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Antineutrophil Cytoplasmic Antibodies Preferentially Engage FcRIIIb on Human Neutrophils¹

Markus Kocher^2,*, Jeffrey C. Edberg^2,*, Howard B. Fleit and Robert P. Kimberly^3,*

^* 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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antineutrophil cytoplasmic Abs (ANCA) are found in the circulation of many patients with systemic vasculitis. ANCA bind to ANCA target, such as proteinase 3 and myeloperoxidase, and activate neutrophils in an FcR-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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antineutrophil cytoplasmic Abs (ANCA)⁴ directed against granule contents are commonly found in patients with systemic vasculitis (1, 2, 3). ANCA are distinguished by two common indirect immunofluorescence staining patterns (1, 2, 3). ANCA that show a granular cytoplasmic pattern are called cANCA (mostly anti-proteinase 3 (PR3) autoantibodies) and ANCA that show a perinuclear staining pattern are called pANCA (most commonly anti-myeloperoxidase (MPO)). The correlation of cANCA titers with disease activity in some studies suggests that ANCA may be directly involved in the pathogenesis of Wegener’s granulomatosis (WG) (1, 2, 3). ANCA targets displayed on the cell surface, due to cell priming and/or apoptosis, allow ANCA to bind to neutrophils (4, 5, 6, 7, 8). Binding of ANCA to ANCA target and subsequent engagement of Fc 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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies

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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The effects of 1,10-phenanthroline on neutrophil FcRIIIb 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. 1A) 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.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Expression of ANCA-target (PR3 and MPO) on the surface of neutrophils from disease-free donors. A, Purified neutrophils were incubated as described in Materials and Methods in the presence of anti-PR3 or anti-MPO mAb and binding was detected with PE-labeled F(ab')2 goat anti-mouse IgG. Binding of a mIgG1 isotype control is also shown (dotted line). B, Purified neutrophils were incubated with human cANCA or pANCA containing IgG preparations (prepared as described in Materials and Methods) and binding was detected with PE-labeled F(ab')2 goat anti-human IgG. Binding of human IgG isolated from a healthy control donor is shown as a negative control (dotted line).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Inhibition of the binding of anti-FcRIIa mAb IV.3 to neutrophils by anti-PR3. Purified neutrophils from donors homozygous for FcRIIa-R131 were incubated in the presence of anti-PR3 mAb ANCA or mIgG1. The binding of FITC-labeled mAb IV.3 was determined, and the results are expressed as the % inhibition of binding induced by the anti-PR3 mAb. Expression of ANCA target was measured in parallel samples and is expressed as MFI of the anti-PR3 binding after subtraction of the MFI from the isotype control.

ANCA engagement of FcRIIIb

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.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Binding of anti-FcRIIIb mAb on neutrophils. Purified neutrophils were incubated in the presence of ANCA (anti-PR3 mAb) or an isotype control at the same concentration (10 µg/ml). In this representative experiment, the binding of the biotinylated mAb 214.1 was then detected with PE-labeled streptavidin (A) and mAb 3G8 was a direct FITC-conjugate (B). The binding of the reporting anti-FcRIII mAb after incubation of the cells with mIgG1 (solid line) or anti-PR3 mAb (filled) is shown. In each panel, binding of streptavidin-PE (A) or IgG1-FITC (B) alone is shown (dotted line).

View this table: [in this window] [in a new window]   Table I. Inhibition of anti-FcRIII mAb binding to neutrophils by murine ANCA1

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Inhibition of binding of anti-FcRIIIb mAb on neutrophils. Purified neutrophils from each donor were incubated in the presence of anti-PR3 mAb (A) or anti-MPO mAb (B). The binding of the biotinylated mAb 30.2, 135.9, and 214.1 was then detected with PE-labeled streptavidin; mAb 3G8 was a FITC-conjugate. In each experiment, the percent inhibition of anti-FcRIIIb mAb binding is shown relative to the level of expression of PR3 or MPO (MFI). The inhibition of binding of mAb 135.9 by the Fc region of anti-PR3 in these experiments is shown for comparison.5

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).

View this table: [in this window] [in a new window]   Table II. Inhibition of anti-FcRIII mAb binding to neutrophils by human ANCA1

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.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Differential kinetics of CD11b up-regulation and CD62L shedding induced by anti-PR3 on neutrophils. mIgG1 anti-PR3 (10 µg/ml) was added to isolated neutrophils, and the time dependent changes in expression of CD11b and CD62L were determined at t = 0 min (A), 30 min (B), and 60 min (C). The quadrants are drawn to demonstrated the time-dependent changes in expression of these adhesion molecules relative to their expression on resting cells (A). A representative experiment from a total of four experiments is shown. The mIgG1-FITC + mIgG2a-PE isotype control, shown at the t = 0 min point in A (gray lines), did not change over the time course of the experiment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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.

	Acknowledgments

 
We thank Patsy Redecha for the purification of human IgG from serum, Andy Beavis for his expert assistance with flow cytometry, and Drs. E. Csernok and W. Gross for the IgM anti-PR3 mAb. We are grateful for the advice and discussions with our colleagues at the Hospital for Special Surgery where much of this work was performed.

	Footnotes

 
¹ This work was supported by grants from the Swiss National Science Foundation (823A-046672) and from the National Institutes of Health (RO1-AR42476, RO1-AR33062). The Flow Cytometry Core at the Hospital for Special Surgery was supported by National Institutes of Health Grant P60-AR38320.

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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