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J Am Soc Nephrol 15:796-808, 2004
© 2004 American Society of Nephrology

CLINICAL SCIENCE

Activation of Syk in Neutrophils by Antineutrophil Cytoplasm Antibodies Occurs via Fc{gamma} Receptors and CD18

Peter Hewins*, Julie M. Williams*, Michael J.O. Wakelam{dagger} and Caroline O.S. Savage*

*Renal Immunobiology and {dagger}Institute for Cancer Studies, MRC Centre for Immune Regulation, The Medical School, University of Birmingham, Birmingham, United Kingdom

Correspondence to Prof. Caroline O.S. Savage, MRC Centre for Immune Regulation, The Medical School, Birmingham B15 2TT. Phone: +44 121 414 6841/0121 697 8427; Fax: +44 121 414 6840; E-mail: C.O.S.Savage{at}bham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Antineutrophil cytoplasm antibodies (ANCA) activate TNF-{alpha}–primed neutrophils to undergo a respiratory burst. The intracellular signals that mediate activation have not been studied extensively but could increase the understanding of the pathogenesis small vessel vasculitis. It was demonstrated that ANCA-IgG induced phosphorylation of the tyrosine kinase Syk in TNF-{alpha}–primed neutrophils from healthy donors. Syk was not phosphorylated in response to ANCA F(ab')2. Furthermore, Syk phosphorylation was attenuated by blockade of both low-affinity Fc{gamma} receptors and CD18. Similarly, low-affinity Fc{gamma} receptor blockade reduced ANCA-induced superoxide production. In patient-derived neutrophils, the high-affinity Fc{gamma} receptor Fc{gamma}RI was also demonstrated to be involved in ANCA-induced superoxide production. However, Syk phosphorylation was not attenuated by blockade of the Fc{gamma}RI, present on neutrophils from vasculitis patients. The tyrosine kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine inhibited the ANCA-induced respiratory burst and Syk phosphorylation, suggesting that Src kinases lie upstream of Syk activation but downstream of ANCA engagement of Fc{gamma} receptors. Piceatannol, another tyrosine kinase inhibitor, also inhibited ANCA-induced Syk phosphorylation and the ANCA-stimulated respiratory burst, supporting the proposed functional role for Syk in ANCA signaling. ANCA-induced phosphorylation of Cbl and intracellular calcium transients, potential downstream mediators of Syk activation, were also blocked by tyrosine kinase inhibitors. While it has previously been shown that pertussis toxin diminishes the ANCA-induced respiratory burst, indicating heterotrimeric G protein involvement, Syk phosphorylation and calcium transients were unaffected by pertussis toxin. Collectively, these data show that Syk phosphorylation is induced during ANCA-triggered neutrophil activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antineutrophil cytoplasm antibodies (ANCA) are IgG autoantibodies detected in the plasma of patients with Wegener’s granulomatosis and microscopic polyangiitis, small vessel vasculitides characterized by endothelial damage and perivascular leukocyte infiltration. Neutrophils are believed to be central to disease pathogenesis. Although the exact role of ANCA is uncertain, accumulating evidence suggests that they have a causative role in vascular inflammation, and collectively the diseases are frequently referred to as ANCA-associated vasculitides (ASV) (1). In vasculitis, ANCA are directed against the antigens proteinase 3 (PR3) and myeloperoxidase (MPO), which are detectable on the plasma membrane of isolated neutrophils. ANCA titers correlate with disease activity in many patients, and rising titers may predict clinical relapse (2). However, not all studies have confirmed an association between ANCA titers and disease activity (3). The precise relationship between the autoantibody and neutrophil activation in vivo remains to be determined, although other factors, such as PR3 expression by circulating leukocytes, are very probably influential (4). Furthermore, there are numerous reports of in vitro ANCA activation of neutrophils from healthy donors, usually after priming with TNF-{alpha} (5–10). Cell surface expression of PR3 and MPO by neutrophils is upregulated by TNF-{alpha} and other proinflammatory mediators (5, 11). Circulating neutrophils from patients with active ANCA-associated vasculitis are primed and express these antigens (12). Evidence favoring the pathogenicity of ANCA has recently been strengthened considerably by animal models of MPO-ANCA–associated vasculitis. In one model, pathogenic transfer of anti-MPO IgG into RAG2-/- mice was demonstrated (13).

The mechanism of ANCA-induced leukocyte activation has remained controversial. In particular, there have been conflicting reports over the necessity of Fc{gamma} receptor engagement for the induction of a respiratory burst. Some reports conclude that ANCA F(ab')2 fragments are sufficient for leukocyte activation (5, 9), whereas others find that ANCA must be intact (6–8). Fc{gamma}RIIa (CD32) blockade has been shown to diminish ANCA-mediated activation (9, 10), and Fc{gamma}RIIIb (CD16) is also important in ANCA binding to neutrophils (10, 14). Previously, it was reported that blockade of the common {beta}2 integrin antigen CD18 completely inhibits the ANCA-induced respiratory burst (8). Mac-1 (CD11b/CD18) may cooperate with Fc{gamma}RIIa and Fc{gamma}RIIIb in neutrophil activation, and specific Mac-1 cross-linking directly induces a respiratory burst in a manner augmented by TNF-{alpha} (15, 16). Another area of interest, which has thus far received little attention, is the involvement of the high-affinity Fc{gamma} receptor Fc{gamma}RI (CD64) in neutrophil activation by ANCA. Neutrophils obtained from patients with active vasculitis have been shown to express Fc{gamma}RI (17).

A number of models could explain how ANCA activate neutrophils. Multiple ANCA may bind target antigens on one neutrophil and cross present oligomerized Fc moieties to surrounding neutrophils. However, in a flow assay examining ANCA-induced adhesion of neutrophils to an endothelial monolayer, it was clear that cell–cell contact between neutrophils was not necessary for ANCA-mediated effects (18). Alternatively, ANCA may heterodimerize Fc{gamma} receptors and target antigens on the surface of individual neutrophils. Fc{gamma} receptor heterodimerization has been proposed to account for macrophage and platelet activation by anti-CD9 antibodies (19, 20). A third possibility is that degranulated PR3 and MPO bind ANCA in solution, forming immune complexes that then conventionally bind and activate Fc receptors.

Recently, this laboratory demonstrated tyrosine phosphorylation of multiple proteins during ANCA activation of neutrophils (7). Membrane translocation of the classical protein kinase C isoenzyme {beta}II also occurs, and ANCA-induced calcium transients have been demonstrated (21). Importantly, ANCA-stimulated neutrophils do not exhibit activation of phospholipase D or type Ia/b p85-phosphatidylinositol 3-kinase (p85-PI3K), whereas activation of PI3K-{gamma} does occur and superoxide (SO) anion release is inhibited by pertussis toxin, indicating clear divergence from the usual pathways recruited during conventional Fc{gamma} receptor cross-linking (10). Furthermore, both ANCA-IgG and ANCA-F(ab')2 induce pertussis toxin–inhibited GTPase activity in neutrophil membranes (22). This and other evidence, including ANCA-F(ab')2–induced gene transcription, indicate that ANCA-antigen engagement has direct effects in its own right and further suggests that ANCA-induced activation is not simply the result of Fc{gamma} receptor engagement by immune complexes (23).

The cytosolic tyrosine kinase Syk is essential for Fc{gamma}RIIa-mediated leukocyte functions, and Syk antisense oligonucleotides ameliorated damage in an animal model of macrophage-induced lung injury (24, 25). Syk is recruited to Fc{gamma}RIIa after phosphorylation of critical immunoreceptor tyrosine activation motif (ITAM) tyrosine residues by a Src kinase constitutively associated with the receptor at the plasma membrane (26). Syk itself is then tyrosine phosphorylated, leading to its activation. Downstream targets of activated Syk can include PLC{gamma} and Cbl as well as phospholipase D and p85-PI3K (27, 28).

Therefore, we wished to determine the involvement of Syk in ANCA-mediated neutrophil activation. PR3-ANCA and MPO-ANCA phosphorylated Syk in primed neutrophils from healthy donors, whereas IgG from healthy donors did not. Syk phosphorylation was dependent on intact ANCA-IgG and diminished by blockade of Fc{gamma}RIIa, Fc{gamma}RIIIb, and CD18. Fc{gamma}RIIa and Fc{gamma}RIIIb blockade also reduced ANCA-induced SO production. Furthermore, Fc{gamma}RI was shown to be involved in ANCA-induced SO production by neutrophils isolated from patients with ASV, although the influence of this receptor on ANCA-induced Syk phosphorylation was uncertain. Tyrosine kinase inhibitor studies reveal that Src kinases are proximal to Syk in the ANCA-induced signaling cascade and confirm the importance of Src kinases in SO production. Furthermore, there are differential effects of tyrosine kinase inhibitors on Syk phosphorylation induced by ANCA and conventional Fc{gamma}RIIa receptor cross-linking. ANCA-induced calcium transients are sensitive to tyrosine kinase inhibition but resistant to pertussis toxin, indicating the participation of PLC{gamma} not PLC{beta} despite the involvement of pertussis toxin–sensitive G proteins in the ANCA-respiratory burst. ANCA also induced tyrosine phosphorylation of Cbl.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
HBSS containing calcium, Iscove’s modified Dulbecco’s medium, diisopropylfluorophosphate (DFP), fMLP, pertussis toxin, cytochrome c, goat anti-mouse antibody F(ab')2 fragment, and goat anti-human (Fab specific) antibody F(ab')2 fragment were obtained from Sigma-Aldrich (Poole, Dorset, UK). Percoll, Dextran T-500, Sephadex G-10, Hi-Trap columns, and ECL-Plus were from Amersham Biosciences (Little Chalfont, Bucks, UK). Recombinant human TNF-{alpha} was from NIBSC (Potters Bar, Herts, UK). Anti-Syk (4G10 and rabbit polyclonal) and anti-Cbl (7G10) antibodies were from Upstate Biotechnology (Botolph Claydon, Bucks, UK). Anti-phosphotyrosine (PY99) monoclonal antibody was from Santa Cruz (Autogen Bioclear, Mile Elm, Calne, UK). Anti-Fc{gamma}RIIa (Fab IV.3) and anti-Fc{gamma}RIII (F(ab')2 3G8) antibodies were from Medarex (Annandale, NJ). Anti-CD18 antibody (MHM23), anti-CD45 antibody, and murine irrelevant IgG1 were from Dako (Ely, Cambs, UK). Anti-CD64 F(ab')2 (10.1) was from Ancell (Alexis Corp., Nottingham, UK). Fura-2 AM, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and piceatannol were from Calbiochem (CN Biosciences, Nottingham, UK).

Neutrophil Isolation
Neutrophils were isolated as described previously (7). Briefly, peripheral blood from healthy volunteers was anticoagulated with acid citrate dextrose, diluted with 2% dextran solution, and centrifuged to pellet red blood cells. Leukocyte-rich plasma was then loaded onto Percoll gradients and centrifuged. Neutrophils were recovered from the gradients, washed twice in PBS, and suspended in HBSS buffered with 10 mM Hepes (HBH) at 2 x 107/ml. Before immunoprecipitation, neutrophils were preincubated with 2 mM DFP for 20 min at room temperature, then washed and resuspended in HBH. When necessary, neutrophils were then incubated with inhibitors, piceatannol, PP2, or pertussis toxin before use.

Induction of Fc{gamma}RI Expression on Neutrophils
Freshly isolated neutrophils were suspended (2 x 106/ml) in Iscove’s modified Dulbecco’s medium supplemented with 10% autologous serum and 400 IU/ml IFN-{gamma} and incubated overnight (16 h) at 37°C in 5% CO2. Neutrophils were then washed, resuspended in HBH, and used straight away.

ANCA-Containing IgG Isolation and Preparation of F(ab')2 Fragments
ANCA-containing IgG was isolated from stored sera of patients with active ANCA-associated vasculitis by Hi-Trap protein G Sepharose column affinity chromatography as described previously (7). All patients from whom ANCA were isolated were judged to have active Wegener’s granulomatosis or microscopic polyangiitis on clinical and serologic grounds (29). Control IgG was isolated from sera of healthy volunteers by the same method. F(ab')2 fragments of patient IgG were then prepared by pepsin digestion as described previously (7). Dose equivalence of IgG and F(ab')2 was determined using PR3- or MPO-specific ELISA (Binding Site, UK). ANCA-IgG and F(ab')2 were endotoxin-free as assessed by Limulus amoebocyte assay.

Immunoprecipitation and Western Blotting
Immunoprecipitation from neutrophil lysates was performed according to the method of Al-Shami et al. (30) using a denaturing buffer because preservation of phosphotyrosine was unreliable using other methods. Briefly, 500 µl aliquots of neutrophils (2 x 107/ml) in HBH were warmed to 37°C, primed with TNF-{alpha} 80 IU/ml for 15 min, and stimulated by adding the appropriate stimulus and agitating the cell suspension for 5 s. When necessary, in the final 5 min of priming, blocking antibodies against Fc{gamma} receptors or CD18 were added. In other experiments, neutrophils were preincubated with pertussis toxin (2 h), PP2 (30 min), or piceatannol (30 min) before TNF-{alpha} priming. Stimulation was stopped by adding neutrophil aliquots to equal volumes of boiling lysis buffer (final concentrations: 62.5 mM Tris-HCl [pH 6.8], 3% SDS, 1.5% {beta}-mercaptoethanol, 8.5% glycerol, 2.5 mM Na3VO4, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). After boiling for 7 min, lysates were transferred to ice then filtered through Sephadex G10 columns by centrifugation at 1500 x g for 2 min. Filtered lysates were supplemented with buffer containing BSA, Nonidet P-40, Na3VO4, leupeptin, and aprotinin (final concentrations: 0.05%, 1%, 2 mM, 10 µg/ml, and 10 µg/ml, respectively), and immunoprecipitating antibodies were added. Immunoprecipitation was performed overnight at 4°C on a rotary wheel, then protein G Sepharose beads were added for an additional 1.5 h. Beads were centrifuged, washed four times, and then boiled in sample buffer. Samples were resolved on 8 or 10% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, blocked with 5% BSA in TBS containing 0.1% Tween 20, and incubated overnight with PY99 antibody at 4°C. After washing, membranes were incubated with horseradish peroxidase–conjugated anti-mouse Ig antibody in 5% nonfat milk TBS containing 0.1% Tween 20 for 1 h at room temperature. Detection was performed using ECL-Plus. Membranes were stripped and reprobed to confirm equal protein loading.

Intracellular Calcium Flux Measurements
Calcium fluxes were measured in neutrophils preloaded with 2 µM FURA-2AM for 30 min and suspended at 2 x 106/ml in HBH as described previously (21). Neutrophils were primed with TNF-{alpha} 80 IU/ml for 15 min after loading and immediately before use. Using a Cairns fluorimeter, fluorescence was measured at 510 nm following alternating excitation at 340 nm and 380 nm, and the 340/380 ratio was determined as a measure of intracellular free calcium.

SO Anion Release
SO anion release was measured by cytochrome c reduction as described previously (7). Each stimulus was tested in triplicate, and values quoted represent mean SO anion released ± SEM in response to the specified stimulus after subtracting SO released by primed but unstimulated cells at the corresponding time point.

Luminol-Derived Chemiluminescence
Chemiluminescence was measured in luminol-loaded neutrophils. Briefly, neutrophils (2 x 106/ml in HBH) were treated with piceatannol or vehicle (DMSO) for 30 min, primed as for the SO anion release assay, then supplemented with luminol (0.156 mM) and catalase (2000 U). After allowing the cell suspensions to equilibrate for 1 min in the luminometer maintained at 37°C, 200 µg/ml of the relevant IgG fraction (ANCA or normal) or an equal volume of PBS was added. Readings were obtained every 30 s for 10 min. Results are expressed as a percentage of chemiluminescence detected in unstimulated cells.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Syk Phosphorylation Follows Incubation of TNF-{alpha}–Primed Neutrophils with ANCA
Immunoprecipitation of Syk from TNF-{alpha}–primed neutrophils obtained from healthy donors revealed that priming alone did not cause an increase in Syk tyrosine phosphorylation. On some occasions, low levels of phosphorylated Syk were obtained from unstimulated cells irrespective of priming. Exposure to PR3-ANCA or MPO-ANCA but not IgG from healthy donors caused marked, transient Syk phosphorylation (five MPO-ANCA, five PR3-ANCA, and five normal IgG were tested). The time course of ANCA-induced Syk phosphorylation varied somewhat between IgG fractions but was consistently slower and more sustained than that induced by cross-linking Fc{gamma}RIIa using monoclonal antibodies. Fc{gamma}RIIa cross-linking triggered Syk phosphorylation within 15 s that decreased again within 5 min (Figure 1A), whereas ANCA-induced Syk phosphorylation was typically evident at 1 min and sustained for >10 min (Figure 1B). ANCA antigen (MPO or PR3) specificity did not influence the time course of Syk phosphorylation (data not shown).



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  		Figure 1. Syk phosphorylation in response to Fc{gamma}RIIa cross-linking by monoclonal anti-CD32 antibody or antineutrophil cytoplasm antibodies (ANCA). (A) Fresh neutrophils, isolated from healthy donors, were suspended in HBSS buffered with 10 mM Hepes (HBH; 2 x 107/ml) at 37°C and primed with TNF-{alpha} (80 IU/ml) for 10 min, then incubated with Fab IV.3 anti-CD32 (Fc{gamma}RIIa) antibody (5 µg/ml) or an equal volume of PBS for an additional 5 min. Finally, F(ab')2 goat anti-mouse antibody (50 µg/ml) was added for the indicated length of time. Stimulation was stopped by adding boiling lysis buffer, and Syk immunoprecipitation and phosphotyrosine Western blotting were performed as described in the Materials and Methods section. Finally, blots were stripped and reprobed for total Syk to confirm equal loading. Experiments were performed on three separate occasions using different neutrophil donors, and a representative blot is shown. (B) Freshly isolated neutrophils from healthy donors were suspended in HBH (2 x 107/ml) at 37°C, primed with TNF-{alpha} (80 IU/ml) for 15 min, then stimulated with ANCA-IgG or normal IgG from healthy donors (200 µg/ml). Reactions were terminated, and samples were processed as in A. Experiments were performed on a total of five proteinase 3 (PR3)-ANCA, five myeloperoxidase (MPO)-ANCA, and five normal IgG fractions using a panel of seven neutrophil donors. Representative blots for PR3-ANCA and normal IgG are shown.

 
In TNF-{alpha}–primed neutrophils, homotypic Fc{gamma}RIIIb cross-linking also induced some Syk phosphorylation. It is interesting that in unprimed neutrophils, cross-linking Fc{gamma}RIIIb did not induce Syk phosphorylation, whereas Fc{gamma}RIIa cross-linking was effective (not shown). Concordant with the kinetics of Syk phosphorylation, SO anion release after Fc{gamma} receptor cross-linking using monoclonal anti-Fc{gamma} receptor antibodies was rapid and reached a plateau within 10 min, whereas ANCA-induced SO release did not plateau before 30 min (data not shown) (10). SO anion release at 45 min by three representative IgG fractions was as follows: PR3-ANCA = 15.4 ± 1.30 nmol SO/105 cells; MPO-ANCA = 5.23 ± 0.881 nmol SO/105 cells; normal IgG = 0.431 ± 0.451 nmol SO/105 cells (SO anion ± SEM for each IgG fraction is based on five separate experiments and use of at least three different neutrophil donors).

Syk Phosphorylation Requires Intact ANCA-IgG and Is Not Seen with ANCA-F(ab')2
We have previously reported that ANCA-F(ab')2 do not induce SO anion release, although there have been conflicting reports by other investigators (5–9). Few studies have examined intracellular pathways recruited by ANCA-IgG or ANCA-F(ab')2. We found that ANCA-F(ab')2 derived from four different patients (two PR3-ANCA and two MPO-ANCA) did not induce tyrosine phosphorylation of Syk, whereas the corresponding intact IgG did so, suggesting that ANCA-induced Syk phosphorylation is linked to Fc{gamma} receptor engagement (Figure 2). Furthermore, Syk phosphorylation was not increased by cross-linking ANCA-F(ab')2 fragments using an anti-human antibody F(ab')2 fragment. SO anion release in response to ANCA-F(ab')2 was minimal: ANCA-IgG = 10.6 ± 2.5 nmol SO/105 cells at 45 min versus corresponding ANCA-F(ab')2 = 0.20 ± 0.17 nmol SO/105 cells at 45 min (mean of two separate experiments using a representative fraction). ANCA-F(ab')2 retained their ability to bind to ANCA antigens coated onto ELISA plates (data not shown).



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  		Figure 2. Syk phosphorylation in response to ANCA-IgG but not ANCA-F(ab')2. Freshly isolated neutrophils from healthy donors were primed with TNF-{alpha} (80 IU/ml) for 15 min, then stimulated with ANCA-IgG (200 µg/ml) or the equivalent dose of ANCA-F(ab')2 as determined by binding to anti-PR3 or anti-MPO ELISA plates (data not shown). After 10 min, reactions were stopped as described. In the left lane, after an initial 10-min incubation with ANCA F(ab')2, goat anti-human (GAH) F(ab')2 antibody fragment (50 µg/ml) was added for an additional 10 min. Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were then stripped and reprobed for total Syk. F(ab')2 fragments and intact IgG from two PR3-ANCA and two MPO were compared. A representative blot is shown.

 
ANCA-Induced Syk Phosphorylation Is Diminished by Fc{gamma}RIIa, Fc{gamma}RIIIb, and CD18 Blockade
We and others have previously reported that ANCA-induced SO anion release can be reduced by Fc{gamma} receptor blockade (6, 9, 10 ). Further evidence for Fc{gamma} receptor involvement in ANCA-mediated Syk phosphorylation was sought by preincubating TNF-{alpha}–primed neutrophils with anti-Fc{gamma}RIIa (Fab IV.3) or anti-Fc{gamma}RIIIb (F(ab')2 3G8) antibody fragments. Preincubation with either antibody fragment attenuated ANCA-induced Syk phosphorylation, supporting the involvement of both low-affinity receptors in this response, although neither fragment alone nor both in combination completely inhibited Syk phosphorylation (Figure 3A). Similarly, we confirmed that ANCA-induced SO production was partially inhibited by blockade of either Fc{gamma}RIIa or Fc{gamma}RIIIb (Figure 3B). SO anion release at 30 min induced by a representative PR3-ANCA IgG fraction = 5.58 ± 1.75 nmol SO/105 cells; with Fc{gamma}RIIa blockade SO release = 2.30 ± 0.821 nmol SO/105 cells (60% ± 4.9% inhibition); with Fc{gamma}RIIIb blockade SO release = 2.50 ± 1.04 nmol SO/105 cells (57 ± 12% inhibition); and with combined Fc{gamma}RIIa and Fc{gamma}RIIIb blockade SO release = 1.00 ± 1.04 nmol SO/105 cells 0.522 nmol (84 ± 5.0% inhibition). SO anion ± SEM is based on the mean of three experiments using different neutrophil donors.



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  		Figure 3. (A) ANCA-induced Syk phosphorylation is diminished by low-affinity Fc{gamma} receptor blockade, whereas homotypic Fc{gamma} receptor cross-linking phosphorylates Syk. Freshly isolated neutrophils from healthy donors were primed with TNF-{alpha} (80 IU/ml) for 10 min, then Fab IV.3 anti-CD32 (Fc{gamma}RIIa) antibody (5 µg/ml), F(ab')2 3G8 (Fc{gamma}RIII) antibody (5 µg/ml), or an equal volume of PBS was added and incubation continued for an additional 5 min. Finally, PR3-ANCA (200 µg/ml) or F(ab')2 goat anti-mouse (GAM; 50 µg/ml) were added. The reaction was stopped after the indicated time, and Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were stripped and reprobed for total Syk. (B) ANCA-induced superoxide (SO) anion release is diminished by low-affinity Fc{gamma} receptor blockade. Freshly isolated neutrophils from healthy donors were primed with TNF-{alpha} (80 IU/ml) for 10 min, then Fab IV.3 anti-CD32 (Fc{gamma}RIIa) antibody (5 µg/ml), F(ab')2 3G8 (Fc{gamma}RIII) antibody (5 µg/ml), or an equal volume of PBS was added and incubation continued for an additional 5 min. Cells were then transferred to flat-well plates and stimulated with PR3-ANCA IgG (200 µg/ml). SO anion release was measured by cytochrome c reduction as described.

 
Syk is also involved in integrin-mediated leukocyte responses and associates with the cytoplasmic domain of CD18 in adherent neutrophils (31). Furthermore, the {beta}2 integrin Mac-1 (CD11b/CD18) may transduce signals from Fc{gamma}RIIIb that has no cytoplasmic domain (15). ANCA activation of neutrophils is reported to be CD18 dependent (8). We therefore examined the effect of CD18 blockade on Syk activation. Preincubation with the murine anti-CD18 blocking antibody (MHM23) reduced Syk phosphorylation in response to ANCA (Figure 4A). Fragments of CD18 antibody were not available. To attempt to exclude the possibility that MHM23 exerted its inhibitory effect by blocking Fc{gamma} receptors rather than CD18, we tested murine IgG1 directed against another neutrophil surface marker, CD45, as a control (Figure 4A). FACS analysis confirmed that there was considerably greater binding to neutrophils of both anti-CD18 IgG1 and anti-CD45 IgG1 than control IgG1 (data not shown). Neither anti-CD45 IgG1 nor irrelevant murine IgG1 (not shown) treatment attenuated ANCA-induced Syk phosphorylation, supporting a specific role for CD18 in ANCA-induced signaling. Thus, ANCA-induced Syk phosphorylation involves two classes of low-affinity Fc{gamma} receptor and the {beta}2 integrin antigen CD18. To determine whether the effects of low-affinity Fc{gamma} receptor and CD18 blockade were additive, we preincubated neutrophils with a combination of blocking antibodies before being exposing them to ANCA. However, this combination did not increase the attenuation of ANCA-induced Syk phosphorylation above that seen with either Fc{gamma} receptor or CD18 blockade alone (Figure 4B).



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  		Figure 4. ANCA-induced Syk phosphorylation requires CD18 ligation. (A) Freshly isolated neutrophils from healthy donors were primed with TNF-{alpha} (80 IU/ml) for 10 min, then anti-CD18 IgG1 (10 µg/ml) or anti-CD45 murine IgG1 (10 µg/ml) were added and incubation continued for an additional 5 min. Finally, ANCA (200 µg/ml) were added. The reaction was stopped after 10 min, and Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were stripped and reprobed for total Syk. Two different ANCA were tested, and a representative blot is shown. (B) Freshly isolated neutrophils from healthy donors were primed with TNF-{alpha} (80 IU/ml) for 10 min, then PBS, anti-CD18 IgG1 (10 µg/ml), anti-CD32 Fab plus anti-CD16 F(ab')2 (5 µg/ml each), or a combination of all three antibodies was added and incubation continued for an additional 5 min. Finally, ANCA (200 µg/ml) were added. The reaction was stopped after 10 min, and Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were stripped and reprobed for total Syk. Two different ANCA were tested, and a representative blot is shown. The ratio of tyrosine phosphorylated Syk to total Syk (PY:Syk) was calculated after densitometric measurement of relevant bands.

 
Phosphorylation of Syk has also been reported in direct response to rising intracellular concentrations of reactive oxygen species (32). For excluding the possibility that ANCA-induced Syk phosphorylation was secondary to activation of the respiratory burst, Syk was immunoprecipitated from neutrophils that were pretreated with 5 µM diphenyleneiodonium chloride (an NADPH oxidase inhibitor) before exposure to ANCA. Under these conditions, which completely inhibited SO anion release, Syk still became tyrosine phosphorylated (data not shown), suggesting that the response resulted directly from receptor activation.

Fc{gamma}RI Blockade Attenuates ANCA-Induced SO Release by Patient Neutrophils but Does Not Modulate ANCA-Induced Syk Phosphorylation
Neutrophils express the high-affinity Fc{gamma} receptor Fc{gamma}RI (CD64) after overnight culture with IFN-{gamma} and in vivo in various inflammatory conditions, including active ANCA-associated vasculitis (17, 33 ). We therefore explored the involvement of Fc{gamma}RI in ANCA-mediated signaling. Neutrophils from three separate patients with active ASV were isolated and immediately primed with TNF-{alpha} in the presence or absence of anti-Fc{gamma}R antibody fragments before being exposed to PR3-ANCA isolated from a different patient (Figure 5). ANCA-induced SO anion release from patient neutrophils was diminished by Fc{gamma}RI blockade (40 ± 5.9% inhibition for 30 min of ANCA stimulation). Importantly, ANCA-induced SO anion release from patient-derived neutrophils was also partially inhibited by combined low-affinity Fc{gamma} receptor (RIIa and RIIIb) blockade (64 ± 12% inhibition for 30 min of ANCA stimulation). We next assessed the effects of Fc{gamma}RI blockade on ANCA-induced Syk phosphorylation. Freshly isolated neutrophils from healthy donors exhibited variable, low-level expression of Fc{gamma}RI by FACS analysis, which was not increased by TNF-{alpha} priming (data not shown). Cross-linking Fc{gamma}RI using mouse anti-Fc{gamma}RI F(ab')2 antibody and goat anti-mouse F(ab')2 fragments did not induce Syk phosphorylation on these cells (Figure 6A). Fc{gamma}RI expression in this instance probably reflects minor degrees of priming/activation during isolation but suggests that the surface-expressed receptor is not functionally active. When cells were cultured overnight with IFN-{gamma}, Fc{gamma}RI expression was upregulated. Trypan blue staining plus dual-color FACS analysis for Annexin V and Fc{gamma}RI indicated that Fc{gamma}RI high-expressing cells remained viable (data not shown). Furthermore, directly cross-linking Fc{gamma}RI induced Syk phosphorylation in these cells (Figure 6A). In these circumstances, Fc{gamma}RI would also be available for ligation by ANCA-Fc, and our data on SO anion release from patient neutrophils indicate that Fc{gamma}RI participates in the ANCA response. However, we did not detect any inhibition of ANCA-induced Syk phosphorylation after preincubation of IFN-{gamma}–treated neutrophils with the F(ab')2 anti-Fc{gamma}RI antibody (Figure 6A). Similarly, using freshly isolated neutrophils from a patient with active ANCA-associated vasculitis, direct Fc{gamma}RI cross-linking induced Syk phosphorylation but Fc{gamma}RI blockade did not diminish ANCA-induced Syk phosphorylation (Figure 6B). The cause of this discrepancy between SO anion release and Syk phosphorylation is discussed below (see the Discussion section).



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  		Figure 5. ANCA-induced SO anion release from fresh patient-derived neutrophils is diminished by both low- and high-affinity Fc{gamma} receptor blockade. Neutrophils were isolated from patients with active ANCA-associated vasculitides (ASV), suspended in HBH (2 x 106/ml), and immediately primed with TNF-{alpha} (80 IU/ml) at 37°C for 10 min. Cells were then treated with PBS, Fab anti-CD32 plus F(ab')2 anti-CD16 (5 µg/ml each), or F(ab')2 anti-CD64 (10 µg/ml) for an additional 5 min. Finally, cells were transferred to flat-well plates and stimulated with PR3-ANCA IgG (200 µg/ml). SO anion release was measured by cytochrome c reduction. Results are expressed as mean percentage inhibition of the ANCA response and SEM of the mean for three experiments using different neutrophil donors, each activated with the same PR3-ANCA.

 


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  		Figure 6. Syk phosphorylation is unaffected by Fc{gamma}RI blockade. (A) Neutrophils from healthy donors were used fresh or incubated overnight in Iscove’s modified Dulbecco’s medium containing 10% autologous serum and 400 IU/ml IFN-{gamma}. Subsequently, neutrophils (2 x 107/ml) were suspended in HBH at 37°C and primed with TNF-{alpha} (80 IU/ml) for 10 min, then F(ab')2 anti-CD64 (Fc{gamma}RI) antibody (10 µg/ml) or an equal volume of PBS was added and incubation continued for an additional 5 min. Finally, ANCA (200 µg/ml) or GAM F(ab')2 antibody fragment (50 µg/ml) was added. The reaction was stopped after 10 min, and Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were then stripped and reprobed for total Syk. Two different ANCA were tested, and a representative blot is shown. In IFN-{gamma}–treated polymorphonuclear leukocytes (PMN), F(ab')2 anti-CD64 had no effect on Syk phosphorylation in the absence of GAM (not shown). (B) Freshly isolated neutrophils (2 x 107/ml) from a patient with active ASV were suspended in HBH at 37°C, primed with TNF-{alpha} (80 IU/ml), and treated as in A.

 
Src Kinases Lie Upstream of Syk in ANCA-Induced Signaling: Syk Phosphorylation Is Inhibited by the Tyrosine Kinase Inhibitor PP2
We have reported that genistein diminishes ANCA-induced SO production (7). Therefore, we sought to examine the influence of PP2, a more selective inhibitor of Src family kinases, on ANCA-induced SO anion release (34). In separate experiments, using PR3-ANCA and MPO-ANCA, PP2 consistently inhibited ANCA-induced SO anion release over 1 h. For PR3-ANCA, SO release at 45 min was reduced from 8.3 ± 1.1 nmol SO/105 cells to 3.5 ± 0.48 nmol SO/105 cells by 1 µM PP2 (58% inhibition) and to 0.57 ± 0.87 nmol SO/105 cells by 10 µM PP2 (93% inhibition; results represent the mean of two separate experiments using different neutrophil donors). MPO-ANCA–induced SO release was similarly inhibited 47% by 1 µM PP2 and 90% by 10 µM PP2 (Figure 7A). Concurrent with its inhibitory effect on SO release, PP2 (10 µM) completely inhibited ANCA-induced Syk phosphorylation. Similarly, PP2 (10 µM) inhibited Syk phosphorylation induced by homotypic Fc{gamma}RIIa cross-linking using monoclonal antibody (Figure 7B). The importance of Src kinases in ANCA-mediated tyrosine phosphorylation was further suggested by the effect of PP2 on Cbl phosphorylation. Cbl, an adapter protein with negative regulatory functions in immunoreceptor and integrin signaling, is a substrate for Syk and Src family kinases. ANCA induced tyrosine phosphorylation of Cbl, which, moreover, was inhibited by 10 µM PP2 (Figure 7C).



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  		Figure 7. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibits ANCA-induced SO anion release and blocks ANCA-induced Syk and Cbl phosphorylation. (A) Freshly isolated neutrophils from healthy donors were preincubated with PP2 or vehicle for 30 min at 37°C in HBH, then primed with TNF-{alpha} (80 IU/ml) for 15 min. Cells were then transferred to flat-well plates and stimulated with PR3-ANCA IgG (200 µg/ml). SO anion release was measured by cytochrome c reduction as described. Result is the mean of two separate experiments using different neutrophil donors. (B) Freshly isolated neutrophils from healthy donors were treated for 30 min with PP2 (10 µM) or vehicle. Subsequently, cells were either primed with TNF-{alpha} (80 IU/ml) for 15 min then treated with ANCA (200 µg/ml) for 10 min or primed with TNF-{alpha} (80 IU/ml) for 10 min then treated with Fab IV.3 anti-CD32 (Fc{gamma}RIIa) antibody (5 µg/ml) for an additional 5 min before F(ab')2 GAM antibody (50 µg/ml) was added for 60 s. The reactions were stopped, and Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were stripped and reprobed for total Syk. Three experiments were performed, and a representative blot is shown. (C) Freshly isolated neutrophils from healthy donors were treated for 30 min with PP2 (10 µM) or vehicle, primed with TNF-{alpha} (80 IU/ml) for 15 min, and then treated with PR3-ANCA (200 µg/ml) for 10 min. The reactions were stopped, and Cbl immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were stripped and reprobed for total Cbl. Three experiments were performed, and a representative blot is shown

 
ANCA-Induced Syk Phosphorylation Is Pertussis Toxin Insensitive
We have previously reported that ANCA-induced SO release is sensitive to pertussis toxin and that both ANCA-IgG and ANCA-F(ab')2 activate pertussis toxin–sensitive GTPase activity in neutrophil membranes (10, 22). Therefore, it was pertinent to examine the sensitivity of ANCA-induced Syk phosphorylation to pertussis toxin. Preincubation of neutrophils with 2 µg/ml pertussis toxin, which maximally inhibited ANCA-induced SO production (data not shown) (10), did not influence ANCA-induced Syk phosphorylation (Figure 8). The effects of pertussis toxin on ANCA-mediated SO production are therefore likely to be parallel to or downstream of Fc{gamma} receptor recruitment and Syk activation.



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  		Figure 8. Syk phosphorylation is not affected by pertussis toxin. Freshly isolated neutrophils from healthy donors were treated with pertussis toxin (2 µg/ml) or an equal volume of vehicle for 2 h, then washed and resuspended in fresh HBH (2 x 107/ml). Neutrophils at 37°C were then primed with TNF-{alpha} (80 IU/ml) for 15 min before addition of ANCA (200 µg/ml) or PBS for 10 min. The reaction was stopped, and Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were stripped and reprobed for total Syk. Two different ANCA were tested, and a representative blot is shown.

 
Functional Consequences of ANCA-Induced Syk Activation
To explore further the functional relevance of Syk activation, we examined the effect of piceatannol, a semispecific inhibitor of Syk, on the ANCA-induced respiratory burst. Piceatannol interfered with cytochrome c reduction and was not suitable for use in the SO anion release assay. Using a chemiluminescence assay, however, we confirmed that the ANCA-induced respiratory burst was fully sensitive to piceatannol at 1 to 10 µM (Figure 9A). In addition, we determined that ANCA-induced Syk phosphorylation was partially sensitive to the tyrosine kinase inhibitor piceatannol (maximal inhibition occurred at 10 µM), whereas Syk phosphorylation in response to Fc{gamma}RIIa cross-linking was unaffected by 40 µM piceatannol (Figure 9B). This suggests that Syk phosphorylation in response to ANCA differs from Fc{gamma}RIIa cross-linking (see the Discussion section).



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  		Figure 9. ANCA-induced SO anion release and Syk phosphorylation are sensitive to the tyrosine kinase inhibitor piceatannol. (A) Freshly isolated neutrophils from healthy donors were treated for 30 min with piceatannol (1 to 50 µM) or vehicle. Subsequently, cells were primed with TNF-{alpha} (80 IU/ml) for 15 min, then treated with normal IgG, PR3-ANCA, or MPO-ANCA (200 µg/ml each) for 10 min. Luminol-derived chemiluminescence was measured as described in the Materials and Methods section. Results, expressed as % chemiluminescence compared with unstimulated neutrophils, are the mean and SEM from three separate experiments. (B) Freshly isolated neutrophils from healthy donors were treated for 30 min with piceatannol (10 to 40 µM) or vehicle. Subsequently, cells were either primed with TNF-{alpha} (80 IU/ml) for 15 min then treated with ANCA (200 µg/ml) for 10 min or primed with TNF-{alpha} (80 IU/ml) for 10 min then treated with Fab IV.3 anti-CD32 (Fc{gamma}RIIa) antibody (5 µg/ml) for an additional 5 min before F(ab')2 GAM antibody (50 µg/ml) was added for 60 s. The reactions were stopped, and Syk immunoprecipitation followed by phosphotyrosine Western blotting was performed. Blots were stripped and reprobed for total Syk. Three experiments were performed, and a representative blot is shown.

 
Tyrosine Kinases Instigate ANCA-Induced Intracellular Calcium Fluxes
Various neutrophil effector functions, including activation of the respiratory burst, are dependent on intracellular calcium concentrations (35). ANCA stimulation initiates intracellular calcium fluxes in neutrophils, and we sought to confirm whether these were tyrosine kinase-dependent because classical Fc{gamma} receptor ligation activates tyrosine kinase–regulated phospholipase C{gamma} (21, 36). ANCA-induced calcium fluxes were fully inhibited by previous incubation with PP2 (10 µM) and by piceatannol (10 µM; Figure 10, A and B). In contrast, ANCA-induced calcium fluxes were resistant to treatment with 2 µg/ml pertussis toxin (Figure 10C). Conversely, when neutrophils were activated with fMLP, a classical G protein–linked receptor ligand, the pattern of inhibition was reversed, pertussis toxin abolished the calcium flux (Figure 10D), whereas PP2 had no effect (data not shown). Tyrosine phosphorylation of phospholipase C{gamma} has been shown to require Syk, and our data suggest that the same relationship may also apply in ANCA-mediated signaling (27, 37). Despite the activation of heterotrimeric G proteins by ANCA-IgG and ANCA-F(ab')2, we found no evidence to suggest that ANCA-induced calcium fluxes involved phospholipase C{beta}, the isozyme recruited by fMLP.



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  		Figure 10. ANCA-induced calcium fluxes are inhibited by tyrosine kinase inhibitors but resistant to pertussis toxin. (A and B) Freshly isolated neutrophils from healthy donors were loaded with 2 µM FURA-2AM, washed, and suspended in HBH (1 x 106/ml). Thereafter, cells were treated with PP2 (10 µM) or piceatannol (10 µM) or an equal volume of vehicle for 30 min, then primed with TNF-{alpha} (80 IU/ml) for 15 min before being transferred to the fluorimeter. Once a stable baseline emission reading was obtained, 500 µg/ml ANCA was added. Calcium flux is represented by the 340/380 nm ratio. Each stimulus was tested in two separate experiments, and a representative trace is shown. (C and D) Freshly isolated neutrophils from healthy donors were treated with pertussis toxin (2 µg/ml) or an equal volume of vehicle for 2 h, washed, and resuspended in fresh HBH. Cells were then loaded with 2 µM FURA-2AM, washed, and suspended in HBH (1 x 106/ml). After priming with TNF-{alpha} (80 IU/ml) for 15 min, cells were transferred to the fluorimeter. Once a stable baseline emission reading was obtained, 500 µg/ml ANCA (C) or 100 nM fMLP (D) was added. Calcium flux is represented by the 340/380 nm ratio. Each stimulus was tested in three separate experiments, and a representative trace is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inappropriate neutrophil activation at intravascular and perivascular sites, with release of reactive oxygen species and granule contents, is central to the pathogenesis of endothelial damage in the small vessel vasculitides. In vitro, ANCA are clearly potent activators of primed neutrophils and very probably exert similar effects in vivo, where priming may well be initiated by concurrent inflammatory events such as infection. To date, the intracellular signaling events recruited during ANCA activation have not been studied extensively, but they are relevant to our understanding of disease pathogenesis and ultimately may direct novel therapeutic strategies. We have demonstrated that ANCA recruit the tyrosine kinase Syk and the adapter protein Cbl and trigger tyrosine kinase–dependent intracellular calcium transients. Tyrosine phosphorylation of Syk is a robust marker of its activation after immunoreceptor ligation (27, 38). Collectively, our data support the hypothesis that ANCA-mediated Syk activation is Fc{gamma} receptor dependent, because ANCA-F(ab')2 did not phosphorylate the kinase and Fc{gamma} receptor blockade attenuated it. The failure of ANCA-F(ab')2 fragments to induce Syk phosphorylation even after cross-linking with a secondary anti-human F(ab')2 fragment further supports the concept that ANCA-antigen clustering alone is insufficient to activate Syk.

Residual Syk phosphorylation after ANCA stimulation in neutrophils that were pretreated with anti-Fc{gamma} receptor antibodies probably represents receptor turnover and, in the case of Fc{gamma}RIIIb, receptor shedding, which are well recognized in neutrophils. Similarly, Fc{gamma} receptor blockade was unable to inhibit completely ANCA-induced SO anion release. The failure of ANCA-F(ab')2 fragments to phosphorylate Syk mitigates against the alternative possibility that ANCA induce Fc{gamma} receptor–independent Syk phosphorylation.

Fc{gamma}RIIa and Fc{gamma}RIIIb blockade were both effective in reducing Syk phosphorylation. Although Fc{gamma}RIIa-induced signaling is well characterized, the precise mechanism by which Fc{gamma}RIIIb signals remains unclear. Previously, it has been reported that Fc{gamma}RIIIb cross-linking augments Syk phosphorylation triggered via Fc{gamma}RIIa in a manner dependent on endogenous reactive oxygen species (32). However, ANCA-induced Syk phosphorylation was not diminished when NADPH oxidase was inhibited by diphenyleneiodonium. Initial binding of immune complexes to Fc{gamma}RIIIb, the more abundant neutrophil Fc{gamma} receptor, may also upregulate the affinity of Fc{gamma}RIIa for its ligand and therefore permit more intense activation (39). A similar mechanism could operate with regard to ANCA and would be consistent with a previous report that ANCA preferentially engage Fc{gamma}RIIIb (14). However, we found that homotypic Fc{gamma}RIIIb cross-linking led directly to Syk phosphorylation in TNF-{alpha}–primed neutrophils, which concurs with the results of other investigators who previously primed neutrophils with a higher dose of TNF-{alpha} (16, 35). The explanation for this phenomenon remains unknown, but it suggests that Fc{gamma}RIIIb can mediate Syk phosphorylation independent of Fc{gamma}RIIa and that ligation of Fc{gamma}RIIIb by ANCA-Fc may directly induce Syk phosphorylation.

The relative inhibitory effects of Fc{gamma}RIIa and Fc{gamma}RIIIb blockade on ANCA-induced Syk phosphorylation displayed some variation between donors (not shown), suggesting that one or the other receptor may be more important for a given individual. This may reflect receptor polymorphisms or other unidentified variables. It has previously been reported that monoclonal anti-PR3 and anti-MPO antibodies can activate human neutrophils derived from an Fc{gamma}RIIIb-deficient donor (40). Our data on the attenuation of ANCA-induced SO anion release and Syk phosphorylation by Fc{gamma}RIIIb blockade suggest that this receptor is functionally important. Other receptors may substitute for Fc{gamma}RIIIb when it is deficient. We used a very brief incubation with anti-Fc{gamma}RIIIb antibody fragments, which may have minimized loss of receptor blockade through shedding, and this may explain the difference between our results and earlier studies that reported little inhibitory effect from Fc{gamma}RIIIb blockade (6, 8).

Fc{gamma}RIIIb has been suggested to cooperate with Mac-1 (CD11b/CD18) in the production of the respiratory burst (15). CD18 blockade completely inhibits ANCA-induced neutrophil SO production, and CD11b blockade reduces ANCA-induced neutrophil adherence to platelets (8, 41). Our finding that CD18 blockade diminished ANCA-induced Syk phosphorylation is consistent with these reports. We cannot completely exclude the possibility that MHM23 anti-CD18 antibody was able to inhibit Syk phosphorylation by ligating Fc{gamma} receptors, but experiments using anti-CD45 antibody, which was not inhibitory, suggest that this is unlikely and favors a specific role for CD18 in ANCA signaling.

In previous studies, Syk has been co-precipitated with CD18 from adherent neutrophils and shown to redistribute to the cytoskeleton when {beta}2 integrins are cross-linked using soluble antibodies (31, 42, 43 ). A consequence of ANCA-induced antigen/receptor ligation could be the intracellular association of Syk with either the Fc{gamma}RIIa ITAM or CD18 or both. CD18 blockade might prevent ANCA-IgG–induced clustering of {beta}2 integrins with target antigens and Fc{gamma} receptors or inhibit homotypic adhesion of neutrophils. It was of interest that the combination of Fc{gamma}RIIa/RIIIb and CD18 blockade did not provide additional reduction in ANCA-induced Syk phosphorylation. Examining the capacity of ANCA to induce Syk phosphorylation in CD18-deficient neutrophils would be informative, because we could not confirm that CD18 blockade was complete.

The high-affinity Fc{gamma} receptor Fc{gamma}RI was functionally active on IFN-{gamma}–treated neutrophils from healthy donors and on freshly isolated patient neutrophils because homotypic cross-linking induced Syk phosphorylation. Moreover, Fc{gamma}RI blockade diminished ANCA-induced SO anion release from patient neutrophils. To our knowledge, this is the first report to identify a role for Fc{gamma}RI in ANCA signaling, although it has previously been demonstrated that the receptor is upregulated on patient neutrophils (17, 44 ). It is noteworthy that low-affinity Fc{gamma} receptor blockade continued to have a significant inhibitory effect on ANCA-induced SO anion release in patient neutrophils. However, Fc{gamma}RI blockade failed to diminish ANCA-induced Syk phosphorylation, raising a number of possibilities. There may be redundancy among Fc{gamma} receptors for Syk activation, such that ANCA is able to overcome Fc{gamma}RI blockade and maximally phosphorylate Syk via Fc{gamma}RIIa or Fc{gamma}RIIIb. This does not preclude a role for Fc{gamma}RI in ANCA-induced Syk phosphorylation but suggests that its role is not an obligate one in contrast to Fc{gamma}RIIa and Fc{gamma}RIIIb. Fc{gamma}RIIIb remains the most abundant Fc{gamma} receptor on neutrophils, even after IFN-{gamma} treatment (33). It may be that Fc{gamma}RI is unable to participate in ANCA-induced Syk phosphorylation because of the nature of the interaction between ANCA antigens and Fc{gamma} receptors. If ANCA antigens do form unique heterodimers with Fc{gamma} receptors, then there may be specific features of the Fc{gamma} receptor or its location that Fc{gamma}RI does not share with Fc{gamma}RIIa and Fc{gamma}RIIIb. However, Fc{gamma}RI-induced Syk phosphorylation and the association of Syk with the Fc{gamma}-chain that transduces Fc{gamma}RI signals have been clearly documented (44). Therefore, ANCA-induced Fc{gamma}RI signaling might well be expected to involve Syk, and it is more likely that we were unable to discern the effect of Fc{gamma}RI blockade on ANCA-induced Syk phosphorylation because Western blotting is only semiquantitative and we could not separate {gamma}-chain–associated Syk from Fc{gamma}RIIa/RIIIb-associated Syk.

Studies that have used the specific Src tyrosine kinase inhibitor PP2 indicated that ANCA-induced Syk phosphorylation is dependent on Src family kinases. This is analogous to the classical model of Fc{gamma}RIIa-mediated signaling, wherein Lyn is held to be the Src kinase constitutively associated with the receptor and responsible for tyrosine phosphorylation of the ITAM and probably of Syk itself.

The functional importance of Src kinase activation was confirmed by the inhibition of ANCA-induced SO production by PP2. Furthermore, this extends previous data on the effects of another tyrosine kinase inhibitor, genistein, on ANCA-induced neutrophil activation (7). Genistein may have nonspecific effects through its action as a competitive antagonist for ATP binding, whereas PP2 is a highly specific Src kinase inhibitor (34).

We next sought to test the functional significance of ANCA-induced Syk activation. Piceatannol is a stilbene, which is believed to compete with substrate for binding to the catalytic site of Syk. At a concentration equivalent to the IC50 for Syk (10 µM), piceatannol has been demonstrated to have minimal effect on Src family kinase activity (45, 46). Therefore, piceatannol is an appropriate tool with which to study the consequences of Syk activation, although doubts have been raised over its specificity, which may limit the interpretation of its effects (47). Other tools that are often used to examine the influence of Syk on leukocyte responses could not be readily applied in our investigations. Interspecies’ variation in neutrophil Fc{gamma} receptor expression and neutrophil resistance to antisense oligonucleotide uptake restrict the utility of genetic approaches to modulating Syk activity when studying human neutrophils. Accepting these limitations, it remains probable that Syk is functionally important in ANCA-induced neutrophil activation, and we attempted to examine its influence.

To this end, low concentrations of piceatannol (1 to 10 µM) were shown to inhibit completely the respiratory burst triggered by PR3-ANCA and MPO-ANCA. Furthermore, ANCA-induced Syk phosphorylation was partially sensitive to piceatannol (10 µM), whereas it had no effect on Syk phosphorylation resulting from homotypic Fc{gamma}RIIa cross-linking by monoclonal anti-receptor antibody. This suggests that Syk may be partially autophosphorylated during ANCA signaling but is exclusively tyrosine phosphorylated by Src kinases during homotypic Fc{gamma}RIIa cross-linking. Complete inhibition of ANCA-induced Syk phosphorylation was not evident, even with 100 µM piceatannol (not shown), indicating that non–piceatannol-sensitive mechanisms are also involved. Whereas the ANCA-induced respiratory burst was inhibited by 1 µM piceatannol, we detected only marginal effects of this concentration of piceatannol on ANCA-induced Syk phosphorylation (not shown). There is good evidence that piceatannol inhibits Syk activity at lower concentrations, but we cannot exclude the alternative possibility that its effects on the respiratory burst represent inhibition of some other unidentified kinase in addition to Syk (48). Syk phosphorylation, in neutrophils stimulated by urate crystals, has also been reported to be differentially sensitive to piceatannol (48). Urate crystals are believed to recruit Fc{gamma}RIIIb and CD18 to signal. In platelets activated by anti-CD9 monoclonal antibodies, Fc{gamma}RIIa phosphorylation is sensitive to piceatannol, whereas it is piceatannol insensitive when Fc{gamma}RIIa is directly cross-linked (19). Anti–CD-9 antibodies constitute an interesting parallel because it is suggested that they signal by forming heterodimers of CD9 with Fc{gamma}RIIa (19). ANCA may heterodimerize PR3 or MPO with Fc{gamma} receptors. This could also be consistent with our previous finding that ANCA signaling differed from Fc{gamma} receptor cross-linking in terms of phospholipase D and phosphatidylinositol 3-kinase activation (10).

Pertussis toxin attenuates ANCA-induced O production, and we have determined that ANCA stimulate membrane-associated, pertussis toxin–sensitive heterotrimeric G proteins (10, 22). Classical, pertussis toxin–sensitive G protein–linked receptor ligands, such as fMLP, are known to activate NADPH oxidase and recruit Src family tyrosine kinases, although not Syk (47). However, it has also been reported that the G protein–linked thrombin receptor can trigger delayed phosphorylation of Syk and Fc{gamma}RIIa in platelets (49). Therefore, it was conceivable that ANCA-induced Syk phosphorylation would be pertussis toxin sensitive. However, this was not the case, and Syk phosphorylation was equivalent in untreated neutrophils and those that were incubated with pertussis toxin at doses that maximally inhibited ANCA-induced SO production and completely inhibited fMLP-induced SO production. ANCA-induced GTPase activity is also triggered by both ANCA-IgG and ANCA-F(ab')2, whereas Syk phosphorylation was induced only by ANCA-IgG. Therefore, our data suggest that engagement of ANCA antigens on the neutrophil plasma membrane and ANCA-Fc engagement of Fc{gamma} receptors induce distinct but complementary signals, which both contribute to maximal NADPH oxidase activation.

Given the dual activation of Syk via Fc{gamma} receptors and of heterotrimeric G proteins via antigen engagement, the previously demonstrated ANCA-induced intracellular calcium transients could have been activated by either phospholipase C{beta} or phospholipase C{gamma} (21). The specific sensitivity of ANCA-induced calcium fluxes to both PP2 and piceatannol coupled with their resistance to pertussis toxin suggests that ANCA activate phospholipase C{gamma} and not phospholipase C{beta}. The mechanism by which pertussis toxin–sensitive heterotrimeric G proteins contribute to the ANCA-induced NADPH oxidase activation remains to be determined. The effects of PP2 and pertussis toxin on fMLP–induced calcium transients, which are known to be solely phospholipase C{beta} dependent, support the specificity of these inhibitors in this context. We did not directly demonstrate that ANCA-induced calcium fluxes depended on Syk, but the effects of piceatannol and the established involvement of PLC{gamma} and Syk in Fc{gamma} receptor–mediated fluxes support the hypothesis that ANCA do indeed recruit PLC{gamma} via Syk (37, 50).

In summary, we report that ANCA-IgG induce phosphorylation of the tyrosine kinase Syk in human neutrophils, via Fc{gamma} receptors and CD18. Activity of Src family tyrosine kinases is a prerequisite for ANCA-induced stimulation of NADPH oxidase as evidenced by the effects of PP2 on SO anion release. Furthermore, PP2 blocks ANCA-induced Syk phosphorylation. Given the central role of Syk in both the Fc{gamma} receptors and {beta}2 integrin–mediated respiratory burst, it is likely that Syk represents a central component of the signaling pathways recruited by ANCA that lead to inappropriate neutrophil activation. Tyrosine phosphorylation of the regulatory protein Cbl and tyrosine kinase–dependent calcium transients also occur downstream of the ANCA-Fc{gamma} receptor interaction. Co-ligation of antigens and receptors by ANCA may form novel heteromers and confer different characteristics on Syk recruitment compared with conventional Fc{gamma} receptor cross-linking. Identifying the ANCA-activated neutrophil signaling pathways provides new insights into the pathogenesis of small vessel vasculitis and could suggest new treatments that will interrupt ANCA–neutrophil interactions.


   Acknowledgments
 
P.H. was funded by a Sheldon Fellowship awarded by West Midlands NHS Research & Development Executive, UK. M.J.O.W. and C.O.S.S. are supported by the Wellcome Trust, UK.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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