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*Renal Immunobiology, Department of Biochemistry, and Institute for Cancer Studies, MRC Centre for Immune Regulation, The Medical School, University of Birmingham, Birmingham, UK
Correspondence to Professor C.O.S. Savage, Renal Immunobiology, MRC Centre for Immune Regulation, The Medical School, University of Birmingham, Birmingham, B15 2TT, UK. Phone: 44-121-414-7042; Fax: 44-121-414-6840;
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receptors (FcR). The contribution of antigen binding via ANCA F(ab')2 fragments to signaling has been unclear. This study shows that both ANCA IgG and F(ab')2 fragments of ANCA IgG induce significant GTPase activity, which could be blocked with pertussis toxin and anti-Gi protein antibodies. Pertussis toxin inhibited ANCA IgG-induced superoxide generation but was without effect on superoxide production after conventional FcR ligation. ANCA F(ab')2 fragments did not induce superoxide generation. ANCA IgG activated PI 3-kinase–generating PIP3, activated protein kinase B (PKB), and p21ras; activation of each mediator was inhibited with pertussis toxin, but PI3K and PKB were not activated by ANCA IgG F(ab')2 fragments. Intact ANCA IgG induced tyrosine phosphorylation, whereas F(ab')2 fragments did not, and ANCA IgG-mediated superoxide generation was inhibited with genistein. Both genistein and pertussis toxin together completely abrogated the ANCA-induced oxidative burst. Genistein also inhibited ANCA IgG-induced PIP3 generation and p21ras activation. These data implicate a novel ANCA IgG stimulated signaling pathway that involves both F(ab')2-mediated antigen binding and Fc-mediated FcR ligation in cooperative interactions between Gi proteins and tyrosine kinases that facilitates activation of downstream mediators. E-mail: C.O.S.Savage@bham.ac.uk. |
Introduction |
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–primed neutrophils, causing an oxidative burst, accelerated apoptosis, and damage to vascular endothelial cells (1), suggesting that ANCA IgG plays a role in disease pathogenesis or amplification of vascular injury (2).
Priming of neutrophils with cytokines such as TNF-, as is likely to occur in vivo during episodes of infection or inflammatory disease, induces translocation of target antigens (PR3 and MPO) from cytoplasmic granules to the extracellular surface, where they are accessible to autoantibody binding (3–4). Binding of the antibody triggers signaling events that lead to neutrophil activation. It is contentious whether these signaling events are initiated by ANCA F(ab')2 binding to MPO or PR3 (2,5) or proceed via antibody binding to FcRIIa and FcRIIIb receptors (6–8) or both. We and others have reported that Fc receptor ligation is important for ANCA-mediated neutrophil activation (6–9), and ANCA F(ab')2 fragments are not activating (6–7,9); whereas blocking FcR engagement by pretreating with anti-FcR antibodies inhibits ANCA IgG-induced superoxide production (10).
Despite this key role for FcR ligation, we have recently demonstrated that ANCA IgG activate neutrophils in a manner distinct from activation induced by conventional FcR ligation using either cross-linking antibodies or aggregated IgG (10). ANCA IgG activation of TNF-–primed neutrophils, unlike conventional FcR cross-linking, does not activate phospholipase D (PLD) or generate phosphatidate or diacylglycerol (10). Unlike conventional FcR cross-linking, stimulation with ANCA IgG did not activate the phosphotyrosine-associated p85/p110 isoform of phosphatidylinositol (PI) 3-kinase, although there was significant production of PIP3 after ANCA IgG stimulation (10). Both ANCA IgG and conventional FcR cross-linking also activated protein kinase B (PKB/Akt), although the kinetics of the response were different (10). In this study, we have investigated the ability of ANCA IgG F(ab')2 fragments to contribute to signal transduction. The results demonstrate that ANCA IgG activate Gi-proteins, induce hydrolysis of GTP, and activate p21ras. However, ANCA IgG F(ab')2 are insufficient to recruit further downstream mediators such as PIP3 and PKB and, as previously observed (9), are unable to generate superoxide production at equivalent antigen-binding concentrations to intact ANCA IgG. Therefore, neutrophil activation by ANCA IgG seems to require binding of the intact molecule to both antigen and Fc receptors for effective signal transduction that leads to significant functional events.
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Materials and Methods |
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Preparation of ANCA and Normal IgG
Serum samples were obtained from four MPO-ANCA IgG–positive patients with microscopic polyangiitis (MPA), four PR3-ANCA IgG–positive patients with Wegener granulomatosis (WG), and three healthy volunteers. The patients fulfilled the Chapel Hill definitions for MPA and WG (12); all samples were obtained at first acute presentation. IgG was prepared from these serum samples using selection on Protein G-sepharose columns (Amersham Pharmacia Biotech, Little Chalfont, UK) with endotoxin-free materials. The protein content of these samples was estimated, and the IgG used at either 200 or 250 µg/ml (unless otherwise stated) concentrations of ANCA, but not normal, IgG that were able to induce substantial superoxide generation in 105 TNF-–primed neutrophils (200 µg/ml) or 2 x 107 TNF--primed neutrophils (250 µg/ml; data not shown). IgG samples were free of endotoxin contamination as assessed by a Limulus amoebocyte assay (Sigma-Aldrich, Poole, UK). All assays were done in the absence of serum to avoid endotoxin-induced binding and activation.
Preparation of F(ab')2 Fragments
F(ab')2 fragments were prepared from three MPO-ANCA, three PR3-ANCA, and three normal IgG samples as described previously (9), using endotoxin-free materials. F(ab')2 fragments were still able to bind to alcohol-fixed neutrophils, as shown by indirect immunofluorescence. An enzyme-linked immunosorbent assay (ELISA) using MPO- and PR3-coated wells (Binding Site, Birmingham, UK) was used to compare the binding activity of the parent ANCA IgG and F(ab')2 fragments as described previously (13) and used in subsequent assays at concentrations corresponding to equivalent antigen-binding capacity.
Treatment with Pertussis Toxin or Genistein
Freshly isolated neutrophils were resuspended at a concentration of 107 cells/ml and treated with 0.1 to 2 µg/ml pertussis toxin (Calbiochem-Novabiochem, Nottingham, UK) for 2 h or 150 µM genistein (Sigma) for 30 min at 37°C. The cells were resuspended at an appropriate concentration and primed with 2 ng/ml TNF- (National Institute for Biological Standards and Control, Potters Bar, UK) for 15 min at 37°C before stimulation. These concentrations of inhibitors have been shown previously to abolish ANCA IgG-induced tyrosine phosphorylation (genistein) (9) and fMLP-induced superoxide generation (pertussis toxin) (10) with no associated cytotoxicity.
Superoxide Assay
Pertussis toxin and genistein-treated or untreated neutrophils were resuspended at a concentration of 5 x 105 cells/ml in Hanks’ Balanced Salt Solution (Sigma-Aldrich) containing 10 mM HEPES pH 7.4 (HBH) and primed with 2 ng/ml TNF- for 15 min at 37°C. Aliquots of 105 cells were then stimulated either with 1 µM fMLP (Sigma-Aldrich), 200 µg/ml MPO-ANCA, PR3-ANCA, or normal IgG or F(ab')2 fragments at concentrations found to give equivalent antigen-binding compared with the corresponding whole parent antibody. Superoxide release was measured over 15 min using a microtitre plate assay based on the superoxide dismutase inhibitable reduction of ferricytochrome c as described previously (14).
All samples were tested in triplicate and all the experiments were repeated three times using different neutrophil donors.
Generation of Neutrophil Membrane Samples
Pertussis toxin-treated or untreated neutrophils were resuspended at a concentration of 2 x 107/ml in HBH and primed with 2 ng/ml TNF- for 15 min at 37°C. Neutrophil membrane preparations were then generated as described previously (9). The protein content was assessed using the Bio-Rad method (Bio-Rad, Hemel Hempstead, UK), and the samples were stored at -80°C.
GTPase Activity Assay
The rate of hydrolysis of GTP was assessed in aliquots of 10 µg membrane samples after incubation with fMLP, MPO-ANCA, PR3-ANCA, normal IgG, or F(ab')2 fragments for 20 min at 37°C as described previously (15).
In other experiments, membrane samples were pre-incubated for 1 h at 37°C with 1/100 dilutions of either an anti-G0 -subunit antibody or an anti-Gi-1,2,3 antibody (both rabbit polyclonal antisera, Calbiochem-Novabiochem) before assessment of GTPase activity.
All samples were tested in triplicate, and experiments were repeated three times using membranes that were prepared on separate occasions using different neutrophil donors and with whole ANCA or normal IgG or F(ab')2 fragments prepared from different patients and healthy volunteers.
Measurement of PIP3 Generation
Pertussis toxin or genistein treated or untreated neutrophils were resuspended at a concentration of 5 x 107/ml in HBH containing 0.1% fatty acid–free BSA (Sigma-Aldrich). The cells were labeled with 2 mCi/ml [32P]orthophosphate (Amersham Pharmacia Biotech), primed with TNF-, and stimulated with 1 µM fMLP, 250 µg/ml MPO-ANCA, PR3-ANCA, or normal IgG or F(ab')2 fragments at concentrations found to give equivalent antigen-binding compared with the corresponding whole parent antibody. PIP3 generation was determined as described previously (10). All samples were tested in duplicate, and the experiment was repeated twice.
Preparation of Soluble Cell Lysates
Pertussis toxin-treated or untreated neutrophils were resuspended at a concentration of 2 x 107/ml in HBH and primed with 2 ng/ml TNF- and stimulated with 1µM fMLP, 250 µg/ml MPO-ANCA, PR3-ANCA, normal IgG or F(ab')2 fragments at concentrations found to give equivalent antigen-binding compared with the corresponding whole parent antibody. Cell lysates were prepared as described previously (10).
Assessment of PKB Activation
Pertussis toxin-treated or untreated neutrophils were resuspended at a concentration of 5 x 106 cells, primed with 2 ng/ml TNF-, and then treated with fMLP for 30 s or 250 µg ANCA IgG/normal IgG or equivalent F(ab')2 for 15 min at 37°C. Cells were pelleted and resuspended in an ice-cold solution of 10% TCA. After incubation on ice for 15 min, precipitated material was recovered by centrifugation at 13,000 rpm for 5 min at 4°C. Pellets were washed twice with ice-cold acetone and then dried. Samples were resuspended in 2x sample buffer (0.125 M Tris, 20% vol/vol glycerol, 4% wt/vol SDS, 0.2 M dithiothreitol, 0.025 mg/ml bromophenol blue) and boiled for 10 min. Proteins were resolved on an 8% separating gel and blotted onto polyvinylidene fluoride (PVDF). After blocking for 1 h in 5% BSA/Tris–buffered saline/0.1% Tween 20, blots were probed with an anti-phosphoPKB antibody raised against phosphoserine 473 (New England Biolabs, Hitchin, UK) overnight at a concentration of 1:1000. After application of a secondary sheep anti-rabbit HRP-conjugated antibody, bands were visualized by ECL-Plus (Amersham-Pharmacia Biotech). Blots were then stripped and reprobed for total PKB (anti-Akt, New England Biolabs) at 1:1000 overnight at 4°C. After application of a secondary sheep anti-rabbit HRP-conjugated antibody, bands were visualized by ECL plus. Experiments were repeated five times using neutrophils that were prepared on separate occasions using different donors and with intact ANCA IgG, normal IgG and F(ab')2 fragments prepared from different patients and healthy volunteers.
Ras Activation Assay
The Ras-binding domain (RBD) of human Raf-1 (amino acids 1 to 149; a generous gift from Dr. S.J. Cooke, Babraham Institute, Cambridge) was expressed in the GST-containing vector pGEX-KG using BL21 Escherichia coli. The IPTG-induced GST-fusion protein (GST-RBD) was purified from lysed bacteria using glutathione agarose.
Neutrophils were purified as described and resuspended at 5 x 106 cells/ml. Cells were treated with pertussis toxin, genistein, or vehicle followed by priming with TNF- as above. Aliquots of 2.5 x 106 cells were stimulated with either 1 µM fMLP for 30 s at 37°C or 250 µg ANCA IgG/normal IgG or equivalent ANCA F(ab')2 for 15 min at 37°C. Pelleted cells were snap frozen then resuspended on ice in lysis buffer with protease and phosphatase inhibitors (50 mM Tris [pH 7.5], 100 mM NaCl, 1% Triton X-100, 12 mM MgCl2, 1 mM EDTA [pH 8.0], 1 mM EGTA [pH 8.0], 20 mM NaF, 1 mM Na3VO4, 1 mM PNPP, 10 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml trypsin inhibitor). Extraction proceeded for 30 min, insoluble matter was removed, and lysates were incubated with 50 µl of a 50% slurry of glutathione agarose bound to GST-RBD for 30 min at 4°C. Beads were subsequently washed with PBS containing 5 mM MgCl2 and 0.1% Triton X-100, and active Ras was eluted by boiling in 2x sample buffer (0.125 M Tris, 20% vol/vol glycerol, 4% wt/vol SDS, 0.2 M dithiothreitol, 0.025 mg/ml bromophenol blue). Samples were resolved on 12% separating gel, transferred to PVDF membrane, blocked with 5% milk solution/Tris-buffered saline/0.1% Tween 20 for 3 h and exposed to anti-Ras (Upstate, Botolph Claydon, Bucks) at 0.5 µg/ml overnight at 4°C. After application of a secondary sheep anti-mouse HRP-conjugated antibody, bands were visualized by ECL plus (Amersham-Pharmacia Biotech). Blots were then stripped and reprobed with anti-GST antibody (Sigma-Aldrich) 1:5000 overnight at 4°C. After application of a secondary sheep anti-mouse HRP-conjugated antibody, bands were visualized by ECL (Amersham-Pharmacia Biotech).
Western Blotting for Phosphotyrosine Residues
Neutrophils in HBH at 2 x 107/ml were treated with 2 mM diisopropylfluorophosphate (Sigma-Aldrich) for 20 min at room temperature. After being washed once, neutrophils were resuspended in fresh HBH and primed with 2 ng/ml TNF- for 15 min at 37°C. Aliquots (100 µl) were then stimulated with ANCA-IgG (400 µg/ml) or ANCA-F(ab')2 (at the equivalent dose). At relevant time points, equal volumes of hot 2x Laemmli sample buffer (125 mM Tris-HCl [pH 6.8]; 8% SDS; 10% 
-mercaptoethanol; 17% glycerol; 5 mM orthovanadate; 20 µg/ml leupeptin, 20 µg/ml aprotinin, 0.025% bromophenol blue) were added, and samples were boiled for 7 min. Samples were subjected to SDS-PAGE and transferred to PVDF membranes for Western blotting. Blots were first blocked with 5% BSA in Tris-buffered saline (TBS)-Tween for 30 min at room temperature and then incubated overnight at 4°C with anti-phosphotyrosine antibody (PY99; Santa Cruz, Autogen Bioclear, Caln, UK) in 5% BSA in TBS-Tween. After washing, blots were incubated for 1 h at room temperature with HRP-conjugated anti-mouse Ig antibody, washed again, and visualized using ECL-Plus (Amersham Biosciences, Balfont, UK). To confirm equal protein loading, blots were stripped and reprobed with anti-p38 MAPK antibody (New England Biolabs Inc, Hitchin, Herts, UK).
Statistical Analyses
Results are expressed as means ± SEM. For each data set, results from all the replicate experiments were pooled. Statistical significance was evaluated using ANOVA (Minitab v.13.1) to assess whether there was a significant overall effect of treatment and time. Where significant effects were found, individual analyses were also performed using Tukey-Kramner multiple comparison tests (Minitab), with these results presented as probability values (P). P 
0.05 was considered to be statistically significant.
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–primed neutrophils. Stimulation of these membranes with both fMLP and ANCA IgG lead to GTPase activity (Table 1). F(ab')2 fragments of ANCA IgG at concentrations that gave equivalent antigen binding to intact ANCA IgG also induced significant membrane GTPase activity (Table 1). Stimulation with normal IgG or F(ab')2 fragments of normal IgG did not induce significant GTPase activity.
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Pertussis Toxin and Anti-Gi Antibodies Inhibit ANCA-Induced GTPase Activity
The oxidative burst stimulated by fMLP and ANCA IgG was inhibited by pretreatment of TNF-–primed neutrophils with 0.1 to 2 µg/ml pertussis toxin in a dose-dependent manner, confirming previous studies (10). Thus, treatment with 0.1 µg/ml pertussis toxin lead to 66% inhibition of the fMLP response and 55 to 70% inhibition of the ANCA IgG response (P < 0.01). Treatment with 2 µg/ml pertussis toxin gave complete inhibition of the fMLP response and up to 86% inhibition of the ANCA IgG response (P < 0.001; data not shown).
The effect of pertussis toxin on ANCA IgG-induced responses was not due to nonspecific inhibition of TNF-–induced cell surface expression of MPO or PR3, as there was no alteration detected by flow cytometry after pertussis toxin pretreatment (data not shown). This suggested a direct involvement of Gi/o proteins in ANCA IgG-mediated neutrophil activation. In contrast, pertussis toxin was without effect on the stimulation of superoxide generation by conventional FcR engagement of TNF-–primed neutrophils, either using anti-FcR cross-linking antibodies or aggregated IgG for 15 min (data not shown). Thus stimulation of neutrophils by fMLP and ANCA IgG, but not by conventional FcR engagement, recruits pertussis toxin-sensitive G-proteins.
Agonist-induced GTPase activity was inhibited by pretreatment with pertussis toxin in a dose-dependent manner (Figure 1A); 2 µg/ml pertussis toxin inhibited the fMLP response by 85% (P < 0.01), the intact ANCA IgG-induced responses by 84 to 98% (P < 0.01), and the ANCA F(ab')2 responses by 89 to 97%(P < 0.02). Pertussis toxin ADP ribosylates and thereby inhibits both G0 and Gi proteins (16). To examine which pertussis toxin-sensitive G protein was being activated, anti-G protein antibodies were included in the GTPase activity assays. Using the anti-G0 antibody, a significant reduction of the fMLP-stimulated GTPase activity was observed (inhibition by 24 ± 7%; P < 0.02; Figure 1B), but there was no significant reduction of that induced by intact ANCA IgG or ANCA IgG F(ab')2 fragments. Pretreatment with the anti-Gi1,2,3 antibody led to almost complete abrogation of the fMLP response (inhibition by 92 ± 8%; P < 0.001) and significant inhibition (79 to 88%; P < 0.02) of the GTPase activity induced by both intact and F(ab')2 fragments of ANCA IgG (Figure 1B).
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PKB is Activated in a Pertussis Toxin-Inhibitable Manner by ANCA IgG but Is not Activated by ANCA IgG F(ab')2 Fragments
Stimulation of TNF--primed neutrophils with fMLP induced rapid (30 s) phosphorylation of PKB, which then decreased to background levels by 15 min (data not shown). Phosphorylation of PKB was also stimulated by ANCA IgG, although this was slower, being detectable after 1 min (data not shown) and sustained for 15 min (Figure 3A). The F(ab')2 fragments of ANCA IgG failed to induce PKB phosphorylation at times when intact ANCA IgG induced significant PKB activation (Figure 3A), as did normal intact IgG (Figure 3B) and normal F(ab')2 fragments (data not shown). Pretreatment with 2 µg/ml pertussis toxin substantially inhibited both the fMLP stimulation at 30 s (data not shown) and the ANCA IgG-induced PKB phosphorylation at 15 min (Figure 3B). Thus, while ANCA F(ab')2 fragments can recruit pertussis toxin-sensitive Gi/o proteins, they are not adequate to activate PI 3-kinase, PKB, or indeed to mediate superoxide production.
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-primed neutrophils with fMLP induced Ras activation at 30 s (Figure 4A). Intact ANCA IgG induced active Ras at 15 min as did ANCA IgG F(ab')2 but not normal IgG. Pretreatment with pertussis toxin and genistein decreased both the ANCA IgG and the ANCA IgG F(ab')2 response (Figure 4B).
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R engagement. The overall intensity of ANCA IgG induced tyrosine phosphorylation was, however, weaker than that seen after FcR cross-linking using monoclonal antibodies (not shown), and it is possible that ANCA F(ab')2 mediate phosphorylation of certain proteins that are not detected by whole cell lysate western blotting.
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-primed neutrophils with 150 µM genistein abrogated both the fMLP and ANCA IgG-induced PIP3 generation. Consequently, both tyrosine phosphorylation and heterotrimeric G proteins are necessary for ANCA IgG-induced PI 3-kinase activation and subsequent superoxide generation.
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Discussion |
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-primed neutrophils and induce superoxide generation via a signaling pathway that involves Gi proteins. Stimulation with both ANCA F(ab')2 fragments and intact ANCA IgG induces pertussis toxin-sensitive membrane-bound GTPase activity, although only the intact ANCA IgG can induce PI 3-kinase activation, PKB activation, and superoxide generation. The generation of PIP3 by PI 3-kinase activation and the generation of superoxide can be inhibited by pretreatment with pertussis toxin and the tyrosine kinase inhibitor genistein, suggesting a cooperative stimulation through both Gi proteins and tyrosine kinases occurs. p21ras could be activated by both intact ANCA IgG and by ANCA F(ab')2 fragments and was inhibitable by both pertussis toxin and genistein, further supporting this proposal.
Superoxide generation after ANCA IgG stimulation, but not conventional FcR ligation, can be inhibited with pertussis toxin, implicating heterotrimeric G proteins in the ANCA IgG-induced respiratory burst. This is supported by both intact ANCA IgG and ANCA F(ab')2 fragments stimulating membrane-associated GTPase activity inhibitable by pertussis toxin and antibodies to Gi. Thus the G protein effects are mediated through recruitment and activation of Gi by ANCA F(ab')2 binding to its antigens (MPO or PR3) on the neutrophil cell surface. The manner by which ANCA F(ab')2 binding to its antigens recruits G proteins is unclear. Such binding may result in antigen aggregation and internalization, which could lead to Gi recruitment. Alternatively, because both MPO and PR3 are highly charged molecules, they may associate covalently with other as yet unidentified membrane components or receptors, which then recruit G proteins. Once activated by receptor-catalyzed GTP binding, the GGTP subunit dissociates from the G subunits (reviewed in reference 19), the latter can activate a large number of signaling molecules, including PI 3-kinase (20), Ras, and the MAP kinases (21–22); they are also postulated to activate various SH2-containing tyrosine kinases (reviewed in reference 19). We have shown previously that the ANCA IgG-induced oxidative burst requires activation of PI 3-kinase; however, no recruitment ANCA IgG of the phosphotyrosine-associated Class IA p85 PI 3-kinase was observed, implying a role for the G subunits-activated Class IB, isoform p101/p110 (PI 3-kinase ) (23,10). MAP kinases have also been reported to be activated by ANCA IgG (24), and the results presented here suggest this is mediated by activating p21ras.
It is of particular interest that the F(ab')2 fragments of ANCA IgG are not inert, because they activate Gi proteins and p21ras; however, they do not activate PI 3-kinase (as assessed by PIP3 generation and PKB phosphorylation) or initiate superoxide generation at concentrations that give equivalent antigen binding as intact antibody. Although ANCA IgG F(ab')2 activation of Gi proteins and p21ras may play a role in the ANCA IgG-induced respiratory burst, other signaling pathways must be recruited. Tyrosine phosphorylation of a number of neutrophil proteins follows ANCA stimulation, and genistein can fully inhibit tyrosine phosphorylation and partially inhibit the ANCA IgG-induced respiratory burst (9). However, ANCA IgG F(ab')2 fragments have less marked effects on tyrosine phosphorylation as determined by Western blotting. These observations are in accordance with the requirement for Fc receptor ligation during ANCA-mediated neutrophil activation, because FcR typically recruit tyrosine kinases (reviewed in reference 25). Figures 2 and 7 show that pertussis toxin and genistein can each abolish ANCA IgG-induced PIP3 generation and together can abolish superoxide generation. A likely interpretation of these observations is that ANCA IgG activation of neutrophils requires cooperative signaling between Gi proteins and tyrosine kinases.
How ANCA IgG-activated G proteins cooperate with tyrosine kinases is not clear, although there are precedents for cooperative signaling between G proteins and tyrosine kinases (26–30). In the monocytic U937 cell line, aggregation of FcRI led to the recruitment of heterotrimeric G proteins and activation of PI 3-kinase (31). Moreover, aggregation of FcRI was associated with p85 PI 3-kinase activity, and all PI 3-kinase activity was blocked by pretreating cells with genistein. This suggested that activation of PI 3-kinase occurred downstream of tyrosine kinases. However, we find that Gi activation occurs after antigen binding, suggesting a role for F(ab')2 rather than Fc/FcR interactions. The inhibitory effects of both genistein and pertussis toxin on PI 3-kinase activity suggest a cooperative rather than linear relationship between Gi and tyrosine kinases, this could possibly involve p21ras, which can be activated by both G protein- and tyrosine kinase-dependent routes; indeed, ANCA IgG and ANCA IgG F(ab')2 were both able to activate p21ras in a pertussis toxin- and genistein-sensitive manner.
In summary, ANCA IgG F(ab')2 activate Gi proteins, thus binding of ANCA IgG to its antigen ligands at the neutrophil cell surface is not an inert event. Whereas G-proteins are necessary for development of a full ANCA-stimulated respiratory burst, as shown by the ability of pertussis toxin to partially inhibit superoxide generation by intact ANCA IgG, activation of G proteins by F(ab')2 fragments is insufficient for development of such a respiratory burst. Both the current study as well as previous studies (6–9) indicate an important role for ligation of FcRIIa/IIIb to enable appropriate tyrosine kinase phosphorylation events and activation of PI 3-kinase and PKB. These data implicate a novel signaling pathway stimulated by ANCA IgG that involves both F(ab')2-mediated antigen binding and Fc-mediated FcR ligation in cooperative interactions between Gi proteins and tyrosine kinases. p21ras appears critical for permitting this cooperation because it was activated by both ANCA IgG and ANCA IgG F(ab')2 in a pertussis toxin- and genistein-inhibitable manner.
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subunits. Nature 369: 418–420, 1994[CrossRef][Medline]
activation of p21ras. Proc Nat Acad Sci USA 91: 12706–12710, 1994RI) activates both tyrosine kinase and G protein-coupled phosphoinositide 3-kinase isoforms. Proc Nat Acad Sci USA 95: 2169–2174, 1998This article has been cited by other articles:
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  S. L. Nolan, N. Kalia, G. B. Nash, D. Kamel, P. Heeringa, and C. O.S. Savage Mechanisms of ANCA-Mediated Leukocyte-Endothelial Cell Interactions In Vivo J. Am. Soc. Nephrol., May 1, 2008; 19(5): 973 - 984. [Abstract] [Full Text] [PDF]
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S. Brachemi, A. Mambole, F. Fakhouri, L. Mouthon, L. Guillevin, P. Lesavre, and L. Halbwachs-Mecarelli Increased Membrane Expression of Proteinase 3 during Neutrophil Adhesion in the Presence of Anti Proteinase 3 Antibodies J. Am. Soc. Nephrol., August 1, 2007; 18(8): 2330 - 2339. [Abstract] [Full Text] [PDF]
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