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RIIIb on Human Neutrophils1
*
Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294; and
Department of Pathology, State University of New York, Stony Brook, NY 11794
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R-dependent manner. Human neutrophils constitutively express
FcRIIa (CD32) and FcRIIIb (CD16), and there is clear in vitro
experimental evidence of ANCA-mediated engagement of FcRIIa.
However, direct experimental evidence of ANCA engagement of neutrophil
FcRIIIb has been obscured by technical problems related to
activation-induced receptor shedding and activation-induced expression
of receptor on the surface of neutrophils. In this study, by blocking
receptor shedding and using appropriate reporter anti-FcR mAb,
we show that human cANCA and pANCA, and murine mAb with corresponding
reactivities, can indeed engage FcRIIIb. Furthermore, our data
suggest that FcRIIIb is preferentially engaged by ANCA relative to
FcRIIa presumably due to the nearly 10-fold excess of FcRIIIb
expression relative to FcRIIa expression. These results clearly
demonstrate that the Fc region of ANCA bound to an ANCA target on the
neutrophil surface engage FcRIIIb and indicate that FcRIIIb and
FcRIIa may both be active participants in ANCA-induced neutrophil
activation. However, given the low levels of ANCA target expression on
neutrophils from patients with systemic vasculitis, FcRIIIb is
likely to play a critical role in initiating and perpetuating
ANCA-induced neutrophil activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors results in
activation of an oxidative burst and cytokine secretion in neutrophils
and monocytes (6, 7, 9, 10). While these observations do not preclude
direct ANCA-induced cell activation (11), the importance of FcR
engagement by ANCA in WG is highlighted by our recent observation that
neutrophil FcR are a genetic risk factor for severity of disease in
patients with WG (12)5.
Human neutrophils express two structurally and functionally
distinct Fc receptors, the transmembrane FcRIIa and the
glycosyl-phosphatidylinositol (GPI)-anchored FcRIIIb which is
expressed at 10-fold higher density than FcRIIa (13). While both
receptors are capable of independently inducing cell activation, recent
data have highlighted differences in the signaling properties and in
the functional responses induced by these receptors (14, 15, 16, 17). Of
particular interest in the pathogenesis of WG, signaling through
FcRIIIb induces a different neutrophil adhesive phenotype than
signaling through FcRIIa (16), and FcRIIIb is the dominant Fc
receptor responsible for the immune complex-induced oxidative burst in
neutrophils (18, 19).
Several groups have demonstrated engagement of FcRIIa by ANCA
(6, 7, 20). However, direct experimental evidence of FcRIIIb
engagement by ANCA has been obscured by activation-induced receptor
shedding and activation-induced expression of receptor on the surface
of neutrophils. To address these issues, we have used a panel of
anti-FcRIII mAb to examine the ability of neutrophil-bound ANCA
to engage and block the ligand binding site of FcRIIIb under
conditions that limit activation-induced shedding of FcRIIIb. Human
cANCA and pANCA, and murine anti-PR3 and anti-MPO mAb, do
indeed engage FcRIIIb. The ability to detect this interaction is
inversely proportional to the relative affinity of the reporting
anti-FcRIII mAb. Our results indicate that multiple FcR may
participate in neutrophil activation by ANCA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phycoerythrin (PE)-labeled F(ab')2 goat
anti-mouse IgG, anti-mouse µ-chain, and anti-human IgG
were from Biosource (Burlingame, CA) and Jackson ImmunoResearch (West
Grove, PA). The anti-PR3 mAb CLB-702 (mIgG1) was from Research
Diagnostics (Flanders, NJ), and the anti-MPO mAb MPO-7 (mIgG1) was
from Dako (Carpinteria, CA). The anti-PR3 IgM mAb WGM3 (21) was
provided by Drs. W. L. Gross and E. Csernok (Medizinische
Universität zu Lübeck, Lübeck, Germany). IgG from
serum (normal and ANCA positive) was prepared by protein G affinity
chromatography (Pharmacia, Piscataway, NJ). The columns were eluted
with 0.1 M glycine-HCL (pH 2.7) according to the manufacturer’s
instructions. ANCA-positive sera included two patients positive for
cANCA with biopsy-proven WG and one patient positive for
pANCA with polyarteritis nodosa. The concentration of IgG in all
preparations was determined by a radial immunodiffussion assay (The
Binding Site, Birmingham, U.K.). FITC-labeled anti-FcRII mAb
IV.3 and anti-FcRIII mAb 3G8 were from Medarex (Annandale, NJ).
Anti-CD43 (clone DF-T1) was obtained from Dako. Anti-CD62L mAb
DREG-56-FITC (mIgG1) was from Immunotech (Miami, FL), and the
anti-CD11b mAb F6.2-PE (mIgG2a) was from ExAlpha (Boston, MA).
Isotype controls (labeled and unlabeled) were obtained from Sigma (St.
Louis, MO). The anti-FcRIIIb mAbs 214.1, 135.9, and 30.2 (22)
were biotinylated using NHS-LC-Biotin from Pierce (Rockford, IL)
using standard protocols (23). PE-conjugated streptavidin for the
detection of bound primary Ab was from Caltag (San Francisco, CA).
Reagents
1,10-Phenanthroline,N-
-p-tosyl-L-lysine
chloromethyl ketone (TLCK), diisopropyl fluorophosphate (DFP),
leupeptin, pepstatin A, and aprotinin were from Sigma. Pefabloc was
from Boehringer Mannheim (Indianapolis, IN). All other standard
chemicals were from Fisher Scientific (Pittsburgh, PA) except for BSA,
which was from Boehringer Mannheim.
Isolation of cells
Blood was collected into heparinized tubes (Becton Dickinson,
Franklin Lakes, NJ) using a protocol approved by the Institutional
Committee on Human Rights in Research. All donors were healthy
volunteers genotyped for the FcRIIa allelic polymorphism
(FcRIIa-H131/R131) using an allele-specific PCR-based assay as we
have described (24). Neutrophils were isolated by Ficoll gradient
centrifugation at room temperature (15) and immediately used in the
experiments. All washes were performed in 125 mM sodium chloride, 10 mM
sodium phosphate, 5 mM potassium chloride, and 5 mM glucose, pH 7.35
(buffer 1). Cells were resuspended at a concentration of 5 x
106 cells/ml in buffer 1 containing 0.1% BSA and 1 mM
1,10-phenanthroline. Before use, 1.09 mM CaCl2 and 1.62 mM
MgCl2 was added.
ANCA competition assay
The ability of unlabeled ANCA to engage the ligand binding site
of FcR was measured by the inhibition of binding of
receptor-specific anti-ligand binding site mAb by ANCA. Inhibition
was defined as the percent decrement in anti-FcR mAb binding
induced by ANCA compared with isotype control and is expressed as a
positive number. Total ANCA binding was independently determined with a
PE-labeled F(ab')2 goat anti-mouse IgG or
anti-human IgG incubated for 15 min at 4°C.
A total of 50 µl of neutrophils in buffer 1 with 0.1% BSA, 1 mM
1,10-phenanthroline, 1.09 mM CaCl2, and 1.62 mM
MgCl2 chloride were placed in polypropylene tubes (Falcon
Labware, Franklin Lakes, NJ) and incubated with 10 µg/ml of
anti-PR3 mAb, anti-MPO mAb, or isotype control (mIgG1) for 10
min at 37°C, followed by 20 min at 4°C. For the experiments with
human ANCA the final concentration of total IgG was 1 mg/ml. The cells
were then washed and centrifuged at 350 x g for 5 min
with 2 ml of buffer 1 at 4°C containing 0.1% BSA, 1 mM
1,10-phenanthroline, 1 mM EDTA, and 1 mM sodium azide. In the
inhibition assay, a saturating concentration (2 µg/ml) of
biotinylated anti-FcRIIIb mAb (135.9, 214.1, or 30.2), mAb
3G8-FITC (2 µg/ml), or mAb IV.3-FITC (2 µg/ml) was incubated for 8
min at 4°C, followed by two washes. Separate control experiments
indicated that this was the minimum incubation time required for
optimal mAb binding. Binding of the biotinylated mAbs was detected with
PE-labeled streptavidin incubated for 15 min at 4°C.
ANCA-induced neutrophil activation
A total of 3–4 ml of blood was collected into heparinized Vacutainer tubes (Becton Dickinson) and immediately chilled to 4°C, washed twice in buffer 1 (see above), and resuspended in the original volume. Aliquots (50 µl) of this washed whole blood were used per tube (Becton Dickinson Labware, Lincoln Park, NJ). Stimulation with mIgG1 anti-PR3 at various concentrations was performed at 37°C for the indicated times as we have previously described (6, 16). Negative control samples were incubated with an isotype control mAb, and positive controls included cells stimulated with 1 µM FMLP (Sigma). All samples were then treated with 1 ml FACS Lysing Solution (Becton Dickinson Immunocytometry, San Jose, CA) for 10 min at room temperature, washed once with 2 ml PBS, and analyzed by flow cytometry.
Flow cytometry
Data was collected using a FACScan (Becton Dickinson
Immunocytometry) that was routinely calibrated using fluorescent beads
(Sphero Rainbow; Spherotech, Libertyville, IL). Neutrophil doublets and
higher aggregates (typically <5%) were detected by analysis with
anti-CD43 (which allowed elimination of events with fluorescence
greater than the dominant singlet peak) and light scatter properties.
The results are expressed as mean fluorescence intensity (MFI) of the
histogram data. Data are presented as 
MFI above the MFI of the
appropriate isotype control.
Statistical analysis
Analysis of flow cytometry listmode data was done using CellQuest (Becton Dickinson Immunocytometry). The mean channel fluorescence of histogram data was compared using Student’s t test and analysis of variance. Two-tailed paired-sample t tests or Wilcoxon’s paired-sample test were performed for the analysis of the inhibition of binding data, and a probability of 0.05 was used to reject the null hypothesis that there is no difference between the groups or pairs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RIIIb
expression
Several reports have shown that ANCA bound to
neutrophil-associated ANCA target can engage FcRIIa via their Fc
region (6, 7, 20). Data documenting ANCA engagement of the other
constitutive neutrophil FcR (FcRIIIb) have been limited by
activation-induced, metalloprotease-dependent shedding of FcRIIIb
and the mobilization of preformed intracellular stores of the receptor
to the cell surface (25, 26). To minimize these effects, we tested a
panel of protease inhibitors and found that shedding of FcRIIIb
induced by FMLP was inhibited completely by the metalloprotease
inhibitor 1,10-phenanthroline (27). Other protease inhibitors such as
Pefabloc, DFP, and TLCK were less effective, and no inhibition was
found with aprotinin, pepstatin A, and leupeptin (data not shown). The
inhibition of FMLP-induced FcRIIIb shedding by 1,10-phenanthroline
was dose dependent with an IC50 of 
0.4 mM and the
maximum inhibition reached at 1 mM (results not shown). The
quantitative expression of FcR surface expression and ANCA
engagement of FcR (see below) was not different in the presence of 1
mM and 5 mM 1,10-phenanthroline. Expression of FcRIIa was unaltered
by activation in the presence or absence of 1,10-phenanthroline.
Therefore, 1 mM 1,10-phenanthroline was used in all subsequent
experiments with no effect on cell viability, as detected by trypan
blue exclusion, over the time course of these studies.
ANCA engagement of FcRIIa on neutrophils
We have previously shown that anti-PR3 and anti-MPO engage
and block the ligand binding site of FcRIIa. To determine whether
the presence of 1,10-phenanthroline alters the interaction between cell
bound ANCA and neutrophil FcR, we determined the inhibition of the
binding of the anti-FcRIIa mAb IV.3 by anti-PR3 mAb in the
presence of 1,10-phenanthroline. PR3 and MPO expression was induced by
repletion of divalent cations (Ca2+ and Mg2+)
at 37°C (Fig. 1
A) as we have
previously described (6). Freshly isolated neutrophils, or cells that
were incubated in parallel at 4°C, did not bind anti-PR3 or
anti-MPO. Using an mIgG1 monoclonal anti-PR3, a correlation
between the amount of PR3 expression and inhibition of mAb IV.3 binding
was found for neutrophils from donors homozygous for R131 allele (the
mIgG1 binding allele) of FcRIIa (Fig. 2). In all of the experiments over a
range of PR3 target densities, the binding of the anti-PR3 mAb
resulted in significant inhibition of the binding of mAb IV.3
(p < 0.05, n = 6). As a
control, IgM anti-PR3 mAb did not inhibit the binding of
anti-FcRIIa mAb IV.3 to neutrophils (1.2 ± 2.4%
inhibition (n = 4), p > 0.05). These
results are in agreement with our earlier study (6) and confirm that
ANCA bound to neutrophils can engage FcRIIa in the presence of
1,10-phenanthroline.
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RIIIb
The sensitivity to detect ANCA binding to FcRIIIb will be
dependent on the relative affinity of the reporting anti-FcRIII
mAb. The commonly used anti-FcRIII mAb 3G8 has high affinity for
the ligand binding site of FcRIIIb and blocks immune complex binding
to neutrophils completely (28). We considered the possibility that an
anti-FcRIII mAb with lower relative affinity might be more
suitable in this assay and selected three mAb with relatively low
affinities for the ligand binding site of FcRIIIb (mAbs 214.9,
135.9, and 30.2) (22). Within this group of mAb, the relative
affinities are mAb 214.9 < 135.9 < 30.2 based on their
differential capacity to inhibit the binding of immune complexes to
neutrophils and on the various degrees by which their binding could be
competitively inhibited by an excess of other high affinity
anti-FcRIIIb mAb (such as mAb 3G8) (22). Using these mAbs as
reporters, binding of anti-PR3 mAb to the surface of neutrophils
resulted in inhibition in the binding of these anti-FcRIII mAb
relative to cells incubated with an isotype control (Fig. 3A, Table I). In a panel of normal donors, the
binding of the anti-FcRIIIb reporter mAbs was significantly
decreased by anti-PR3 (Fig. 4A, Table I) and by
anti-MPO (Fig. 4B, Table I). In the same panel of normal
donors, there was a range of binding of the high affinity mAb 3G8 after
binding of anti-PR3 (Figs. 3B and 4A) or
anti-MPO (Fig. 4B), but the average binding was not
altered by binding of either anti-PR3 (Table I) or anti-MPO to
the neutrophil surface (Table I) presumably due to the ability of mAb
3G8 to displace ANCA Fc region engagement of FcRIIIb. It is also
important to note the ability of anti-PR3 and anti-MPO, at low
ANCA target density (MFI < 100), to significantly inhibit the
binding of the reporter anti-FcRIII mAb 214.1, 135.9, and 30.2
but not the anti-FcRIIa reporter mAb IV.3 (Fig. 2
). This finding
indicates that ANCA may preferentially engage FcRIIIb on the surface
of neutrophils.
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RIIIb binding
(mAb 214.1 
135.9 > 30.2 > 3G8) is inversely
proportional to the relative affinity of the reporting
anti-FcRIIIb mAb. However, there was no correlation between the
magnitude of PR3 or MPO expression and the magnitude of the reduction
in net anti-FcRIII mAb binding (Fig. 4). Even with blockade of
receptor shedding, translocation of intracellular FcRIIIb to the
cell surface precludes a precise quantitative relationship between ANCA
binding and net reduction of anti-FcRIIIb binding. Such
translocation of new receptor to the membrane should result in an
underestimation of the actual amount of FcRIIIb engagement by ANCA.
Indeed, the binding of mAb 3G8 was found on average to be slightly
increased after binding of anti-PR3 (Fig. 3B, Table I)
or anti-MPO (Table I) indicating that there was a net increase in
receptor number after incubation with these murine ANCA.
Using a monoclonal anti-PR3 IgM ANCA as a negative control, no
significant inhibition of the binding of any anti-FcRIII mAb was
observed (Table I). Finally, there was no significant difference in
binding of any of the anti-FcRIII mAbs to neutrophils incubated
with an isotype control (murine IgG1) compared with the samples
incubated with buffer alone.
Human pANCA and cANCA engage FcRIIIb on neutrophils
We next determined if engagement of FcRIIIb was
generalizable to human ANCA. Accordingly, IgG from two plasmas
containing cANCA activity, one plasma containing pANCA activity and one
plasma from a disease-free healthy control donor was isolated as
described in Materials and Methods. Both cANCA and pANCA
containing IgG fractions bound to ANCA target positive neutrophils
relative to normal serum human IgG (Fig. 1B). We have
previously shown that human cANCA binding to neutrophils significantly
blocks the detection of the FcRIIa ligand binding site with the
anti-FcRIIa mAb IV.3 (6). Likewise, cANCA binding to neutrophils
significantly inhibited the binding of the two lowest affinity
anti-FcRIIIb mAbs 214.1 and 135.9 (Table II) when compared directly with normal
human serum IgG, which did not inhibit the binding of these mAb. cANCA
also inhibited the binding of the higher affinity mAb 30.2, but the
magnitude of inhibition did not reach statistical reliability with our
sample size (Table II). As with cANCA, we observed significant
engagement of FcRIIIb by pANCA. Indeed, binding of all three lower
affinity anti-FcRIIIb mAb was significantly inhibited by pANCA
binding to neutrophils (Table II).
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To determine whether the binding of ANCA to the surface of
neutrophils triggers cell activation in a manner that is consistent
with engagement of FcRIIIb, we quantitated the time-dependent
changes in surface expression of the ß2 integrin CD11b
and CD62L (L-selectin) induced by ANCA. Cross-linking of FcRIIa
results in the simultaneous up-regulation of CD11b and shedding of
CD62L whereas FcRIIIb results in the up-regulation of CD11b without
altering CD62L expression (16). Accordingly, ANCA engagement of
FcRIIIb before FcRIIa should elicit an initial phenotype
characterized by increased CD11b and preserved CD62L expression
followed by CD62L shedding. Binding of anti-PR3 mAb to neutrophils
in washed whole blood, performed as we have previously described (29),
resulted in the time-dependent change in expression of both CD11b and
CD62L (Fig. 5). After 30 min incubation,
we observed significant up-regulation of CD11b expression relative to
unstimulated cells (Fig. 5, A and B) and only at
the later time points did we observe a significant loss of CD62L
expression (Fig. 5C). Addition of an isotype control mAb to
whole blood did not induce any significant changes in the expression of
either CD11b or CD62L. These results are consistent with a model of
neutrophil activation in which ANCA initially engage FcRIIIb
followed by engagement of FcRIIa.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R have been shown to play a role in ANCA-induced neutrophil
and monocyte activation (6, 7, 9, 20), perhaps in conjunction with
direct ANCA-induced stimulation (4, 11, 30). However, it is not clear
which FcR are involved in ANCA-induced activation. Engagement of
only FcRIIa on neutrophils and monocytes has been clearly shown (6, 7, 9, 20). This study shows that ANCA on neutrophils engage FcRIIIb
as well as FcRIIa and that engagement of FcRIIIb is observed for
both cANCA (anti-PR3) and pANCA (anti-MPO). The importance of
engagement of FcRIIIb as well as FcRIIa is underscored by the
observation that FcRIIIb has distinct functional properties relative
to FcRIIa (15, 16), that FcRIIIb can modulate the quantitative
functional capacities of other cell surface receptors such as FcRIIa
and CD11b/CD18 (15, 31, 32), that FcRIIIb and FcRIIa engage
distinct signal transduction pathways (14, 17), and that alleles of
FcRIIIb are a genetic risk factor for the severity of disease in
patients with WG (12)5.
The ability to detect FcRIIIb engagement by both human and murine
ANCA is dependent 1) on the relative affinity of the reporter
anti-FcRIIIb mAb, 2) on the degree of receptor shedding, and 3)
on surface expression of pre-formed intracellular receptor stores. We
have used a panel of lower relative affinity anti-FcRIII mAb
(22) in this study to address the first issue and the inclusion of the
metalloprotease inhibitor 1,10-phenanthroline to prevent
activation-induced FcRIIIb shedding. The affinity of 3G8 is
particularly high and greater than any of the other mAb used in this
study (28). mAb 3G8 is capable of competing effectively with
multivalent immune complexes and most probably it is capable of
displacing the Fc portion of ANCA from the low affinity binding site of
FcRIIIb. This competitive advantage results in no inhibition of
binding of mAb 3G8 by ANCA, and due to up-regulation of FcRIIIb
surface expression induced by priming and/or by ANCA during the
incubation, a net increase in mAb 3G8 binding to the receptor was found
in some experiments (Fig. 3B and Table I). This increase
illustrates that the quantitative inhibition of binding found with
other antireceptor mAb clearly underestimates the actual engagement of
FcRIIIb by ANCA on the neutrophil surface.
We have shown that both cANCA and pANCA, as well as murine mAb
with corresponding reactivities, can engage FcRIIIb. However, it is
important to note the ability of the anti-MPO and anti-PR3, at
low ANCA target density (MFI < 100), to significantly inhibit the
binding of the reporter anti-FcRIIIb mAbs 214.1, 135.9 and 30.2
(Fig. 4). Since engagement of FcRIIa apparently requires higher ANCA
densities (Fig. 2), this observation suggests that FcRIIIb might be
preferentially engaged by ANCA when ANCA target is limiting. Such
preferential engagement is consistent with the nearly 10-fold higher
density of surface expression of FcRIIIb relative to FcRIIa (33).
This numerical advantage in surface receptor expression could be
further amplified by activation-induced translocation of a pre-formed
intracellular pool of FcRIIIb to the cell surface with a net gain in
expression even in the context of receptor shedding. Together, these
points also suggest that initial neutrophil triggering of cell
responses may occur via FcRIIIb. Indeed, anti-PR3 binding to
neutrophils induced a transient phenotype characterized by increased
CD11b and preserved CD62L expression (Fig. 5), a phenotype that is
induced by cross-linking of FcRIIIb but not FcRIIa (16).
Furthermore, we have recently shown that the more functionally active
allele of FcRIIIb, the NA1 allele, is a significant risk factor for
the development of renal disease in patients with WG (12)5.
It is important to note that the significance of engagement of
FcRIIIb by ANCA bound to ANCA target on the cell surface would be
the transient induction of a qualitatively different adhesive phenotype
not necessary a difference in the kinetics of neutrophil activation.
Our previous data have shown that the kinetics of FcRIIa- and
FcRIIIb-induced up-regulation of CD11b expression is identical (16).
It is also worth noting that the level of membrane associated ANCA
target on neutrophils from patients with WG (5, 34) is considerably
lower than the levels achieved after maximal induction in this study, a
finding that favors FcRIIIb in ANCA-induced neutrophil activation.
The ability of ANCA to engage FcR is influenced by the subclass of
the ANCA and by the allelic variants of FcR expressed by the host.
For example, only the FcRIIa-H131 allele binds human IgG2 well (24).
FcRIIIa-V176 binds IgG1 and IgG3 with 10-fold greater efficiency
than the FcRIIIa-F176 allele (35), and some evidence suggests that
FcRIIIb is responsible for neutrophil responses triggered by IgG3
(36). Our finding of a significant association between FcRIIIb
alleles and renal disease in patients with WG does not preclude a role
for polymorphisms in FcRIIa expressed on neutrophils and monocytes
and FcRIIIa expressed on monocytes that can alter the binding of
IgG. These polymorphisms may impact on the development of other
ANCA-associated vasculitides and are clearly important for further
study in the context of ANCA-associated vasculitis. The nature of the
ANCA target may also play a role in the neutrophil activating potential
of ANCA. For example, studies by Csernok et al. (5) have shown that PR3
preferentially binds to neutrophil membranes relative to MPO, a finding
that is consistent with the higher level of PR3 target expression
relative to MPO on neutrophils from patients with WG (5, 34).
In conclusion, we find that ANCA engage FcRIIIb on the surface
of neutrophils as detected by the inhibition of binding of
anti-FcRIIIb mAb when the shedding of this receptor is blocked,
even at relatively low detectable levels of ANCA-target expression. The
initial and perhaps preferential engagement of FcRIIIb will clearly
influence the signal transduction pathways and the specific cellular
responses induced by ANCA. For example, cross-linking of FcRIIIb has
been shown to induce a pro-adhesive neutrophil phenotype (16) and
FcRIIIb is the dominant Fc receptor responsible for the immune
complex-induced oxidative burst in neutrophils (18, 19). It is also
likely that ANCA binding to monocytes will lead to engagement of the
available repertoire of available FcR (FcRI, FcRII, and
FcRIIIa). A better understanding of the dynamics of ANCA-FcR
interactions on both neutrophils and monocytes may lead to new
therapeutic approaches in the systemic vasculitides such as the use of
soluble FcR and/or peptide mimics to block ANCA engagement of FcR
on the cell surface.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Robert P. Kimberly, Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294. E-mail address:
4 Abbreviations used in this paper: ANCA, antineutrophil cytoplasmic Abs; PR3, proteinase 3; MPO, myeloperoxidase; WG, Wegener’s granulomatosis; PE, phycoerythrin; TLCK, 1,10-phenanthroline,N--p-tosyl-L-lysine chloromethyl ketone; DFP, diisopropyl fluorophosphate; MFI, mean fluorescence intensity.
5 E. Wainstein, M. Kocher, J. C. Edberg, J. Wu, E. Csernok, M. Sneller, G. Hoffman, E. Keystone, W. L. Gross, J. E. Salmon and R. P. Kimberly. Fc receptor alleles associate with renal dysfunction in Wegener’s granulomatosis. 1998. Submitted for publication.
Received for publication March 6, 1998. Accepted for publication August 26, 1998.
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M ß2, CD11b/CD18) and FcRIII cooperate in generation of a neutrophil respiratory burst: requirement for FcRIII and tyrosine phosphorylation. J. Cell Biol. 125:1407. receptor IIIb enhances Fc receptor IIa function in an oxidant-dependent and allele-sensitive manner. J. Clin. Invest. 95:2877.
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 O. J. Florey, M. Johns, O. O. Esho, J. C. Mason, and D. O. Haskard Antiendothelial cell antibodies mediate enhanced leukocyte adhesion to cytokine-activated endothelial cells through a novel mechanism requiring cooperation between Fc{gamma}RIIa and CXCR1/2 Blood, May 1, 2007; 109(9): 3881 - 3889. [Abstract] [Full Text] [PDF]
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 A. P. van Rossum, A. A. Rarok, M. G. Huitema, G. Fassina, P. C. Limburg, and C. G. M. Kallenberg Constitutive membrane expression of proteinase 3 (PR3) and neutrophil activation by anti-PR3 antibodies J. Leukoc. Biol., December 1, 2004; 76(6): 1162 - 1170. [Abstract] [Full Text] [PDF]
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 P. Hewins, J. M. Williams, M. J.O. Wakelam, and C. O.S. Savage Activation of Syk in Neutrophils by Antineutrophil Cytoplasm Antibodies Occurs via Fc{gamma} Receptors and CD18 J. Am. Soc. Nephrol., March 1, 2004; 15(3): 796 - 808. [Abstract] [Full Text] [PDF]
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 S. Tanaka, J. C. Edberg, W. Chatham, G. Fassina, and R. P. Kimberly Fc{gamma}RIIIb Allele-Sensitive Release of {alpha}-Defensins: Anti-Neutrophil Cytoplasmic Antibody-Induced Release of Chemotaxins J. Immunol., December 1, 2003; 171(11): 6090 - 6096. [Abstract] [Full Text] [PDF]
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A. A. Rarok, P. C. Limburg, and C. G. M. Kallenberg Neutrophil-activating potential of antineutrophil cytoplasm autoantibodies J. Leukoc. Biol., July 1, 2003; 74(1): 3 - 15. [Abstract] [Full Text] [PDF]
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D. Reumaux, M. de Boer, A. B. Meijer, P. Duthilleul, and D. Roos Expression of myeloperoxidase (MPO) by neutrophils is necessary for their activation by anti-neutrophil cytoplasm autoantibodies (ANCA) against MPO J. Leukoc. Biol., June 1, 2003; 73(6): 841 - 849. [Abstract] [Full Text] [PDF]
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J. M. Williams, A. Ben-Smith, P. Hewins, S. K. Dove, P. Hughes, R. McEwan, M. J.O. Wakelam, and C. O.S. Savage Activation of the Gi Heterotrimeric G Protein by ANCA IgG F(ab')2 Fragments Is Necessary but not Sufficient to Stimulate the Recruitment of Those Downstream Mediators Used by Intact ANCA IgG J. Am. Soc. Nephrol., March 1, 2003; 14(3): 661 - 669. [Abstract] [Full Text] [PDF]
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A. Ben-Smith, S. K. Dove, A. Martin, M. J. O. Wakelam, and C. O. S. Savage Antineutrophil cytoplasm autoantibodies from patients with systemic vasculitis activate neutrophils through distinct signaling cascades: comparison with conventional Fc{gamma} receptor ligation Blood, September 1, 2001; 98(5): 1448 - 1455. [Abstract] [Full Text] [PDF]
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K. Hattar, U. Sibelius, A. Bickenbach, E. Csernok, W. Seeger, and F. Grimminger Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis J. Leukoc. Biol., January 1, 2001; 69(1): 89 - 97. [Abstract] [Full Text]
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C. Hess, S. Sadallah, and J.-A. Schifferli Induction of neutrophil responsiveness to myeloperoxidase antibodies by their exposure to supernatant of degranulated autologous neutrophils Blood, October 15, 2000; 96(8): 2822 - 2827. [Abstract] [Full Text] [PDF]
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 D. Vassilopoulos and G. S. Hoffman Clinical Utility of Testing for Antineutrophil Cytoplasmic Antibodies Clin. Vaccine Immunol., September 1, 1999; 6(5): 645 - 651. [Full Text] [PDF]
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