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The Journal of Immunology, 2003, 171: 6090-6096.
Copyright © 2003 by The American Association of Immunologists

Fc{gamma}RIIIb Allele-Sensitive Release of {alpha}-Defensins: Anti-Neutrophil Cytoplasmic Antibody-Induced Release of Chemotaxins 1

Sumiaki Tanaka2,*, Jeffrey C. Edberg3,*,{dagger}, Winn Chatham*, Giorgio Fassina and Robert P. Kimberly*,{dagger}

* Division of Clinical Immunology and Rheumatology, Department of Medicine and {dagger} Department of Microbiology, University of Alabama, Birmingham, AL 35294; and XEPTAGEN SpA, Pozzuoli, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Antineutrophil cytoplasmic Abs (ANCA) can activate neutrophils in an Fc{gamma}R-dependent manner, but the link between this ANCA-induced effect and mononuclear cell activation with the characteristic granuloma formation of Wegener’s granulomatosis is unclear. Human {alpha}-defensins, small cationic antimicrobial peptides, are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and monocytes. In this study, we quantitated the release of {alpha}-defensins (human neutrophil peptides 1–3) from human neutrophils after targeted Fc{gamma}R cross-linking (XL). Homotypic XL of Fc{gamma}RIIa, Fc{gamma}RIIIb, or heterotypic XL of both receptors resulted in significant release of {alpha}-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-proteinase 3). This release of {alpha}-defensins, as well as of other granule constituents (ANCA targets anti-proteinase 3 and myeloperoxidase and elastase), was significantly greater in donors homozygous for the NA1 allele of Fc{gamma}RIIIb than in donors homozygous for NA2. Interestingly, the ANCA-induced release was completely inhibited by the IgG Fc-binding peptide TG19320, which blocks the IgG-Fc region from binding to Fc{gamma}R. Based on their chemotactic properties, {alpha}-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma formation. The greater activity of the Fc{gamma}RIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human neutrophils contain many distinct granule populations that can be mobilized on inflammatory stimulation (1, 2). The release of these granules is a highly regulated process with a gradient of responsiveness to stimulation. For example, extracellular release of azurophilic granules requires a higher threshold of activation than release of specific or secretory vesicles (3, 4, 5). This is in keeping with a primary role for azurophilic granule enzymes in intracellular degradation of phagocytosed particles.

The control of azurophilic granule release is important in limiting neutrophil-mediated tissue destruction at sites of inflammation. These granules contain key enzymatic mediators of inflammation such as myeloperoxidase (MPO),4 elastase, collagenase, and various acid hydrolases. More recently, it has become clear that among the subpopulations of azurophilic granules expressed in neutrophils (1), there is a subpopulation of granules that contain the {alpha}-defensin antibacterial/chemotactic peptides (6, 7). The receptor-mediated regulation of {alpha}-defensin release has not been explored.

{alpha}-Defensins are a family of small (3.5- to 4.5-kDa) cationic antimicrobial peptides with three to four intramolecular cysteine disulfide bonds and are widely distributed in mammals, insects, and plants (8). In addition to their antimicrobial properties, some of these peptides are potent chemotaxins for mononuclear cells (9), including dendritic cells and CD45RA+ and CD8 T lymphocytes (10, 11)). Based on the pattern of their cysteine residues and disulfide connections, six {alpha} and two {beta} forms have been characterized in humans. Human {alpha}-defensins 1, 2, 3, and 4 are found in azurophilic granules in human neutrophils and thus are termed human neutrophil peptides (HNP) (12), whereas human {alpha}-defensins 5 and 6 (HNP 5 and 6) are generated by small intestine Paneth cells (13). Based on their chemotactic activity, human neutrophil {alpha}-defensins contribute to modification of acquired immune responses by mobilization of mononuclear cells, T cells, and dendritic cells (9, 10, 11).

Activation of human neutrophils by antineutrophil cytoplasmic Abs (ANCA) is at least partially dependent on engagement of Fc{gamma}Rs on the surface of neutrophils. We hypothesized that engagement of Fc{gamma}R on neutrophils would mobilize the {alpha}-defensin-positive azurophilic granule compartment and provide a link between ANCA-induced neutrophil activation in ANCA-associated vasculitis (in particular Wegener’s granulomatosis (WG)) and granuloma formation. Human neutrophils express two structurally distinct Fc{gamma}R, Fc{gamma}RIIa and Fc{gamma}RIIIb (14). Both of these receptors are functionally polymorphic with the H131/R131 single-nucleotide polymorphism of Fc{gamma}RIIa altering the binding of hIgG2 (15) and mIgG1 (16) and the NA1/NA2 single-nucleotide polymorphisms of Fc{gamma}RIIIb altering the quantitative functional capacity of the receptor (17, 18, 19). ANCA have been shown to engage both neutrophil Fc{gamma}R upon binding to cell-associated ANCA-target Ag with preferential engagement of Fc{gamma}RIIIb presumably due to its numeric predominance over Fc{gamma}RIIa (20, 21, 22).

In this study, we show that cross-linking of neutrophil Fc{gamma}R results in significant release of {alpha}-defensins. The magnitude is influenced by the host receptor genetics, with donors homozygous for the NA1 allele of Fc{gamma}RIIIb displaying significantly greater release. This release is induced by both monoclonal and human cytoplasmic staining ANCA (cANCA) (anti-proteinase 3 (PR3)) from patients with WG, and importantly, its blockade by the Fc-specific TG19320 peptide suggests an intervention strategy that can be carried to the in vivo setting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Blood donors

Whole blood anticoagulated with EDTA was obtained from healthy volunteers. All donors were genotyped for both neutrophil Fc{gamma}Rs (Fc{gamma}RIIa and Fc{gamma}RIIIb) by allele-specific PCR (23, 24). In some studies, neutrophils were isolated by centrifugation through a discontinuous Ficoll-Hypaque density gradient as previously described (25). The protocol for phlebotomy was approved by the Institutional Committee on Human Rights in Research.

Antibodies

Anti-Fc{gamma}RIIIb mAb 3G8 F(ab')2 or IgG, anti-Fc{gamma}RII mAb IV.3 Fab and FITC-conjugated 3G8 (mIgG1) and IV.3 (mIgG2b) were purchased from Medarex (Annandale, NJ). F(ab')2 fragments of F(ab')2-specific goat anti-mouse IgG (G{alpha}M) for anti-Fc{gamma}R mAb cross-linking, Fc-specific PE-conjugated G{alpha}M F(ab')2 for detection of primary Abs, and murine F(ab')2 were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The anti-PR3 mAb CLB 12.8 (mIgG1) was from Research Diagnostics (Flanders, NJ), and the anti-MPO mAb MPO-7 (mIgG1) was from Dako (Carpinteria, CA). Anti-CD66b mAb CLB-B13.9-FITC (mouse IgG1 (mIgG1)) and FITC- or PE-conjugated isotype-matched controls were purchased from Caltag (San Francisco, CA). Human cANCA (anti-PR3) or perinuclear staining ANCA (pANCA) (anti-MPO) plasmas were obtained from five patients with active WG. IgG from the plasmas was purified as previously described (21, 22).

Washed whole blood assay

Neutrophils in washed whole blood, which eliminates isolation-induced neutrophil activation (26), were stimulated as described previously (27). Briefly, EDTA-anticoagulated blood was chilled to 4°C, washed twice in modified PBS (125 mM sodium chloride, 10 mM phosphate, 5 mM potassium chloride, 5 mM glucose, pH 7.35), and then resuspended in the original volume. Washed whole blood was keep at 4°C, and all washes were performed with modified PBS at 4°C. For neutrophil stimulation, aliquots (300 µl) of washed whole blood were incubated with saturating concentrations of the anti-Fc{gamma}RII- and/or anti-Fc{gamma}RIII-specific mAb fragments (1 µg/ml IV.3 Fab and/or 2 µg/ml 3G8 F(ab')2) for 15 min at 4°C, washed twice, and resuspended in buffer with 1.09 mM CaCl2 and 1.62 mM MgCl2. Cell-bound mAb fragments were cross-linked with F(ab')2 G{alpha}M (35 µg/ml) at 37°C for 15 min. Alternately, cells were stimulated with PMA (Sigma-Aldrich, St. Louis, MO; 10 µg/ml) as a positive control or with F(ab')2 G{alpha}M as a negative control. Endotoxin levels in all reagents were below detection limits (Limulus ameobocyte assay; Sigma-Aldrich) as described (22, 27). In some studies, cells were pretreated with 50 ng/ml rTNF-{alpha} (R&D, Minneapolis, MN) for 20 min at 37°C before mAb cross-linking. After stimulation, cell-free supernatants were then collected and stored at -20°C. After two washes, the remaining G{alpha}M binding sites on the cells were blocked with murine F(ab')2 (88 µg/ml). Aliquots of stimulated cells were incubated with primary mAb for flow cytometry for 30 min at 4°C. Unconjugated mAb were detected with PE-conjugated G{alpha}M F(ab')2 for 30 min. Fc{gamma}R expression on neutrophil in washed whole blood was quantitated with FITC-conjugated anti-Fc{gamma}RIIIb mAb 3G8 or anti-Fc{gamma}RII mAb IV.3. All samples were then treated with FACS Lysing Solution (BD Biosciences, San Jose, CA) for 10 min at room temperature (RT), washed once, and analyzed by flow cytometry (FACSCalibur; BD Biosciences).

ELISA for {alpha}-defensin and elastase

The concentration of {alpha}-defensins (HNP 1–3) in diluted cell-free supernatant was measured with the HNP 1–3 ELISA Kit (Cell Sciences, Norwood, MA) according to the manufacturer’s instructions. This assay quantitates the three principal {alpha}-defensins, HNP 1–3, that are unique to neutrophils and account for >99% of the total defensin content of these cells. The concentration of elastase in diluted cell-free supernatants was also measured by ELISA. Microtiter plates were coated with 100 µl of a rabbit polyclonal anti-elastase (Biodesign, Saco, ME) (51 µg/ml) for 2 h at RT, and the wells were blocked with 0.1% BSA in PBS (PBS/0.1%BSA). The diluted supernatants (1:25 or 1:50) or elastase standards (0.5 ng/ml to 100 ng/ml) (Sigma), all diluted in PBS/0.1% BSA, were added to wells and incubated for 2 h at RT followed by four washes with PBS, 0.1% BSA. Biotinylated rabbit polyclonal anti-elastase Ab prepared with the Biotin Protein Labeling Kit (Roche Diagnostics, Mannheim, Germany) was added at 100 µg/ml in PBS, 0.1% BSA and incubated for 2 h at RT. After four washes, plates were incubated for 30 min at RT with avidin-alkaline phosphatase (Sigma-Aldrich) diluted 1:1000 in PBS, 0.1% BSA, followed by an additional four washes. The plates were developed by the addition of 50 µl of 1 mg/ml p-nitrophenyl phosphate (Sigma-Aldrich) in 10% diethanolamine (Sigma-Aldrich). After 30 min at RT, the reaction was stopped with 50 µl of 0.1% EDTA and OD405 was determined (VMAX; Molecular Devices, Sunnyvale, CA).

Functional MPO activity assay

Functional MPO activity was performed as previously described (28). Briefly, isolated neutrophils (2 x 106/ml) were added to wells coated with IgG (100 µg/ml) and incubated at 37°C for 1 h to heterotypically cross-link with Fc{gamma}RIIa and Fc{gamma}RIIIb. After stimulation, cell-free supernatants were collected and stored at -20°C. Released MPO was quantitated by mixing 300 µl of dilutions of cell-free supernatants with 250 µl of 80 mM NaH2Po4 buffer, pH 5.4, and 80 µl of 20 mM 3,3',5,5'-tetramethylbenzidine in dimethylformamide. Then 100 µl of 1.0 mM H2O2 were added, and the {Delta}OD655 was recorded at 15-s intervals at 25°C for 1 min (29). Released functional MPO activity is represented as {Delta}OD655/min during the liner phase of the reaction.

cANCA-induced neutrophil activation

ANCA (mAb or human) stimulation of neutrophils in washed whole blood was performed as described above except that the washed cells were preincubated for 40 min at 37°C to induce cell surface expression of proteinase 3 (PR3) ANCA target. Preliminary studies indicated that near maximal surface PR3 was detected at this time point. Aliquots were then incubated with or without anti-PR3 mAb CLB 12.8 (10 µg/ml) or diluted cANCA-positive plasma for 45 min. After stimulation, supernatants were harvested as described above, and cells in washed whole blood were analyzed for CD66b expression by flow cytometry as described above (30).

In some studies, we used the IgG-Fc region-binding peptide (TG19320) (31) or a scrambled TG19320 peptide to block ANCA-induced neutrophil activation. In these studies, aliquots of washed whole blood were incubated with anti-PR3 mAb or human ANCA pretreated with various concentration of peptides at 37°C at 45 min.

Flow cytometry

Data were collected using a BD Biosciences FACSCalibur. The instrument was routinely calibrated using fluorescent beads (Rainbow Calibration Particles, 3.5 µm; Spherotech, Libertyville, IL). Neutrophils in whole blood were identified by characteristic light scatter properties and confirmed by Fc{gamma}R characteristic expression (14) using FITC-conjugated IV.3 and 3G8. As previously reported, cell surface expression of PR3 displayed a bimodal pattern (32, 33).

Analysis of flow cytometry listmode date was done using CellQuest (BD Biosciences). The results are expressed as mean fluorescence intensity (MFI) of the histogram data.

Data analysis

For quantitation of azurophilic granule release in donors homozygous for Fc{gamma}RIIIb alleles, experiments were performed in a matched paired donor design. The effect of activating stimuli on Fc{gamma}R-mediated degranulation are presented as percent increase relative to control incubations. Data are displayed as mean ± SEM. The effects of stimuli were compared using the t test. p = 0.05 was used to reject the null hypothesis that there is no difference between the groups or conditions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neutrophil Fc{gamma}R-mediated release of {alpha}-defensin

To study the release of {alpha}-defensins in response to Fc{gamma}R cross-linking, we used ELISA to quantitate the concentration of HNP 1–3 in cell-free supernatants after targeted Fc{gamma}R cross-linking on neutrophils in washed whole blood. No significant release of HNP 1–3 above baseline was detected in response to homotypic cross-linking of either Fc{gamma}RIIa or Fc{gamma}RIIIb. However, HNP 1–3 were released from neutrophils after co-cross-linking of both Fc{gamma}RIIa and Fc{gamma}RIIIb together and after stimulation with 10 µg/ml PMA (Fig. 1A). Because TNF-{alpha} can prime neutrophils for enhanced stimulus-mediated degranulation (28, 34, 35), we examined the release of {alpha}-defensins induced by Fc{gamma}R cross-linking in TNF-{alpha}-primed neutrophils to enhance release of azurophilic granules. TNF-{alpha} (50 ng/ml) alone induced significant release of HNP 1–3 and markedly enhanced the Fc{gamma}R-mediated release of HNP 1–3 (Fig. 1B). In the presence of TNF-{alpha}, homotypic cross-linking of Fc{gamma}RIIa or Fc{gamma}RIIIb and heterotypic cross-linking of Fc{gamma}RIIa and Fc{gamma}RIIIb induced significant release of HNP 1–3 relative to control incubation. These results indicated that cross-linking of Fc{gamma}Rs could induce the release of the {alpha}-defensin subpopulation of azurophilic granules in both unprimed and primed neutrophils.



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  		FIGURE 1. Fc{gamma}R cross-linking induces release of {alpha}-defensins. A, Heterotypic cross-linking but not homotypic cross-linking of Fc{gamma}RIIa and Fc{gamma}RIIIb on unprimed neutrophils induced significant release of {alpha}-defensins (HNP 1–3). B, Both heterotypic and homotypic cross-linking of Fc{gamma}RIIa and/or Fc{gamma}RIIIb on TNF-{alpha}-primed neutrophils in washed whole blood induced significant release of {alpha}-defensins (HNP 1–3). Unprimed or TNF-{alpha}-primed neutrophils in aliquots (300 µl) of a washed whole blood were stimulated with specific cross-linking of Fc{gamma}RIIa with IV.3 Fab (1 µg/ml) and/or Fc{gamma}RIIIb with 3G8 F(ab')2 (2 µg/ml) by G{alpha}M F(ab')2 (30 µg/ml) for 15 min at 37°C. The concentrations of HNP 1–3 were measured by ELISA (n = 2–4). Bars, SEM. CTRL, Control incubation of cells with mouse F(ab')2. TNF-{alpha} significantly enhanced release of {alpha}-defensins in all cases (p < 0.03). *, p < 0.05, relative to control incubation, **, p < 0.001 relative to control incubation.

 
Fc{gamma}R-mediated release of {alpha}-defensins from neutrophils is Fc{gamma}RIIIb allele dependent

Donors homozygous for the NA1 allele of Fc{gamma}RIIIb display a quantitatively greater Fc{gamma}R-dependent phagocytic capacity (17) and greater Fc{gamma}RIIIb enhancement of Fc{gamma}RIIa-specific phagocytosis relative to NA2-homozygous donors (19). To test whether the NA1 allele might have a greater capacity to mobilize azurophilic granules relative to the NA2 allele, we assessed the influence of Fc{gamma}RIIIb-NA1/NA2 polymorphism on the quantitative magnitude of release of {alpha}-defensins in response to heterotypic cross-linking of Fc{gamma}RIIa and Fc{gamma}RIIIb on TNF-{alpha}-primed neutrophils. Indeed, heterotypic cross-linking from donors homozygous for the NA1 allele of Fc{gamma}RIIIb showed significantly greater release of HNP 1–3 (Fig. 2).



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  		FIGURE 2. Greater release of {alpha}-defensins from TNF-{alpha}-primed neutrophils from donors homozygous for the NA1 allele of Fc{gamma}RIIIb (NA1/1) relative to donors homozygous for the NA2 allele of Fc{gamma}RIIIb (NA2/2) in response to heterotypic cross-linking of Fc{gamma}RIIa and Fc{gamma}RIIIb. Neutrophils in washed whole blood from normal donors homozygous for the NA1 or NA2 alleles were studied in a matched paired experimental design (n = 7 pairs). Released {alpha}-defensins (HNP 1–3) were measured as described in Materials and Methods. Data are presented as percent release relative to control incubated samples (mean ± SEM). CTRL, Control incubation of cells with mouse F(ab')2. Statistical analysis of the data was performed with a one-tailed paired t test and indicates that the percent release in NA1/1 donors is significant greater than that of NA2/2 donors (p = 0.004).

 
There was no difference in the concentration of released HNP 1–3 in washed whole blood supernatants after incubation with TNF-{alpha} alone in these donors (HNP1–3 (ng/ml): 198.7 ± 26.42 vs 196.1 ± 38.07 for NA1/1 vs NA2/2, p = 0.4340; seven pairs). The surface expression of Fc{gamma}RIIa and Fc{gamma}RIIIb on neutrophils was identical in the two groups (Fig. 3), with the mean MFI for Fc{gamma}RIIa being 56.3 ± 3.2 vs 55.1 ± 2.7 (p = 0.571; seven pairs) and the mean MFI for Fc{gamma}RIIIb being 366.4 ± 47.4 vs 377.6 ± 49.4 (p = 0.688; seven pairs) for NA1/1 and NA2/2 donors, respectively. Fc{gamma}RIIa is also functionally polymorphic. The H131/R131 polymorphism alters the binding of human IgG2 (15) and murine IgG1 (16) which results in differences in ligand-induced Fc{gamma}RIIa-mediated functions (36, 37). Because we have used Fab or F(ab')2 for engagement of Fc{gamma}Rs on neutrophils, Fc{gamma}RIIa alleles should not influence the magnitude of {alpha}-defensin release in this system. Indeed, there was no difference in the release of HNP 1–3 in donors homozygous for the H131 allele and the R131 allele of Fc{gamma}RIIa when donors were matched for the Fc{gamma}RIIIb NA genotype (data not shown).



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  		FIGURE 3. Expression of Fc{gamma}RIIa and Fc{gamma}RIIIb on the surface of neutrophils in washed whole blood from donors homozygous for the NA1 allele (NA1/1) (black line) or the NA2 allele (NA2/2) (gray line). Histograms of a representative experimental pair are shown. Expression of Fc{gamma}R was measured with FITC-conjugated anti-Fc{gamma}RIIa mAb IV.3 or anti-Fc{gamma}RIIIb mAb 3G8 using flow cytometry. Dotted lines, Isotype controls.

 
Fc{gamma}RIIIb allele-dependent release of other azurophilic granule constituents

There was little evidence of elastase release or display of MPO and PR3 on the cell surface after Fc{gamma}R cross-linking on unprimed neutrophils in washed whole blood. However, Fc{gamma}R induced release of elastase was strikingly enhanced by TNF-{alpha} (Fig. 4A). Likewise, cell surface expression of MPO and PR3, both ANCA target Ags found in azurophilic granules, was significantly up-regulated in response to Fc{gamma}R cross-linking after TNF-{alpha} priming (Fig. 4, B and C). Expression of MPO after receptor-specific stimulation was also significantly greater in NA1/1 donors relative to NA2/2 donors (Fig. 5), as was Fc{gamma}R-stimulated release of soluble MPO, measured functionally in the medium (Table I). In contrast to MPO, PR3 remains mainly membrane bound and is released only in minute amounts into the extracellular medium (38). Together, our results demonstrate that Fc{gamma}R-mediated degranulation of azurophilic granules, including the {alpha}-defensin subpopulation, was quantitatively greater in donors homozygous for the NA1 allele relative to donors homozygous for the NA2 allele of Fc{gamma}RIIIb.



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  		FIGURE 4. Release of azurophilic granule constituents in response to Fc{gamma}R cross-linking in the presence of TNF-{alpha}. Neutrophils in aliquots (300 µl) of washed whole blood were primed with TNF-{alpha} (50 ng/ml) for 20 min at 37°C and then stimulated with specific cross-linking of Fc{gamma}RIIa with IV.3 Fab (1 µg/ml) and/or Fc{gamma}RIIIb with 3G8 F(ab')2 (2 µg/ml) by G{alpha}M F(ab')2 (30 µg/ml) for 15 min at 37°C. Released elastase was measured by ELISA (A), and cell surface-associated MPO (B) and PR3 (C) were quantified by flow cytometry. CTRL, Control. *, p < 0.05; n = 5.

 


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  		FIGURE 5. The increase in cell-associated MPO in heterotypic Fc{gamma}R-stimulated TNF-{alpha}-primed neutrophils is greater in donors homozygous for the NA1 allele (NA1/1) relative to donors homozygous for the NA2 allele of Fc{gamma}RIIIb (NA2/2). Histograms from a representative experiment are shown: black thick line, NA1/1 donor; gray thick line, NA2/2 donor. MPO expressions on TNF-{alpha}-primed neutrophil (thin lines) and matched isotype control (IgG1) (dotted lines) after incubation were identical in both donors.

 

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  		Table I. Azurophilic granule release from primed neutrophils

 
ANCA-induced release of {alpha}-defensins

ANCA bind neutrophil-associated ANCA target and engage Fc{gamma}R with preferential binding to Fc{gamma}RIIIb (20, 21, 22). Although resting neutrophils in washed whole blood expressed little to no PR3 on the cell surface, incubation at 37°C for 40 min induced detectable cell surface PR3 expression as measured by the anti-PR3 mAb CLB12.8 (33). Incubation of cells with CLB12.8 for an additional 45 min at 37°C induced further degranulation as assessed by an increase in CD66b expression (22), the magnitude of which was dependent on the density of PR3 expression (Fig. 6A). This activation also showed Fc{gamma}RIIIb allele sensitivity (Fig. 6B).



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  		FIGURE 6. Monoclonal anti ({alpha})-PR3 (cANCA)-induced release of the granule constituent CD66b. A, Quantitative release of CD66b is strongly correlated with PR3-ANCA target expression on the surface of neutrophils in washed whole blood (p = 0.002). Each data point represents a different donor and, as previously reported (38 ), quantitative expression of PR3-ANCA target varied between donors. All data were derived from NA1/1-homozygous donors. B, Anti-PR3-induced granule release varies with the Fc{gamma}RIIIb alleles with donors homozygous for the NA1 allele showing quantitatively greater release of CD66b relative to donors homozygous for the NA2 allele. These donors expressed identical quantitative levels of PR3 on neutrophils after ANCA-target induction at 37°C for 40 min (MFI = 40.7 and 39.9 for the NA1/1 and NA2/2 donors, respectively).

 
Anti-PR3 mAb also induced release of {alpha}-defensins (HNP 1–3) (Fig. 7A). {alpha}-Defensin release by cells incubated alone (44.1 ± 8.8 ng/ml; Fig. 7A) was not different from cell incubated with an isotype control mAb (45.7 ± 7.3 ng/ml). Similarly, purified IgG from cANCA- and pANCA-containing plasmas, but not purified IgG from control donors that lack ANCA activity, resulted in significant release of HNP 1–3 (Fig. 8), and the magnitude of release was comparable with that of the mAb ANCA-induced release (Fig. 7A). This release was further enhanced by pretreatment of the washed whole blood with TNF-{alpha} (Fig. 8).



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  		FIGURE 7. ANCA-induced whole blood neutrophil activation can induce {alpha}-defensins release in an Fc{gamma}R-dependent manner. Neutrophils in washed whole blood were incubated at 37°C for 40 min, followed by incubation for 45 min with anti-PR3 mAb CLB12.8 (10 µg/ml) (A) or human cANCA (anti-PR3)-containing plasma (diluted 1/87 to final IgG concentrations ranging from 113 to 141 µg/ml; B) in the presence or absence of the IgG-Fc region-binding peptide (TG19320) (1000 µg/ml). Released {alpha}-defensins (HNP 1–3) in supernatant were measured by ELISA. p values were analyzed with one-tailed t test. *, Data represent 11 determinations using 4 PR3-ANCA-positive plasmas from 3 patients with WG. CTRL, Control.

 


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  		FIGURE 8. IgG purified from a cANCA-positive WG plasma (anti-PR3+), a pANCA-positive WG plasma (anti-MPO+) but not from an ANCA-negative plasma (ANCA-) from a disease-free control donor induce significant release of {alpha}-defensins. Neutrophils in washed whole blood were incubated at 37°C for 40 min in the absence ({square}) or presence of TNF-{alpha} ({blacksquare}), as described in Materials and Methods, followed by incubation for 45 min with the IgG fractions (diluted 1/87 to final IgG concentrations ranging from 113 to 141 µg/ml). Control cells were incubated for the same periods of time in the absence of any IgG addition. Released {alpha}-defensins (HNP 1–3) in supernatant were measured by ELISA. Data represent four determinations using each IgG preparation. TNF-{alpha} treatment resulted in a significant increase in HNP 1–3 secretion (p < 0.05) in the presence of the IgG preparations from PR3- and MPO-containing plasmas but not IgG from a control plasma or in the nonstimulated control. *, p < 0.05; **, p < 0.005

 
To demonstrate conclusively that the {alpha}-defensin-releasing activity in ANCA-containing plasma is indeed due to IgG-Fc region engagement of Fc{gamma}R, we used the Fc-region binding peptide TG19320, a peptide previously shown to block IgG-Fc region binding to Fc{gamma}R both in vitro and in vivo (31). {alpha}-Defensin release induced by the anti-PR3 mAb 12.8 was completely blocked by this Fc region-blocking peptide (Fig. 7A). Likewise, ANCA-induced release by four cANCA-containing WG plasmas was completely inhibited by the Fc region-binding peptide (Fig. 7B). Pretreatment of the cANCA plasmas, or anti-PR3 mAb 12.8, with a nonfunctional scrambled TG19320 had no effect on the anti-PR3 induced neutrophil activation (results not shown). The TG19320 studies, and the results in Fig. 8 using purified IgG from cANCA and pANCA positive plasma, clearly demonstrate that IgG in WG derived plasma are responsible for inducing {alpha}-defensin release. Thus, ANCA-mediated neutrophil activation, initiated by monoclonal or polyclonal cANCA, induces significant release of the {alpha}-defensins HNP1–3 and is completely dependent on Fc{gamma}R interactions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ANCA-mediated cross-linking of neutrophil Fc{gamma}R induces extracellular release of {alpha}-defensins capable of recruiting mononuclear, dendritic, and T cells to ANCA-induced granuloma. The magnitude of release is affected by the Fc{gamma}RIIIb genotype, which may explain the association of Fc{gamma}RIIIb genotype with disease phenotype (39). The ability of the TG19320 Fc-blocking peptide, which has been used effectively in some models of autoimmunity (31) to block this release, reveals a potential novel therapeutic approach to ANCA-related diseases.

Because isolation procedures can induce neutrophil activation (26, 33), we used a washed whole blood assay system, which avoids density gradient centrifugation and hypotonic lysis. We also avoided cytochalasin B, which enhances release of neutrophil granule constituents but which is clearly nonphysiological. Previously, we have reported that Fc{gamma}R-specific stimulation induces exocytosis of secretory vesicles in isolated neutrophils (27). Our current data demonstrate that Fc{gamma}R engagement initiated release of the {alpha}-defensin subpopulation of azurophilic granules, an effect that was enhanced by TNF-{alpha}. This effect was also sensitive to the functional NA1/NA2 polymorphism of Fc{gamma}RIIIb (Table I), as has been seen with specific granules (30). {alpha}-Defensins HNP1–3 are specific for neutrophils, and elastase, although found in monocytes and basophils, predominates in neutrophils (40). Thus, measurement of release of both {alpha}-defensins and elastase into the supernatant was feasible in the washed whole blood paradigm. Of course, Fc{gamma}RIIIb is expressed by neutrophils and not by monocytes, providing another parameter of cell type specificity.

Because subpopulations of azurophilic granules are found in neutrophils (1) and the dynamics of azurophilic constituent release vary (38), we measured several other azurophilic constituents either on the cell surface or in cell-free supernatant as markers for release of azurophilic granules. Display of cell-associated PR3 occurred more readily than other azurophilic markers, in part because PR3 is contained in secretory vesicles as well as in azurophilic granules and in part because very little is released into supernatant (38). Because myeloperoxidase is also released from monocytes, we used isolated neutrophils for measurement of both cell associated and soluble MPO in supernatants. Our results clearly show that Fc{gamma}R cross-linking is able to induce the release of azurophilic granules. Interestingly, up-regulation of CD63, a commonly used membrane marker for release of azurophilic granules, was less sensitive than other markers (data not shown). TNF-{alpha} was able to enhance release of all azurophilic constituents induced with Fc{gamma}R cross-linking.

The quantitative difference in release of {alpha}-defensins induced by the NA1 and NA2 alleles of Fc{gamma}RIIIb is not due to differential binding of 3G8 F(ab')2 to Fc{gamma}RIIIb alleles (41) or to quantitative differences in Fc{gamma}RIIa or Fc{gamma}RIIIb expression. Furthermore, the Fc{gamma}RIIa-H131/R131 polymorphism does not influence quantitative magnitude of release of {alpha}-defensins in response to receptor-specific cross-linking. Thus, the differences in granule release seen between NA1- and NA2-homozygous donors relates directly to differences in the function of these two different alleles. The same relationship is seen with ANCA-induced release.

In the washed whole blood paradigm, quantitative differences in cell surface PR3 expression correlate with quantitative degranulation induced by anti-PR3 (Fig. 6). We found heterogeneous levels of cell surface PR3 expression on neutrophils among our donors that appeared to be a stable individual characteristic, as reported by others (32, 33). Therefore, to establish the role of Fc{gamma}RIIIb genetics in anti-PR3-induced activation, we had to control for the level of PR3 expression among Fc{gamma}RIIIb-homozygous donors to demonstrate the predicted difference in degranulation (Fig. 6).

Although the contribution of Fc{gamma}RIIIb alleles to ANCA-induced activation support an important role for Fc{gamma}R, we also explored the ability of an IgG-Fc region blocking peptide, TG19320, which blocks IgG binding to Fc{gamma}R (31), to alter ANCA-induced activation. Strikingly, TG19320 peptide, but not its corresponding scrambled peptide, completely blocked cANCA-induced release of {alpha}-defensins. The same results were shown using multiple cANCA-containing plasmas and from isolated IgG preparations from patients with WG (Fig. 7B).

ANCA are typically observed in the circulation of patients with WG, microscopic polyangiitis, and Churg-Strauss syndrome. The two major ANCA targets are PR3 and MPO, and cANCA (anti-PR3) occurs in 80–90% of patients with WG (42) whereas other less common ANCA specificities have been reported (43, 44). ANCA can directly activate neutrophils (20, 21, 22, 45), and some data indicate that ANCA titers may parallel disease activity (46). The Fab components of the ANCA IgG molecule ligate ANCA targets. Bound ANCA IgG triggers neutrophils through Fc{gamma}RIIIb and Fc{gamma}RIIa; Fc{gamma}R-independent pathway(s) of activation may also play a role (47). It is likely that the polyclonal nature of the ANCA autoantibody repertoire results in the direct clustering of multiple Fc{gamma}R and ANCA target, eliciting functional responses. The ability of monoclonal anti-PR3 (so called monoclonal cANCA) to also directly stimulate neutrophils suggests that there are preformed clusters of PR3 (ANCA target) that facilitate Fc{gamma}R cross-linking or possibly that there are small preformed mAb aggregates. It is also possible that the direct cross-linking of Fc{gamma}R-PR3 results in functional responses. It is also interesting to speculate on the role that other Fc{gamma}R might play in ANCA-induced cell activation. ANCA have been demonstrated to activate monocytes in an Fc{gamma}R-dependent manner (48), implicating Fc{gamma}RIIa but also Fc{gamma}RIa. Likewise, activated neutrophils exposed to IFN-{gamma} or IL-10 will also express Fc{gamma}RIa, making this an additional potential neutrophil Fc{gamma}R targeted by ANCA. The recent description of the expression of the inhibitory Fc{gamma}R, Fc{gamma}RIIb, on monocytes (49) raises the intriguing suggestion that the magnitude of ANCA-induced cell activation could be modulated by changes in the balance of expression of the activating and inhibitory Fc{gamma}R. The expression of Fc{gamma}RIIb on human neutrophils is under active investigation.

Our results documenting ANCA-induced {alpha}-defensin release from neutrophils provides a mechanism to link ANCA-Fc{gamma}R interactions with the recruitment of key elements of the acquired immune response to ANCA-induced granuloma. The Fc{gamma}RIIIb allele sensitivity of this is consistent with previous observations that Fc{gamma}R alleles are significantly associated with disease activity in patients with WG (39, 50, 51). Furthermore, the ability of a small stable and soluble peptide inhibitor (TG19320) of IgG-Fc{gamma}R binding presents the possibility of a new strategy in the treatment of WG and also suggests possible approaches to the treatment and study of Ig-mediated diseases.


   Acknowledgments
 
We thank Dr. Richard Jones (University of Alabama Capstone Medical Center, Tuscaloosa, AL) for kindly providing WG plasmas and clinical data and Debbie McDuffie for expert technical assistance with the MPO functional assay and the ELISAs.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants RO1-AR42476 and RO1-AR33062. The University of Alabama Arthritis and Musculoskeletal Center Flow Cytometry Core Facility is supported by National Institutes of Health Grant P60-AR20614. Back

2 Current address: Department of Internal Medicine, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa, 228-8555, Japan. Back

3 Address correspondence and reprint requests to Dr. Jeffrey C. Edberg, Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, 433 Tinsley Harrison Tower, 1900 University Boulevard, Birmingham, AL 35294. E-mail address: jedberg{at}uab.edu Back

4 Abbreviations used in this paper: MPO, myeloperoxidase; HNP, human neutrophil peptide; ANCA, anti-neutrophil cytoplasmic Ab; WG, Wegener’s granulomatosis; G{alpha}M, goat anti-mouse IgG; mIgG1, mouse IgG1; RT, room temperature; MFI, mean fluorescence intensity; PR3, proteinase 3; cANCA, cytoplasmic staining ANCA; pANCA, perinuclear staining ANCA. Back

Received for publication February 19, 2003. Accepted for publication September 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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