated CD18 mAb MHM23 (10 µg/ml) incubated with fluorescein-conjugated streptavidin. The intensity of the image points within the selected region of interest is plotted against the wavelength of both fluorochrome emission curves (channels 1 vs. 2).

 
Moreover, the Ca2+ chelator BAPTA-AM (25 µM) inhibited the enhanced binding of 7D5 mAb and CD18 mAb MHM23 on the neutrophil surface induced by TNF-{alpha} (2 ng/ml) (Table 1 ). Likewise, BAPTA-AM (25 µM) completely inhibited the respiratory burst in TNF-treated neutrophils exposed to 3G8, anti-PR3, or anti-MPO mAb but did not affect the respiratory burst in neutrophils exposed to PMA (Fig. 3 ).


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  		Table 1. Effect of the Ca2+ Chelator BAPTA-AM (25 µM) on the Binding of CD18 mAb MHM23 and 7D5 mAb against gp91phox to Neutrophils upon TNF Priming or Not

 

Figure 3
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  		Figure 3. Effect of the Ca2+ chelator BAPTA-AM on the respiratory burst activation in TNF-treated neutrophils exposed to PMA or 3G8, anti-PR3, or anti-MPO mAb. Neutrophils (2x106/ml) were incubated at 37°C in polypropylene tubes in a shaking water-bath under gentle agitation, in the absence or presence of BAPTA-AM (25 µM). After 30 min of incubation, neutrophils were washed once with incubation medium and centrifuged before incubation at 37°C with DHR in polystyrene tubes, as described in Materials and Methods, in the presence of TNF-{alpha} (2 ng/ml). After 10 min of priming, the cells were stimulated for 15 min with PMA (100 ng/ml) and for 30 min with 3G8, anti-PR3, or anti-MPO mAb at a final concentration of 5 µg/ml. Control cells (–) received PBS only. Samples were processed for flow cytometry to measure rhodamine-1,2,3 fluorescence. Results (MFI) are the mean ± SEM of three independent experiments. With all stimuli except PMA, BAPTA-AM (25 µM) had a significant, inhibiting effect in the presence of TNF-{alpha} (P<0.05).

 
Distribution of Fc{gamma}RIIa and β2 integrins on the neutrophil surface
We next studied the effect of TNF-{alpha} on distribution of Fc{gamma}RIIa and β2 integrins on the neutrophil surface analyzed by confocal LSM. TNF-{alpha} (2 ng/ml) induced clustering of Fc{gamma}RIIa and enhanced the colocalization of Fc{gamma}RIIa and β2 integrins (Fig. 4A ). Moreover, CD18 mAb MHM23 (mIgG1, 10 µg/ml) inhibited the Fc{gamma}RIIa clustering on TNF-treated neutrophils (Fig. 4B) . We observed the same inhibitory effect on the Fc{gamma}RIIa clustering with IB4 mAb (mIgG2a, 20 µg/ml), known to bind to the β2-integrin subunit (not shown). Spectral curves obtained from these TNF-treated merge cells (Fig. 4A) are reported in Figure 4C and indicate a high degree of colocalization between Fc{gamma}RIIa and β2 integrins.


Figure 4
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  		Figure 4. TNF-{alpha} induces CD18-mediated clustering of Fc{gamma}RIIa on neutrophils. (A) Neutrophils (2x106/ml) were incubated at 37°C in polystyrene wells coated with FN (10 µg/ml), as described in Materials and Methods. After 5 min at 37°C, TNF-{alpha} (2 ng/ml) was added to some of the wells. After 40 min, the nonadherent and the loosely attached cells were transferred to tubes and immediately fixed with 1% (w/v) paraformaldehyde in incubation medium and washed. The cells were then resuspended with IV.3 mAb against Fc{gamma}RIIa (5 µg/ml) and after another washing step, were incubated with Alexa-568-labeled GAM-IgG. The cells were resuspended with biotinylated CD18 mAb MHM23 (10 µg/ml), washed again, and incubated with fluorescein-conjugated streptavidin. After centrifugation onto glass slides, the cells were analyzed by confocal LSM. Results are representative of five independent experiments. (B) Neutrophils were incubated in the presence of TNF-{alpha} (2 ng/ml), without or with CD18 mAb MHM23 (10 µg/ml), in FN-coated wells and were fixed with 1% (w/v) paraformaldehyde in incubation medium and washed. The cells were then resuspended with IV.3 mAb against Fc{gamma}RIIa (5 µg/ml) and after another washing step, were incubated with Alexa-568-labeled GAM-IgG. After centrifugation onto glass slides, the cells were analyzed by confocal LSM. Results are representative of three independent experiments. (C) The AOTF-based system is applied to merging of Fc{gamma}RIIa and β2 integrins on neutrophils treated by TNF-{alpha} (A). The related spectral curves are depicted in C, with fluorochrome emission of IV.3 mAb against Fc{gamma}RIIa (5 µg/ml) incubated with Alexa-568-labeled GAM-IgG versus biotinylated CD18 mAb MHM23 (5 µg/ml), incubated with fluorescein-conjugated streptavidin. (D) The AOTF-based system is applied to assess merging of β2 integrins and CD24 on neutrophils treated by TNF-{alpha} (A). The related spectral curves are depicted in D, with fluorochrome emission of CD24 mAb (2.5 µg/ml) incubated with Alexa-568-labeled GAM-IgG versus biotinylated CD18 mAb MHM23 (2.5 µg/ml) incubated with fluorescein-conjugated streptavidin. The intensity of the image points is plotted against the wavelength of both fluorochrome emission curves (channels 1 vs. 2).

 
As a negative control, we used CD24, an antigen present in neutrophils in the plasma membrane and in the membrane of azurophil granules. Spectral curves obtained from TNF-treated CD24-CD18 merge cells (Fig. 4A) are reported in Figure 4D and indicate a low degree of colocalization between CD24 and β2 integrins. The final concentration of CD24 and CD18 MHM23 mAbs (2.5 µg/ml) explains the weak fluorescence intensity of the merge photomicrophotograph.

Distribution of gp91phox and Fc{gamma}RIIa on the neutrophil surface
As TNF-{alpha} induced colocalization of gp91phox and β2 integrins but also enhanced the colocalization of Fc{gamma}RIIa and β2 integrins, we then confirmed the colocalization of gp91phox and Fc{gamma}RIIa induced by TNF-{alpha} (2 ng/ml) on the neutrophil surface analyzed by confocal LSM (Fig. 5A ). Spectral curves obtained from TNF-treated merge cells (Fig. 5A) are reported in Figure 5B . These curves indicate a high degree of colocalization between gp91phox and Fc{gamma}RIIa.


Figure 5
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  		Figure 5. TNF-{alpha} induces colocalization of gp91phox and Fc{gamma}RIIa on the neutrophil surface. (A) Neutrophils (2x106/ml) were incubated at 37°C in polystyrene wells coated with FN (10 µg/ml), as described in Materials and Methods. After 5 min at 37°C, TNF-{alpha} (2 ng/ml) was added to some of the wells. After 40 min, the nonadherent and the loosely attached cells were transferred to tubes and immediately fixed with 1% (w/v) paraformaldehyde in incubation medium and washed. The cells were then resuspended with 7D5 mAb (10 µg/ml) against gp91phox and after another washing step, were incubated with Alexa-568-labeled GAM-IgG. Thereafter, the cells were resuspended with biotinylated IV.3 mAb againt Fc{gamma}RIIa (5 µg/ml), washed again, and incubated with fluorescein-conjugated streptavidin. After centrifugation onto glass slides, the cells were analyzed by confocal LSM. Results are representative of three independent experiments. (B) The AOTF-based system applied to merging of gp91phox with Fc{gamma}RIIa on the surface of neutrophils primed by TNF-{alpha}. Spectral curves are related to the fluorochrome emission of 7D5 mAb against gp91phox incubated with Alexa-568-labeled GAM-IgG versus biotinylated IV.3 mAb againt Fc{gamma}RIIa incubated with fluorescein-conjugated streptavidin. The intensity of the image points within the selected region of interest is plotted against the wavelength of both fluorochrome emission curves (channels 1 vs. 2).

 
Association of Fc{gamma}RIIa with the cytoskeleton by immunoblot analysis
We then studied the effect of TNF-{alpha} on the translocation of Fc{gamma}RIIa to the cytoskeleton by immunoblot analysis. We first analyzed the presence of Fc{gamma}RIIa in TX-100-soluble and -insoluble fractions in the absence or presence of TNF-{alpha} (2 and 20 ng/ml). Treatment of neutrophils with TNF-{alpha} induced redistribution of Fc{gamma}RIIa from TX-100-soluble to -insoluble fractions in a dose- and time-dependent manner (Fig. 6A ).


Figure 6
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  		Figure 6. Association of Fc{gamma}RIIa with the cytoskeleton by immunoblot analysis. (A) Redistribution of Fc{gamma}RIIa from TX-100-soluble to -insoluble fractions in TNF-untreated and -treated neutrophils. Neutrophils (1x107/ml) were pretreated with DFP (5 mM) and resuspended in incubation medium without HSA at 37°C, as described in Materials and Methods. Then, neutrophils were incubated under resting conditions (lanes 1 and 4) and treated with TNF-{alpha} at a final concentration of 2 ng/ml (lanes 2 and 5) or at a final concentration of 20 ng/ml (Lanes 3 and 6) for 10 and 40 min, respectively. Thereafter, samples were spun down, and the TX-100-soluble and -insoluble fractions were prepared, as indicated in Materials and Methods. Protein (12.5 µg/ml) of soluble fractions and the whole pellets of insoluble fractions in reduced Laemmli sample buffer were loaded onto a 10% (w/v) polyacrylamide gel and electrophoresed. Immunoblot analysis was performed as described in Materials and Methods. Blots were probed with the polyclonal rabbit CT10 antibody directed against the cytoplasmic tail of Fc{gamma}RIIa. Similar results were obtained in three other independent experiments. (B) The MHC class I {alpha}-chain in TX-100-insoluble fractions in TNF-untreated or -treated neutrophils. Neutrophils (1x107/ml) were pretreated with DFP (5 mM) and resuspended in incubation medium without HSA at 37°C. Then, neutrophils were incubated under resting conditions or treated with TNF-{alpha}, as described previously. Thereafter, samples were spun down, and the TX-100-soluble and -insoluble fractions were prepared. After the electrotransfer, blots were probed with a polyclonal rabbit-IgG against the human MHC class I {alpha}-chain as a negative control. Similar results were obtained in another experiment. (C) Effect of CD18 mAb MHM23 and β2-integrin-activating CD18 mAb KIM185 on the association of Fc{gamma}RIIa with TX-100-insoluble fractions in TNF-untreated or -treated neutrophils. Neutrophils (1x107/ml), which were pretreated with DFP (5 mM) and resuspended in incubation medium without HSA at 37°C. Then, neutrophils were incubated in the absence or presence of CD18 mAb MHM23 (mIgG1, 10 µg/ml) for 15 min prior to the addition of TNF-{alpha} (2 ng/ml) (lanes 1 and 2) or were incubated in the presence of β2-integrin-activating CD18 mAb KIM185 (K185; Fab fragments, 20 µg/ml) without subsequent addition of TNF-{alpha} (lane 3). After an additional incubation period of 10 min, samples were spun down, and the TX-100-insoluble fractions were prepared. The whole pellets of insoluble fractions in reduced Laemmli sample buffer were loaded onto a 10% (w/v) polyacrylamide gel and electrophoresed. After the electrotransfer, blots were probed with the polyclonal CT10 antibody. Similar results were obtained in two other experiments. (D) Effect of Cyto B and of D609 [an inhibitor of phosphatidylcholine phospholipase C (PC-PLC)] on the association of Fc{gamma}RIIa with TX-100-insoluble fractions in TNF-treated neutrophils. Neutrophils (1x107/ml) were pretreated with DFP (5 mM) and resuspended in incubation medium without HSA at 37°C. Then, neutrophils were left untreated (lanes 1 and 2) or were pretreated with Cyto B (CB) (5 µg/ml) (lane 3) or with D609 (1 mM) (lane 4) for 15 min prior to the addition of TNF-{alpha} (2 ng/ml) (lanes 2–4). After 10 min of priming, samples were spun down, and the TX-100-insoluble fractions were prepared. The whole pellets of insoluble fractions in reduced Laemmli sample buffer were loaded onto a 10% (w/v) polyacrylamide gel and electrophoresed. After the electrotransfer, blots were probed with the polyclonal CT10 antibody. Similar results were obtained in two other experiments. (E) Actin distribution in TX-100-insoluble fractions in TNF-treated neutrophils. Neutrophils (1x107/ml) were pretreated with DFP (5 mM) and resuspended in incubation medium without HSA at 37°C. Then, neutrophils were left untreated (lanes 1 and 2) or were pretreated with Cyto B (5 µg/ml) (lane 3) for 15 min prior to the addition of TNF-{alpha} (2 ng/ml) (lanes 2 and 3). After 10 min of priming, samples were spun down, and the TX-100-insoluble fractions were prepared. The whole pellets of insoluble fractions in reduced Laemmli sample buffer were loaded onto a 10% (w/v) polyacrylamide gel and electrophoresed. After the electrotransfer, blots initially probed with the polyclonal CT10 antibody were stripped and reprobed with a polyclonal goat-IgG against human actin (I-19). Similar results were obtained in two other experiments.

 
As a negative control, blots were probed with an antibody against the MHC class I {alpha}-chain. This membrane protein did not translocate from TX-100-soluble to -insoluble fractions upon TNF treatment of neutrophils (Fig. 6B) .

The blocking CD18 mAb MHM23 (5 µg/ml) interfered efficiently with the Fc{gamma}RIIa-cytoskeleton association induced by TNF-{alpha}, whereas activation of β2 integrins by mAb KIM185 Fab fragments (20 µg/ml) was sufficient per se to cause Fc{gamma}RIIa association with the cytoskeleton (Fig. 6C) .

Cyto B (5 µg/ml), an inhibitor of cytoskeletal changes, inhibited the association of Fc{gamma}RIIa with the cytoskeleton in TNF-treated neutrophils (Fig. 6D) and decreased actin polymerization induced by TNF-{alpha} in the TX-100-insoluble fractions, as detected by reprobing blots with an antibody against actin (Fig. 6E) .

Cyto B (5 µg/ml) inhibited the respiratory burst in TNF-treated neutrophils exposed to 3G8, anti-PR3, or anti-MPO mAb (Fig. 7A ), as described [8 ]. One central TNF-signaling route is the second messenger-like molecule ceramide [24 ], which is generated by sphingomyelin breakdown catalyzed by a sphingomyelinase, and sphingomyelinase activation is secondary to the generation of 1,2-diacylglycerol (DAG) produced by a TNF-responsive PC-PLC. We studied the effect of D609, a selective inhibitor of PC-PLC [25 ], on the association of Fc{gamma}RIIa with the cytoskeleton in TNF-treated neutrophils. D609 (1 mM) completely inhibited this Fc{gamma}RIIa association (Fig. 6D) . D609 also inhibited the respiratory burst in TNF-treated neutrophils exposed to 3G8, anti-PR3, or anti-MPO mAb but did not affect the burst in neutrophils exposed to PMA (Fig. 7B) . Similar results were obtained with purified IgG preparations from sera with PR3-ANCA or MPO-ANCA (not shown).


Figure 7
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  		Figure 7. Effect of (A) Cyto B and (B) D609 (an inhibitor of PC-PLC) on the respiratory burst activation in TNF-treated neutrophils exposed to PMA and 3G8, anti-PR3, or anti-MPO mAb. Neutrophils (2x106/ml) were incubated at 37°C with DHR in polystyrene tubes in a shaking water-bath under gentle agitation, as described in Materials and Methods, in the absence or presence of (A) Cyto B (5 µg/ml) and (B) D609 (1 mM) for 5 min before the addition of TNF-{alpha} (2 ng/ml). After 10 min of priming, the cells were stimulated for 15 min with PMA (100 ng/ml) and for 30 min with 3G8, anti-PR3, or anti-MPO mAb at a final concentration of 5 µg/ml. Control cells (–) received PBS only. Samples were processed for flow cytometry to measure rhodamine-1,2,3 fluorescence. Results (MFI) are the mean ± SEM of three or four independent experiments. With all stimuli except PMA, Cyto B (5 µg/ml) or D609 (1 mM) had a significant, inhibiting effect in the presence of TNF-{alpha} (P<0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our previous study, we described that TNF-{alpha} significantly but weakly increased the binding of an anti-PR3 mAb to human neutrophils (P<0.05), whereas binding of an anti-MPO mAb was hardly detectable, not even after TNF-{alpha} treatment (differences not significant) [6 ]. We obtained similar results with two different anti-PR3 mAbs and two different anti-MPO mAbs (not shown). In the literature, levels of detectable PR3 are usually higher than levels of detectable MPO on the surface of TNF-primed neutrophils, as reported by Porges et al. [10 ], but data about expression of MPO are inconsistent. Among these, Hess et al. [26 ] have described a different expression of PR3 versus MPO, i.e., an increased PR3 expression but no increased MPO expression on TNF-primed neutrophils. The difference in up-regulation of these two proteins might be explained by the difference between their respective locations, i.e., PR3 in the azurophil granules but also in the highly mobilizable secretory vesicles [27 ] and MPO exclusively in the azurophil granules. The fusion of secretory vesicles with the cell plasma membrane leads to an incorporation into the surface membrane of vesicle components [28 ]. In our opinion, PR3 up-regulation by TNF-{alpha} is likely a result of plasma-membrane fusion of these vesicles, which also substantiates a colocalization of PR3 and β2 integrins upon TNF priming ([29 ] and data not shown). In contrast, the mechanism of MPO up-regulation remains unclear, as the azurophil granules are not induced to fuse with the plasma membrane by cytokines [30 ]. Finally, to evaluate the effect of TNF priming on surface-antigen expression, we previously investigated its effect on a number of antigens already present in the plasma membrane of resting neutrophils (CD16, CD24, and CD66) [6 ]. We concluded that the TNF-induced enhancement of neutrophil activation by antibodies directed against neutrophil antigens can take place independently of changes in cell surface expression of these antigens [6 ]. In the present study, we therefore sought for additional and/or alternative explanation(s) of the requirement of TNF priming for neutrophil activation by anti-PR3 or anti-MPO antibodies, mediated by Fc{gamma}RIIa- and β2-integrin-dependent pathways [6 , 7 ].

First, activation of the NADPH oxidase is known to be dependent on translocation of the p47phox, p67phox, p40phox, cytosolic complex and Rac2 to the plasma membrane, where they associate with flavocytochrome b558 and Rap1A, initially located in the membranes of specific granules and secretory vesicles [31 ]. DeLeo et al. [32 ] have suggested that redistribution of NADPH-oxidase components underlies bacterial LPS priming of the neutrophil respiratory burst (LPS described to increase plasma-membrane association of flavocytochrome b558). The amount of flavocytochrome b558 mobilized to the plasma membrane following TNF priming has been documented. Ward et al. [21 ] have reported that one mechanism by which LPS but also TNF-{alpha} primes the neutrophil-respiratory burst activity is to increase the membrane expression of specific markers from secretory vesicles (CD35) and specific granules (CD66b) that contain flavocytochrome b558. Mansfield et al. [33 ] have described more recently that G-CSF primes the NADPH oxidase in neutrophils through translocation of flavocytochrome b558. Mobilization of secretory vesicles transforms the neutrophil to a β2-integrin-presenting cell primed for migration [28 ], and TNF-{alpha} induces activation of β2 integrins [34 ]. We found that TNF-{alpha} (2 ng/ml) induced up-regulation and colocalization of gp91phox and β2 integrins on the neutrophil surface (Fig. 2) . Similarly, David et al. [35 ] have shown up-regulation and colocalization of cytochrome b558 and PR3 on the plasma membrane of TNF-{alpha}-treated neutrophils.

Further, Sengelov et al. [36 ] have suggested that the mobilization of secretory vesicles is important in early neutrophil activation. These vesicles are exceptionally sensitive to cytosolic-free calcium, being completely mobilized after small, cytosolic calcium transients. As intracellular-free calcium ions [Ca2+]i are implicated in certain aspects of the priming response [37 ], we wondered whether the effect of TNF priming on flavocytochrome b558 and β2-integrins is signaled via cytosolic-free calcium. After chelation of [Ca2+]i with BAPTA-AM (25 µM), a decrease in expression of gp91phox and β2 integrins on the neutrophil surface upon TNF priming was observed (Table 1) . These results corroborate the inhibitory effect of BAPTA-AM on the respiratory burst in TNF-treated cells induced by anti-PR3 or anti-MPO mAb (Fig. 3) . Ward et al. [21 ] have used neutrophil cytoplasts and concluded that intracellular storage granules are necessary for priming of neutrophil respiratory burst activity by TNF-{alpha}. Hence, in our model, the early mobilization of the secretory vesicles as a result of a TNF-induced increase in [Ca2+]i and the parallel redistribution of the main component of the NADPH oxidase associated with β2 integrins may account for the TNF priming for neutrophil activation.

As a second part, we analyzed the effect of TNF priming on the distribution of Fc{gamma}RIIa and β2 integrins on the cell surface. The confocal experiments showed that TNF-{alpha} (2 ng/ml) per se induced clustering but did not significantly increase the surface expression of Fc{gamma}RIIa (P>0.05) [6 ], indicating that Fc{gamma}RIIa signaling might be enhanced, and TNF-{alpha} also induced the colocalization of Fc{gamma}RIIa with β2 integrins (Fig. 4A) . Blocking CD18 mAb MHM23, which prevented the respiratory burst induced by anti-PR3 or anti-MPO antibodies, also prevented the Fc{gamma}RIIa clustering induced by TNF-{alpha} (Fig. 4B) . Hackam et al. [38 ], with the murine macrophage cell line J774 and COS-1 cells transfected with Fc{gamma}RIIa, have reported the clustering of Fc{gamma}Rs as an essential step in Fc{gamma}R-induced signaling. The colocalization of gp91phox with Fc{gamma}RIIa under TNF treatment depicted in Figure 5 matched with results depicted in Figures 2 and 4 . Thus, the Fc{gamma}RIIa clustering upon TNF priming appears to be essential for the induction of the neutrophil respiratory burst by anti-PR3 or anti-MPO antibodies, and the colocalization of Fc{gamma}RIIa with β2 integrins suggests that a cooperation between both molecules is required for this type of neutrophil NADPH-oxidase activation.

Yan et al. [39 ] have reported that TNF-{alpha} (20 ng/ml) triggers redistribution of distinct proteins, such as β2 integrins and the four NADPH-oxidase components gp91phox, p67phox, p47phox, and p22phox to TX-100-insoluble cytoskeletal structures. We propose that the association of Fc{gamma}RIIa with the cytoskeleton is relevant for the effect of TNF priming in our model. We found that TNF-{alpha} (2 and 20 ng/ml) per se triggers redistribution of Fc{gamma}RIIa from TX-100-soluble to -insoluble fractions in a dose- and time-dependent manner (Fig. 6A) . This redistribution was enhanced by the exposure to anti-PR3 or anti-MPO mAb (not shown) and was inhibited by the blocking CD18 mAb MHM23 (Fig. 6C) . This last result corroborates the blocking effect of CD18 mAb MHM23 on the Fc{gamma}RIIa clustering induced by TNF-{alpha} (Fig. 4B) . Zhou and Brown [40 ] have hypothesized that ligation of β2-integrin Mac-1 signals an association of Fc{gamma}RIIa with the actin cytoskeleton and that this association is necessary but not sufficient for a Fc{gamma}RII-dependent respiratory burst. Likewise, the presence of the β2-integrin-activating mAb KIM185 is sufficient per se to induce this Fc{gamma}RIIa association in TNF-untreated neutrophils (Fig. 6C) . However, KIM185-induced β2-integrin activation is insufficient for neutrophil activation by anti-PR3 or anti-MPO antibodies [7 ]. Thus, the association of Fc{gamma}RIIa with the cytoskeleton might be necessary but not sufficient, in our model, to induce a Fc{gamma}RIIa-dependent respiratory burst.

As a third and final step in this study, we investigated the mechanism of this priming effect through linkage of β2 integrins and Fc{gamma}RIIa to the cytoskeleton. Berkow et al. [41 ] have reported that TNF-{alpha} increases the F-actin content of neutrophils and suggested that a cytoskeletal effect could be involved in the priming effect of TNF-{alpha}. Cyto B (5 µg/ml) markedly diminished the redistribution of Fc{gamma}RIIa (Fig. 6D) but also hampered the ability of TNF-{alpha} to cause redistribution of actin to the TX-100-insoluble fractions (Fig. 6E) . In addition, Cyto B completely abolished the neutrophil-oxidase activation induced by anti-PR3 or anti-MPO antibodies (Fig. 7A) . Cyto B also slightly enhanced the expression of PR3 and MPO on the neutrophil surface (not shown). Thus, the inhibition of the NADPH-oxidase activation by anti-PR3 or anti-MPO antibodies in the presence of Cyto B cannot be explained by an inhibition of the expression of the relevant antigens but may be related to inhibition of β2 integrin and Fc{gamma}RIIa linkage to the cytoskeleton.

Many of the stimuli that activate neutrophils mediate their effects through the hydrolysis of phosphoinositides by PLC [42 ]. D609 (1 mM), an inhibitor of PC-PLC, had an inhibitory effect on the Fc{gamma}RIIa-cytoskeleton association induced by TNF-{alpha} (Fig. 6D) and on the respiratory burst in TNF-treated neutrophils triggered by 3G8, anti-PR3, or anti-MPO mAb (Fig. 7B) . Hence, we put forward a role for PC-dependent PLC activation in the TNF priming of neutrophils. Superoxide anion production has also been described to require activation of the PLD pathway in primed neutrophils [43 ]. The interrelationship betweeen PLC and PLD is noteworthy, as several metabolites of these two enzymes are second messengers common to both pathways (e.g., phosphatidic acid, DAG) [44 ]. DAG, generated as a result of the hydrolysis induced by PC-PLC, is known to activate protein kinase C (PKC). Thelen et al. [45 ] have reported that TNF-{alpha} primes neutrophils for enhanced PKC-dependent signal transduction.

TNF-mediated translocation of β2 integrins and flavocytochrome b558 to the neutrophil surface has been described to occur through regulation by p38-MAPK [21 , 46 ]. Likewise, we demonstrated that the colocalization of these components upon TNF priming is mediated through p38-MAPK (not shown). Moreover, we described that inhibition of the pathways with the ERK inhibitor PD98059 (50 µM) and more particularly, with the p38-MAPK inhibitor SB203580 (25 µM) resulted in a decreased respiratory burst in TNF-treated neutrophils exposed to anti-PR3 or anti-MPO mAb [47 ]. In these experiments, SB203580 depicted the strongest effect on the respiratory burst when added before TNF priming, further supporting a role for MAPK in TNF priming and subsequent neutrophil activation induced by these antibodies, as also reported by Kettritz et al. [48 ]. These authors have pointed out another signal transduction pathway that controls the effect of TNF-{alpha} on neutrophil activation by ANCA and involves the PI-3K and the serine-threonine PKB/Akt kinase, being an immediate downstream effector of PI-3K [49 ]. The possibility arises that PI-3K products exert their effects on the respiratory burst by activation of downstream protein kinases, which may directly phosphorylate components of the oxidase complex [50 ].

Finally, we provide evidence for direct effects of TNF-{alpha} in facilitating neutrophil activation by anti-PR3 or anti-MPO antibodies through Fc{gamma}RIIa- and β2-integrin-dependent pathways. We hypothesize that the flavocytochrome b558 interacts with β2 integrins and Fc{gamma}RIIa through linkage to the plasma membrane and/or the cytoskeleton, thus establishing mechanism(s) of this priming effect relevant for this activation. Preliminary experiments indicate that at least part of the signals involved in TNF priming of Fc{gamma}R-mediated oxidase activation is also involved in TNF priming of fMLP-mediated oxidase activation. Knowledge and emerging issues about neutrophil priming are of crucial relevance for understanding effects of therapeutic strategies, such as TNF-blockade treatment in primary vasculitides. Beneficial effects of such alternative, therapeutic agents would support the statement that TNF-{alpha} has a central role in the pathological, inflammatory response associated with Wegener’s granulomatosis.


   ACKNOWLEDGEMENTS
 
The authors gratefully thank Dr. A. J. Verhoeven (Sanquin Research, The Netherlands) and Dr. L. A. Boxer (University of Michigan, Ann Arbor) for their helpful discussions, D. Dehay (Centre Hospitalier de Valenciennes, France) and Dr. A. T. J. Tool (Sanquin Research) for their expert technical assistance, A. Fauvel and the late M. Flactif (Service Commun d’Imagerie Cellulaire, IFR 114, IMPRT, Faculté de Médecine, University Lille-2, France) for their contribution to confocal LSM, and G. Espouy and D. Vanderbecq (ICARE, Faculté de Médecine, Univ. Lille-2) for their contribution to reprography.

Received March 6, 2004; revised July 31, 2006; accepted August 10, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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