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Blood, 1 September 2001, Vol. 98, No. 5, pp. 1448-1455
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Antineutrophil cytoplasm autoantibodies from patients
with systemic vasculitis activate neutrophils through distinct
signaling cascades: comparison with conventional Fc
receptor ligation
Anne Ben-Smith,
Stephen K. Dove,
Ashley Martin,
Michael J. O. Wakelam, and
Caroline O. S. Savage
From the Renal Immunobiology, MRC Centre for Immune
Regulation, the Department of Biochemistry, and the Institute for
Cancer Studies, The Medical School, University of Birmingham, United
Kingdom.
 |
Abstract |
In systemic vasculitis, interactions between antineutrophil
cytoplasm autoantibodies (ANCAs) and neutrophils initiate endothelial and vascular injury. ANCAs directed against either myeloperoxidase (MPO) or proteinase 3 (PR3) can activate cytokine-primed neutrophils by
binding cell surface-expressed MPO or PR3, with the concurrent engagement of Fc receptors (FcR). Because roles for phospholipase D (PLD) and phosphatidylinositol 3 kinase (PI3K) have been demonstrated in FcR activation of neutrophils, this study investigated the hypothesis that ANCA stimulation of neutrophils involved a similar engagement of FcR and activation of PLD and PI3K. Pretreatment of
tumor necrosis factor (TNF)  -primed neutrophils with antibodies against FcRII and FcRIII inhibited MPO-ANCA and PR3-ANCA induced superoxide generation, confirming that FcR ligation is involved in
ANCA-mediated neutrophil activation. However, although stimulation of
TNF--primed neutrophils by conventional FcR ligation, either using antibody-mediated cross-linking of FcR or aggregated IgG, induced PLD activation, ANCA stimulation did not. Moreover, although ANCA-induced neutrophil activation results in significant PI3K activation as assessed by phosphatidylinositol 3,4,5-triphosphate generationconventional FcR ligation, but not ANCA, activates the p85/p110 PI3K subtype. Inhibition of ANCA-induced superoxide generation with pertussis toxin suggests that ANCAs activate the p101/p110 PI3K isoform. In addition, the kinetics of activation of
protein kinase B differs between conventional FcR ligation and ANCA
stimulation of neutrophils. These results demonstrate that though
ligation of FcRIIa and FcRIIIb may be necessary, it is likely
that ANCAs require other membrane cofactors for neutrophil activation.
(Blood. 2001;98:1448-1455)
© 2001 by The American Society of Hematology.
| |
Introduction |
Antineutrophil cytoplasm autoantibodies (ANCAs)
directed against azurophilic granule proteins of polymorphonuclear
cells are pathogenic in patients with specific forms of systemic
vasculitis, namely Wegener granulomatosis, microscopic polyangiitis and
Churg-Strauss syndrome.1 Two main types of ANCAs have been
described, those directed against proteinase 3 (PR3) and those directed
against myeloperoxidase (MPO).2 In vitro, ANCAs can
activate cytokine-primed neutrophils, causing an oxidative burst,
degranulation, production of interleukin-1 , and damage to
endothelial cells.3 Thus, they are implicated in
the initiation of the endothelial and vascular damage
associated with these vasculitides.4
Priming of neutrophils with cytokines such as tumor necrosis factor
(TNF)-, as probably occurs in vivo during episodes of infection or
inflammatory disease, induces the translocation of target antigens (PR3
and MPO) from the cytoplasm to the extracellular surface, where they
are accessible to autoantibody binding.5,6 Binding of the
antibodies then triggers signaling events that lead to neutrophil
activation. At present, there is much debate as to whether these
signaling events are initiated by ANCA Fab binding to MPO or
PR37,8 and additionally, or even exclusively, proceed
through antibody binding to FcRIIa and FcRIIIb
receptors.9-11 Although the signaling mechanism(s) by
which ANCAs stimulate neutrophils have not been fully elucidated, we
have recently demonstrated the involvement of tyrosine kinases and
protein kinase C in ANCA-mediated neutrophil activation.12
Freshly isolated human neutrophils express 2 forms of Fc receptors
(FcR), the glycosylphosphatidylinositol-linked FcRIIIb (CD16) and
FcRIIa (CD32), a conventional type I transmembrane protein of 40 kd.13,14 Cross-linking of these receptors in vitro, either
by using aggregates of immunoglobulin (Ig) G as immune complexes or
specific monoclonal antibodies, results in the activation of neutrophil
phagocytosis, degranulation, and respiratory burst.15-17
Although the signaling events have not yet been fully unraveled,
signaling through FcRIIa and FcRIIIb, either independently or in
combination, is accompanied by the activation of phospholipase C (PLC)
by a tyrosine kinase-dependent, G-protein-independent
mechanism18 and a rise in intracellular calcium
level.15,16 It has also been reported that cross-linking of FcR induces the activation of phosphatidylinositol 3 kinase (PI3K)19 and the recruitment of the serine-threonine
kinase protein kinase B (PKB), a major downstream target of
PI3K.20 Moreover, a recent study demonstrated a role for
phospholipase D (PLD) in immune complex-induced FcR activation of
neutrophils.21 Thus, the signaling mechanisms induced by
ANCAs and FcR activation in neutrophils show many similarities,
suggesting that ANCA signaling proceeds through Fc receptor ligation.
Other neutrophil activators that induce superoxide releasesuch as the
chemoattractant formyl-methionyleucylphenylalanine (fMLP)activate
several intracellular phospholipid signaling pathways including
phospholipase A2, PLC, PI3K, and PLD after ligation of
surface receptors.22 PLD catalyzes the hydrolysis of
phosphatidylcholine to phosphatidate (PtdOH) and choline. PtdOH can
then be converted to diacylglycerol (DAG), a potent second messenger in
a variety of cell signaling processes, though recent studies have
suggested that PtdOH may play a more important role than DAG in
the activation of the NADPH oxidase system and subsequent oxidative
burst.23
Two types of PI3K enzymes have been described in neutrophils, a class
IA form consisting of a p110 catalytic subunit and a p85
regulatory subunit that binds phosphorylated tyrosine residues and a
class IB form, PI3K , consisting of a p110 catalytic
subunit and a p101 regulatory subunit stimulated by G-protein
subunits and also by ras.24 These enzymes
catalyze the 3-phosphorylation of phosphatidylinositol 4,5-bisphosphate
(PIP2) to phosphatidylinositol 3,4,5-triphosphate
(PIP3). PIP3 is a potent second
messenger25 and is thought to play an important role in the
activation of the neutrophil NADPH oxidase system26,27 and
in Fc receptor-mediated phagocytosis.18
The aim of this study was, therefore, to determine the Fc receptor
requirements for ANCAs and subsequently to investigate the roles of PLD
and PI3K in ANCA-induced neutrophil activation and superoxide
production, comparing these results with conventional FcR ligation
(using either monoclonal antibody-mediated FcR cross-linking or
aggregated IgG) and fMLP activation of neutrophils. Detailed
understanding of ANCA-induced neutrophil signaling may ultimately
provide targets for therapeutic intervention and control of
neutrophil-mediated acute severe vasculitis.
| |
Materials and methods |
Isolation of neutrophils
Blood was obtained from healthy volunteers, and neutrophils were
separated as previously described using centrifugation over a Percoll
discontinuous density gradient.28
Preparation of ANCAs and normal IgG
Serum samples were obtained from 3 MPO-ANCA-positive patients,
3 PR3-ANCA-positive patients, and 2 healthy volunteers. IgG was
prepared from these serum samples using selection on protein G-Sepharose columns (Amersham Pharmacia Biotech, St Albans, United Kingdom) with endotoxin-free materials. The protein content of these
samples was estimated, and these samples were then used at either 200 or 250 µg/mL in subsequent assays. In previous studies, both these
concentrations of ANCA, but not normal, IgG have been shown to induce
substantial superoxide generation in 105 neutrophils (200 µg/mL) or 4 × 106 neutrophils (250 µg/mL),
respectively, following dose-response analyses (data not shown). IgG
samples were free of endotoxin contamination, as assessed by a Limulus
amebocyte assay (Sigma, Poole, United Kingdom). All assays were
made in the absence of serum to avoid endotoxin-induced binding and
activation and at equivalent cell densities in either polystyrene
microtiter plates or polystyrene sample tubes to ensure identical
conditions for cell adhesion.
Generation of aggregated IgG
IgG samples from 3 MPO-ANCA-positive patients, 3 PR3-ANCA-positive patients, and 2 healthy volunteers were resuspended
at a concentration of 20 mg/mL in phosphate-buffered saline (PBS) and
heated to 63°C for 20 minutes as previously
described.29
Superoxide assay
Freshly isolated neutrophils were resuspended at a concentration
of either 5 × 105 or 107 cells/mL in 10 mM
Hepes-buffered Hanks balanced salt solution (HBH) and primed with 2 ng/mL TNF- for 15 minutes at 37°C. Aliquots of 105
cells (in microtiter plates) or 4 × 106 cells (in sample
tubes) were then stimulated either with 1 µM fMLP (Sigma), 50-250 µg/mL MPO-ANCA, PR3-ANCA, normal IgG, or heat-aggregated IgG samples.
Superoxide release was measured over 15 minutes using an assay based on
the superoxide dismutase inhibitable reduction of ferricytochrome c and
performed as described previously.30 In some experiments,
wortmannin (Sigma) or LY294002 (Sigma) was added to the primed
neutrophil samples, and the cells were equilibrated for 5 to 10 minutes
before stimulation.
Pertussis toxin treatment was performed by treating freshly isolated
neutrophils, at a concentration of 107 cells/mL, with
pertussis toxin (Calbiochem-Novabiochem, Nottingham, United Kingdom) or
vehicle (100 mM NaPO4, 50 mM NaCl, pH 7) for 2 hours at
37°C. The cells were diluted to 5 × 105 cells/mL and
primed with 2 ng/mL TNF- for 15 minutes at 37°C before stimulation.
For the assessment of superoxide production in FcR cross-linked
neutrophils, 5 × 105/mL cells were primed with 2 ng/mL
TNF- for 15 minutes at 37°C and incubated with either 1 µg/mL
IV.3 Fab (recognizing FcRII), 1 µg/mL 3G8 F(ab')2
(recognizing FcRIII) (Medarex, Annandale, NJ), or both. Monoclonal
antibodies were removed by centrifugation, and cells were resuspended
in HBH containing 2 ng/mL TNF-. Cross-linking was achieved by
treating the cells with 10 µg/mL F(ab')2 fragments of
goat anti-mouse Fab (GAM F(ab')2; Sigma). Superoxide
release was then measured over 15 minutes. Optimal concentrations of
IV.3, 3G8, and GAM F(ab')2 antibodies had been previously
determined from dose-response analyses.
To examine anti-FcR monoclonal antibody blocking of ANCA superoxide
production, 5 × 105/mL primed neutrophils were treated
with either 1 µg/mL IV.3 Fab (anti-FcRII), 1 µg/mL 3G8
F(ab')2 (anti-FcRIII), or both. Aliquots of
105 cells were then stimulated with 200 µg/mL MPO-ANCA,
PR3-ANCA IgG, or 200 µg/mL normal IgG, and superoxide release was
measured over 15 minutes. All samples were tested as 6 replicates, and all the experiments were repeated 3 times.
PLD assays
Two methods were used to assess the involvement of PLD signaling
pathways in anti-FcR, fMLP, and ANCA-stimulated neutrophils.
Assessment of phosphatidylbutanol formation.
Freshly isolated neutrophils were resuspended at a concentration of
2 × 107/mL in 25 mM Hepes buffer containing 125 mM NaCl,
10 mM glucose, 1 mM EGTA, and 1 mg/mL bovine serum albumin (BSA). The
cells were labeled with 37 kBq/mL
1-O-[3H]-alkyl-sn-glyceryl-3-phosphorylcholine
(NEN, Hounslow, United Kingdom) for 30 minutes at 37°C, washed, and
resuspended in HBH. Butan-1-ol (0.3%) was added to aliquots of
4 × 106 cells, which were then primed with 2 ng/mL
TNF- for 15 minutes at 37°C and stimulated with 1 µM fMLP,
50-250 µg/mL MPO-ANCA, PR3-ANCA, normal IgG, or heat-aggregated IgG
samples or by cross-linking FcR1 µg/mL IV.3 Fab, 1 µg/mL 3G8 F(ab')2, or both for 5 minutes followed by 10 µg/mL GAM F(ab')2. The reaction was stopped by the
addition of chloroform and methanol (1:2), the lipids were separated,
and levels of [3H]phosphatidylbutanol were assessed as
previously described.31 All samples were tested in
triplicate, and the results were expressed as percentage dpm pbut
(percentage [3H] incorporation in phosphatidylbutanol
fraction/[3H] incorporation in total lipids measured).
All experiments were repeated 3 times.
Determination of PtdOH and DAG generation.
Levels of PtdOH and DAG were assessed after fMLP and ANCA stimulation
of neutrophils. Cells were labeled with 37 kBq/mL
1-O-[3H]-alkyl-sn-glyceryl-3-phosphorylcholine,
primed with 2 ng/mL TNF-, and stimulated as before. Levels of PtdOH
and DAG were then measured as described.31 All samples
were tested in triplicate, and the results were expressed as either % dpm PtdOH or % dpm DAG, (ie, percentage [3H]
incorporation in PtdOH or DAG fraction/[3H] incorporation
in total lipids measured).
Measurement of PIP3 generation.
Freshly isolated neutrophils were resuspended at a concentration of
5 × 107/mL in HBH containing 0.1% fatty acid-free BSA
(Sigma). The cells were labeled with 74 MBq/mL
[32P]orthophosphate (Amersham Life Science, Little
Chalfont, United Kingdom) for 70 minutes at 37°C, washed twice, and
resuspended in HBH-BSA. Aliquots of 2 × 107 cells (400 µL) were then primed with 2 ng/mL TNF- for 15 minutes at 37°C
and were stimulated with either 1 µM fMLP, 250 µg/mL MPO-ANCA, PR3-ANCA, or normal IgG. The reaction was stopped by the addition of
1.5 mL ice-cold chloroform and methanol 1:2, and the lipids were
extracted, diacetylated, and separated by high-performance liquid
chromatography (HPLC) as previously described.32
As an internal standard, the HPLC runs were spiked with
[3H]GroPInsPns
(including
[3H]GroPInsP3) derived
from [3H]inositol-radiolabeled yeast inducibly expressing
a farnesylated p110 subunit.33 All samples were tested in
duplicate, and the experiment were repeated twice.
Assessment of PKB activation.
Freshly isolated neutrophils at a concentration of
2 × 107/mL in HBH were primed with TNF- and
stimulated with 1 µM fMLP, 250 µg/mL MPO-ANCA, PR3-ANCA, and normal
IgG or by FcR cross-linking 1 µg/mL IV.3 Fab or 1 µg/mL 3G8
F(ab')2 (or both) followed by 10 µg/mL GAM
F(ab')2. The reaction was stopped with the addition of
4 × ice-cold PBS. Cells were then lysed in 1% (vol/vol) Triton X-100, 20 mM Hepes (pH 7.2), 2.5 mM EGTA, 2 mM EDTA, 1 mM NaCl, and 5 mM MgCl2 containing 2 mM phenylmethylsulfonyl fluoride, 40 µg/mL leupeptin, 40 µg/mL aprotinin, 2 mM sodium orthovanadate, 50 mM sodium fluoride, and 40 mM sodium pyrophosphate (lysis buffer). Lysates were cleared by centrifugation, and their protein content was
assessed using the Bio-Rad method (Bio-Rad, Hemel Hempstead, United Kingdom).
Levels of PKB and phospho-PKB in anti-PKB immunoprecipitates prepared
from the lysates were assessed as follows: 100 µg protein was
incubated with 5 µL anti-PKB antibody (rabbit polyclonal Akt antibody; New England BioLabs, Hitchin, United Kingdom) overnight at
4°C. An aliquot of 65 µL Protein G-Sepharose (Sigma) was then added to the lysates for 2 hours at 4°C. The immunoprecipitate was
washed at 4°C with 2 × 1 mL lysis buffer, 1 × 1 mL 0.5 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.6, 1 × 1 mL 0.5 M LiCl, 0.1 M Tris-HCl,
pH 8, and 2 × 1 mL lysis buffer. Samples were resuspended in 30 µL
2 × sodium dodecyl sulfate (SDS) loading buffer, heated to 100°C
for 5 minutes, subjected to electrophoresis in 10% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Immunodetection of PKB or phospho-PKB was performed using either the PKB antibody or a
phospho-PKB antibody (rabbit polyclonal phospho-Akt antibody; New
England Bio-Labs) followed by an antirabbit peroxidase-linked secondary
antibody (Amersham Life Science) as described in the data sheets and as
visualized using enhanced chemiluminescence (Amersham). Experiments
were repeated 3 times using lysates prepared on separate occasions with
MPO-ANCA or PR3-ANCA IgG samples from different patients.
Assessment of PI3K activity.
PI3K activity in anti-p85 immunoprecipitates prepared from 100 µg
TNF--primed, unstimulated, and stimulated (with 1 µM fMLP, 50-250 µg/mL MPO-ANCA, PR3-ANCA, normal IgG, heat-aggregated IgG, or by
cross-linking FcR (1 µg/mL IV.3 Fab or 1 µg/mL 3G8
F(ab')2 for 5 minutes followed by 10 µg/mL GAM
F(ab')2) neutrophil lysates was assessed essentially as
previously described34 using 1.5 mg/mL propidium iodide
(Sigma) in 0.5% cholate as substrate. After separation of the lipids
by thin-layer chromatography, PtdIns(3)P levels were quantified using a
PhosphorImager (Molecular Dynamics, Chesham, United Kingdom). All
samples were tested in duplicate, and all experiments were repeated at
least 3 times.
Statistical analysis
Results are expressed as means ± SEM. For each data set,
results from all the replicate experiments were pooled. Statistical significance was evaluated using analysis of variance (Minitab v.13.1;
Minitab, State College, PA) to assess whether there was a
significant overall effect of treatment and time. If 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  .05
was considered statistically significant.
| |
Results |
Superoxide production in fMLP-, ANCA-, and anti-FcR-
stimulated neutrophils
ANCA stimulation of TNF--primed neutrophils has previously
been reported to induce superoxide production.10,12 Our
results, shown in Figure 1, confirmed
that stimulation of such neutrophils with fMLP, MPO-ANCA, PR3-ANCA IgG,
or conventional FcR engagement (using either cross-linking
anti-FcR antibodies or heat-aggregated IgG) lead to significant
superoxide production.
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| Figure 1.
Superoxide production in neutrophils stimulated with
fMLP or ANCA IgG or by conventional FcR ligation using either
cross-linking antibodies or aggregated IgG.
Superoxide production in 105 neutrophils primed with 2 ng/mL TNF- and stimulated with (A) 1 µM fMLP, 200 µg/mL MPO-ANCA
IgG, 200 µg/mL PR3-ANCA IgG, or 200 µg/mL normal IgG, or with (B)
primed, unstimulated cells or cells stimulated with 1 µg/mL IV.3
(FcRII XL), 1 µg/mL 3G8 (FcRIII XL), or both monoclonal antibodies
(FcRII+III XL), followed by cross-linking with 10 µg/mL GAM
F(ab')2, or with (C) primed, unstimulated cells or cells
stimulated with either 1 µM fMLP, 200 µg/mL normal IgG, or 200 µg/mL heat-aggregated IgG for 1 ( ), 5 ( ), or 15 ( ) minutes.
(Note different scales in the 3 panels.) All experiments were repeated
3 times using neutrophils from different donors, 3 different MPO-ANCA
and PR3-ANCA IgG preparations, and 2 different normal IgG samples
(native and heat-aggregated) and 6 replicates of all samples. Results
show mean ± SEM of data pooled from all 3 experiments.
|
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After stimulation with fMLP, superoxide production was evident after 1 minute and significantly increased after 5 minutes (Figure 1A). By
contrast, stimulation with 3 different MPO-ANCA or PR3-ANCA samples
consistently produced lower initial levels of superoxide (0-1 nmol at 1 to 5 minutes), which then increased over 15 minutes. Stimulation with
equivalent amounts of normal IgG produced significantly lower levels of
superoxide than seen with any of the ANCA IgG samples (Figure 1A;
normal IgG, 0.61 ± 0.07 nmol at 15 minutes, P < .05).
Conventional FcR engagement of TNF--primed neutrophils,
either using anti-FcR cross-linking antibodies or aggregated IgG, also led to significant stimulation of superoxide production over the
course of the assay (Figure 1B-C). Incubation of primed neutrophils with either IV.3 Fab (FcRII XL) or 3G8 F(ab')2 (FcRIII XL),
followed by cross-linking with GAM F(ab')2, led to initial
low levels of superoxide production after 1-minute stimulation (less
than 0.3 nmol), which further increased over 15 minutes to 1.2 to 1.5 nmol (P < .001). Stimulation of primed neutrophils with
cross-linked IV.3 Fab and 3G8 F(ab')2 antibodies together
(FcRII+III XL) led to a faster and greater superoxide burst, with
levels of 1.09 ± 0.12 nmol at 5 minutes (P .001;
Figure 1B). Stimulation of neutrophils with the monoclonal antibodies
only or the GAM cross-linking antibody alone produced significantly
lower levels of superoxide over the assay period (maximum, 15-minutes
values: IV.3 Fab 0.47 ± 0.11 nmol superoxide; 3G8
F(ab')2 0.43 ± 0.17 nmol superoxide; GAM
F(ab')2 0.52 ± 0.13 nmol superoxide;
P < .05). Use of 200 µg/mL heat-aggregated normal IgG
resulted in significant superoxide generation after 1 minute compared
with nonaggregated normal IgG, with levels increasing to 2.6 ± 0.27
nmol after 15 minutes (P < .001; Figure 1C). Even at
lower concentrations of heat-aggregated normal IgG (50-100 µg/mL),
significant superoxide was evident after 1-minute stimulation (eg,
stimulation with 50 and 100 µg/mL led to levels of 0.61 ± 0.05 and
0.67 ± 0.19 nmol, respectively, compared to 0 ± 0.14 and
0 ± 0.2 nmol, respectively, in nonaggregated IgG samples;
P < .01). Stimulation of primed neutrophils with 200 µg/mL aggregated MPO-ANCA or PR3-ANCA IgG also led to increased superoxide generation, with levels of 1.5 to 3 nmol (aggregated ANCA
IgG) compared with 1.3 to 1.9 nmol superoxide (native ANCA IgG) after
15 minutes (P < .05).
Anti-FcR monoclonal antibody pretreatment blocks ANCA-induced
superoxide production in primed neutrophils
To clarify the role for FcR in ANCA-mediated neutrophil
activation, TNF--primed neutrophils were pretreated with either IV.3 Fab (anti-FcRII) or 3G8 F(ab')2 (anti-FcRIII)
monoclonal antibodies before stimulation with ANCA. Used individually,
either antibody reduced superoxide production by 15% to 61% in
response to stimulation with 3 different MPO-ANCA and PR3-ANCA samples (P .001). Pretreatment of neutrophils with both
anti-FcR antibodies together resulted in even greater inhibition
(46%-85%) of the superoxide response to all the MPO-and PR3-ANCA
samples (P < .001). These results, shown in Figure
2, confirmed a role for both FcRIIa and FcRIIIb in ANCA-mediated neutrophil activation.
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| Figure 2.
Effect of FcR blocking on ANCA IgG superoxide
response.
Aliquots of 105 neutrophils were primed with 2 ng/mL
TNF- and pretreated with either 1 µg/mL IV.3 (anti-FcRII, ), 1 µg/mL 3G8 (anti-FcRIII, ), or both monoclonal antibodies
(anti-FcRII+III, ) before stimulation with 200 µg/mL MPO-ANCA or
PR3-ANCA IgG for 15 minutes. All experiments were repeated 3 times
using neutrophils from different donors, 3 different MPO-ANCA and
PR3-ANCA preparations, and 6 replicates of all samples. Results show
mean ± SEM of data pooled from all 3 experiments.
|
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ANCA stimulation fails to activate PLD, as measured by either
phosphatidylbutanol or lipid messenger production
Signal transduction pathways activated by ANCAs were compared with
those activated by conventional FcR ligation (using either anti-FcR cross-linking antibodies or aggregated IgG) and fMLP stimulation. PLD activation was measured using an accumulating trap
assay for the detection of both small and slowly accumulating levels of
PLD products. This assay has been fully described
elsewhere,30 is well validated, and is definitive for
cellular PLD activation. fMLP stimulation led to rapid and significant
PLD activation of TNF--primed neutrophils, with [3H]
incorporation in phosphatidylbutanol fraction at 0.23% ± 0.02% after 1 minute (P < .001) and remaining elevated
throughout the assay period (0.52% ± 0.03% at 15 minutes,
P < .001) (Figure 3A). By
contrast, stimulation with 3 different MPO-ANCA or PR3-ANCA preparations or with normal IgG did not result in any PLD activation above background levels (primed, unstimulated cells) or indeed for 100 minutes (data not shown) during the time-course of the assay (Figure 3A), in spite of significant superoxide production (as
shown in Figure 1A).
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| Figure 3.
PLD activation in neutrophils stimulated with fMLP or
ANCA IgG or by conventional FcR ligation using either cross-linking
antibodies or aggregated IgG.
Phosphatidylbutanol production in 4 × 106 neutrophils
primed with 2 ng/mL TNF- and stimulated with (A) 1 µM fMLP, 250 µg/mL MPO-ANCA, PR3-ANCA, or normal IgG, or with (B) primed,
unstimulated cells, or cells stimulated with 1 µg/mL IV.3 (FcRII XL),
1 µg/mL 3G8 (FcRIII XL), or both monoclonal antibodies (FcRII+III
XL), followed by cross-linking with 10 µg/mL GAM F(ab')2,
or with (C) primed, unstimulated cells or cells stimulated with either
1 µM fMLP, 250 µg/mL normal IgG, or 250 µg/mL heat-aggregated IgG
for 1 (), 5 (), or 15 () minutes. All experiments were
repeated 3 times using neutrophils from different donors, 3 different
MPO-ANCA and PR3-ANCA IgG preparations, and 2 different normal IgG
samples (native and heat-aggregated) and triplicates of all samples.
Results show mean ± SEM of data pooled from all 3 experiments.
|
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To confirm PLD activation of neutrophils by fMLP stimulation, PtdOH and
DAG levels were measured. Stimulation of neutrophils with 1 µM
fMLP for 15 minutes led to an initial rise in PLD-induced PtdOH
after 5 minutes ([3H] incorporation in PtdOH fraction,
0.712% ± 0.018%, P < .001) and a subsequent
increase in DAG levels ([3H] incorporation in DAG
fraction at 10 minutes, 2.001% ± 0.152%, P < .001).
By contrast, stimulation with either 250 µg/mL MPO-ANCA, PR3-ANCA, or
normal IgG did not result in significant PLD-induced PtdOH or DAG
production over the same time.
FcR ligation using anti-FcR cross-linking antibodies or
aggregated IgG stimulates PLD activation in primed neutrophils
In contrast to the lack of PLD activation after ANCA stimulation,
cross-linking of either FcRIIa (FcRII XL) or FcRIIIb (FcRIII XL)
activated PLD (Figure 3B), with levels of 0.61% ± 0.03% (FcRII XL), P .001) and 0.79% ± 0.06%
[3H]pbut incorporation (FcRIII XL),
P .001) at 15 minutes. Cross-linking of both FcR
(FcRII+III XL) led to an increase in PLD activation, with levels of
0.52% ± 0.02% [3H]pbut incorporation at 1 minute
(P < .001), increasing to 1.00% ± 0.03% after 15 minutes (P < .001, Figure 3B). Stimulation of neutrophils
with monoclonal antibodies only or GAM cross-linking antibody alone did
not lead to significant PLD activation at any time.
Ligation of FcR with 50 to 250 µg/mL heat-aggregated IgG also
induced significant PLD activity. Use of 250 µg/mL aggregated normal
IgG resulted in increased levels of PLD activity after 1 minute
(0.26% ± 0.03% [3H]pbut incorporation) in contrast
to nonaggregated normal IgG (0.20% ± 0.03%
[3H]pbut incorporation), with significantly elevated
levels at 15 minutes (aggregated IgG, 0.49% ± 0.05%
[3H]pbut incorporation compared to nonaggregated IgG,
0.25% ± 0.01% [3H]pbut incorporation;
P < .001) (Figure 3C). Even at lower concentrations (50-100 µg/mL), aggregated normal IgG was able to induce significant PLD activity (eg, stimulation with 50 and 100 µg/mL aggregated IgG
for 5 minutes resulted in 0.24% ± 0.01 and 0.26% ± 0.02%
[3H]pbut incorporation, respectively, compared to
0.18% ± 0.02% and 0.19% ± .02%, respectively, in
nonaggregated IgG samples; P < .05). Aggregation of both
PR3-ANCA and MPO-ANCA IgG also led to significant PLD activation within
1 minute of stimulation, with levels of 0.24% to 0.26%
[3H]pbut incorporation (aggregated ANCA IgG) compared
with 0.18% to 0.19% [3H]pbut incorporation (native ANCA
IgG, P < .05).
fMLP-, ANCA-, and anti-FcR-induced superoxide production can be
blocked by PI3K inhibitors
Superoxide production by TNF--primed neutrophils stimulated
with fMLP, ANCA IgG, or antibody-mediated FcR cross-linking was
inhibited by pretreatment with LY294002 (Figure
4). Use of this compound led to
dose-dependent inhibition of the fMLP-induced superoxide response at
concentrations of 0.5 to 50 µM (Figure 4A) and significant inhibition
of ANCA IgG-induced superoxide responses (Figure 4A; fMLP response at 5 µM, 23% ± 1.0% inhibition, P < .005; ANCA
response at 0.5 µM, 81%-95% inhibition,
P .01). Pretreatment of neutrophils with LY294002 also
significantly suppressed superoxide production in antibody-mediated
FcR cross-linked neutrophils (Figure 4B). There was 70% to 100%
inhibition of anti-FcRII (FcRII XL) and anti-FcRIII (FcRIII
XL)-induced responses (P .05) and 37% inhibition of
FcRII+III XL responses at 0.5 µM concentrations (P < .01) (Figure 4B). These data suggested that
neutrophil stimulation by fMLP, ANCA, and FcR cross-linking
recruited PI3K because the IC50 for inhibition of PI3K by
LY294002 is 1.4 µM.35
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| Figure 4.
Effect of LY294002 on fMLP, ANCA, and anti-FcR
superoxide production.
Aliquots of 105 neutrophils were primed with 2 ng/mL
TNF- and treated with 0.5 (), 5 (), 10 (), or 50 µM
LY294002 ( ) before stimulation with (A) 1 µM fMLP, 200 µg/mL
MPO-ANCA, or PR3-ANCA IgG or (B) 1 µg/mL IV.3 (FcRII XL), 1 µg/mL
3G8 (FcRIII XL), or 1 µg/mL of both anti-FcR monoclonal antibodies
(FcRII+III XL) followed by 10 µg/mL GAM F(ab')2
cross-linking antibody for 15 minutes. All experiments were repeated 3 times, with 6 replicates of all samples. Results are shown as mean
percentage inhibition of superoxide production ± SEM of data
pooled from all 3 experiments.
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Use of another PI3K inhibitor, wortmannin, which selectively inhibits
PI3K activity at concentrations of 5 to 10 nM,27 confirmed these observations. Pretreatment with wortmannin led to significant inhibition of both fMLP and ANCA IgG-induced superoxide responses at
concentrations of 2 to 25 nM (fMLP response at 5 nM, 56% ± 9.9%
inhibition, P < .001; ANCA response at 5 nM, 56%-62%
inhibition, P .001). Pretreatment of neutrophils with
wortmannin also significantly suppressed superoxide production in
neutrophils stimulated by FcR cross-linking, with 48% to 56%
inhibition of anti-FcRII (FcRII XL) and anti-FcRIII (FcRIII XL)
responses (P .05) and 64% inhibition of
anti-FcRII+FcRIII-induced responses at 5 nM concentration
(P < .03).
ANCA stimulation results in PIP3 formation
To confirm the activation of PI3K, levels of its product,
PIP3, were measured. Stimulation of TNF--primed
neutrophils with fMLP led to significant PIP3 generation at
30 seconds (Figure 5A), with a 3-fold
increase above background levels maintained over 15 minutes (Figure 5B;
P < .03). Stimulation with both MPO- and PR3-ANCA also
resulted in PIP3 generation, but the initial response was
slower, with significantly greater levels (6-fold increase,
P < .01) seen after 15 minutes of stimulation
(Figure 5B).
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| Figure 5.
PIP3 production in fMLP and ANCA-stimulated
neutrophils.
2 × 107 neutrophils were primed with 2 ng/mL TNF- and
stimulated either with 1 µM fMLP, 250 µg/mL MPO-ANCA, 250 µg/mL
PR3-ANCA, or 250 µg/mL normal and PIP3 production
measured. (A) Representative HPLC trace showing inositol phosphates
derived from 30-second fMLP-stimulated 32P-labeled
neutrophils ( ) superimposed on trace 3H internal
standards (). (B) Graph showing percentage dpm in 32P
PIP3 fraction after 30-second and 15-minute stimulation.
All samples were tested in duplicate, and results show pooled data from
2 experiments. indicates cells; , fMLP; , MPO-ANCA;  ,
PR3-ANCA; , normal IgG.
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fMLP, ANCA, and FcR cross-linking activate protein
kinase B
Protein kinase B (PKB) is a downstream target following PI3K
activation. Stimulation of TNF--primed neutrophils with fMLP, MPO-ANCA, and PR3-ANCA and by cross-linking FcR each led to an increase in phospho-PKB levels. However, the kinetics differed with
each stimulus, with fMLP stimulation giving the earliest detectable
activation of PKB, at 30 seconds, with levels returning to background
by 15 minutes (Figure 6A). Activation of
PKB occurred later, after ANCA stimulation, with increased
phosphorylation detectable after 1 minute and sustained at 15 minutes
(Figure 6A). Cross-linking of either FcRIIa or FcRIIIb
individually gave significant phosphorylation of PKB at 1 minute
(FcRIIIb) and 15 minutes (FcRIIa), whereas cross-linking of both
receptors resulted in a greater, but more transient, increase in
phospho-PKB (Figure 6B).
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| Figure 6.
PKB activation in fMLP, ANCA, and FcR cross-linked
neutrophils.
Phospho-PKB (pPKB) levels were assessed in blots of PKB
immunoprecipitates from primed neutrophils stimulated with either (A) 1 µM fMLP, 250 µg/mL MPO-ANCA, PR3-ANCA, or normal IgG or (B) 1 µg/mL anti-FcRII, 1 µg/mL anti-FcRIII, or 1 µg/mL of both
anti-FcR monoclonal antibodies followed by 10 µg/mL GAM
F(ab')2 cross-linking antibody for 30 seconds, 1 minute,
and 15 minutes. Molecular weight (kd), as assessed using Rainbow
markers (Amersham), are shown on the left. Levels of PKB are also shown
to indicate equal amounts of protein within the samples. These results
are representative of 3 independent experiments.
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FcR ligation and fMLP stimulation, but not ANCA, activates
p85 PI3K
To examine the PI3K isoform activated by fMLP, ANCA, and FcR
ligation (using either cross-linking antibodies or aggregated IgG), the
in vitro activity of PI3K in anti-p85 immunoprecipitates was
determined. Stimulation of TNF--primed neutrophils with fMLP induced significant activation of p85 PI3K 30 seconds after stimulation (Figure 7A; P < .04).
Stimulation with 3 different preparations of MPO-ANCA or PR3-ANCA,
however, failed to induce p85 PI3K activation above that seen in cells
stimulated with normal IgG at any time examined (Figure 7A). By
contrast, individual cross-linking of FcRII (FcRII XL) and FcRIII
(FcRIII XL) using monoclonal antibodies led to significant p85 PI3K
activation after 1 minute (Figure 7B; P < .05). Antibody
cross-linking of both FcR together (FcRII+III XL) also induced
significant p85 activity, which was evident after 30 seconds of
stimulation (Figure 7B; P < .04). Ligation of FcR using 250 µg/mL heat-aggregated normal IgG also induced significant p85 activity, which was evident after 30 seconds and increased over 15 minutes (Figure 7C).
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| Figure 7.
p85 PI3K activity in neutrophils stimulated with fMLP or
ANCA IgG or by conventional FcR ligation using either cross-linking
antibodies or aggregated IgG.
p85 PI3K activation measured by in vitro kinase assays in anti-p85
immunoprecipitates from aliquots of 4 × 106 neutrophils
primed with 2 ng/mL TNF- and stimulated with either (A) 1 µM fMLP,
250 µg/mL MPO-ANCA, PR3-ANCA, or normal IgG or (B) 1 µg/mL IV.3
(FcRII XL), 1 µg/mL 3G8 (FcRIII XL), or 1 µg/mL both anti-FcR
monoclonal antibodies (FcRII+III XL) followed by 10 µg/mL GAM
F(ab')2 cross-linking antibody or (C) primed, unstimulated
cells or cells stimulated with either 1 µM fMLP, 250 µg/mL normal
IgG, or 250 µg/mL heat-aggregated IgG for 30 seconds (), 1 minute
(), and 15 minutes (). All experiments were repeated 3 times
using neutrophils from different donors, 3 different MPO-ANCA and
PR3-ANCA IgG preparations, and 2 different normal IgG samples (native
and heat-aggregated) and duplicates of all samples. Results show
mean ± SEM of data pooled from all 3 experiments.
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Compared to native ANCAs or normal IgG samples, aggregation of both
PR3-ANCA and MPO-ANCA IgG also led to significant p85 activity, with
levels of 0.6 to 0.8 PtdIns3P U at 30 seconds and 1.2 to
1.4 U after 15 minutes. (Figure 7C). Even at lower concentrations
(50-100 µg/mL), aggregated ANCA IgG and aggregated normal IgG samples
were able to induce significant p85 activity (eg, stimulation with 50 and 100 µg/mL aggregated IgG for 30 seconds and resulted in 0.55-0.7 and 0.62-0.9 PtdIns(3)P U, respectively, compared to 0.12-0.23 and
0.2-0.3 PtdIns(3)P U, respectively, in nonaggregated IgG samples;
P < .04). These data showed that native ANCAs do not
recruit the p85/p110 isoform of PI3K, suggesting that an alternative
isoform of PI3K, possibly G  -activated p101/p110,
is recruited.
fMLP and ANCA, but not anti-FcR-induced, superoxide generation
can be blocked with pertussis toxin
To determine whether ANCA activation of neutrophils involves
heterotrimeric G-protein activation, which could then lead to G-mediated recruitment of p101/p110, the effect of pertussis toxin (a Gi/o protein inhibitor) on superoxide
generation was investigated. Pretreatment with 0.1 to 2 µg/mL
pertussis toxin led to significant inhibition of both the fMLP and the
ANCA-induced oxidative burstfor example, at 0.1 µg/mL, there is
100% inhibition of fMLP response and 70% to 75% inhibition of
MPO-ANCA and PR3-ANCA responses (P < .005). By contrast,
pretreatment with equivalent concentrations of pertussis toxin had no
significant effect on the superoxide response seen after conventional
FcR cross-linking.
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Discussion |
Systemic vasculitis is an important cause of pulmonary hemorrhage
and rapidly progressive glomerulonephritis with acute renal failure.
Where ANCAs are present in such diseases, these autoantibodies have
been implicated in the initiation of neutrophil-mediated vascular and
endothelial cell damage.1-3 The results presented in this
study demonstrate that the ANCA-induced oxidative burst from primed
neutrophils can be blocked by pretreatment with anti-FcR antibodies.
This implies that FcR engagement is involved in ANCA activation of
primed neutrophils. Despite the involvement of FcR, we demonstrate
for the first time that the activation events evoked by ANCAs are
distinct from those induced by conventional FcR ligation, either by
cross-linking of FcR using monoclonal antibodies or by aggregated
IgG, because ANCAs stimulate neither activation of PLD nor production
of the PLD-induced messengers PtdOH and DAG. Our results also show that
ANCAs activate PI3K and subsequently PKB. However, ANCAs use a PI3K
isoform distinct from conventional FcR ligation, which activates the
p85/p110 isoform.
Blocking either FcRIIa or FcRIIIb with anti-FcR antibodies led
to significant inhibition (up to 65%) of the ANCA-induced superoxide
response, whereas the combined use of both anti-FcRII and
anti-FcRIII antibodies could abolish the response. Our results confirm and extend previous findings that ANCA activation of
neutrophils requires both FcRIIa and FcRIIIb
engagement9-11; indeed, we have previously shown that
F(ab')2 fragments of ANCAs alone are insufficient to
produce a superoxide response.12 Therefore, these
results demonstrate an important role for FcR in the initiation of
ANCA-mediated signal transduction pathways.
The activation of neutrophils by ANCAs was dependent on PI3K. The
fungal metabolite wortmannin, which selectively inhibits PI3K at
concentrations of 5 to 10 nM,27 inhibited ANCA-induced superoxide production at a concentration of 5 nM. Moreover, the structurally and functionally distinct PI3K inhibitor
LY29400235 also blocked ANCA-mediated superoxide
production. Stimulation of neutrophils with ANCAs and fMLP and
cross-linking them with FcR also led to the activation of PKB, but
with differing kinetics. PKB, a major downstream target of PI3K, has
been reported to be involved in the neutrophil respiratory burst and
exocytosis.20 Altogether, these results suggest a role for
PI3K in the ANCA-induced oxidative burst. This is supported by a recent
study showing that PI3K-generated PIP3 enhances superoxide
production in TNF--primed neutrophils.36 PI3K
activation has been shown to mediate the activation of Rac by certain
growth factors,37 resulting in membrane ruffling. Thus,
ANCA-stimulated PI3K could also play a role in the shape change and
actin reorganization reported previously,38 which may lead
to neutrophil sequestration within the microcirculation and contribute
to the vascular damage associated with systemic vasculitis.
Two types of PI3K enzymes have been described in human neutrophils, the
phosphotyrosine-associated class IA p85/p110 isoform and
the G-protein-activated class IB p101/p110
isoform.24 Stimulation with fMLP is known to strongly
activate this latter isoform,39 though there have been
reports that fMLP can also activate p85/p110 PI3K.40 In
this study, stimulation with fMLP led to the activation of p85 PI3K,
though to a lesser degree than stimulation of neutrophils by
conventional FcR ligation using either cross-linking antibodies or
aggregated IgG. Stimulation with ANCA, on the other hand, failed to
activate p85 PI3K, though we could demonstrate significant production
of the PI3K-generated second messenger PIP3. Thus, it is
likely that ANCAs activate the p101/p110 PI3K isoform. In
support of this, pretreatment of neutrophils with pertussis |
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Abstract
Introduction