FcγRIIIb Allele-Sensitive Release of α -: Anti- Cytoplasmic Antibody-Induced Release of Chemotaxins

This information is current as Sumiaki Tanaka, Jeffrey C. Edberg, Winn Chatham, Giorgio of September 24, 2021. Fassina and Robert P. Kimberly J Immunol 2003; 171:6090-6096; ; doi: 10.4049/jimmunol.171.11.6090 http://www.jimmunol.org/content/171/11/6090 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Fc␥RIIIb Allele-Sensitive Release of ␣-Defensins: Anti-Neutrophil Cytoplasmic Antibody-Induced Release of Chemotaxins1

Sumiaki Tanaka,2* Jeffrey C. Edberg,3*† Winn Chatham,* Giorgio Fassina,‡ and Robert P. Kimberly*†

Antineutrophil cytoplasmic Abs (ANCA) can activate in an Fc␥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 ␣-defensins, small cationic , are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and . In this study, we quantitated the release of ␣-defensins (human neutrophil peptides 1–3) from human neutrophils after targeted Fc␥R cross-linking (XL). Homotypic XL of Fc␥RIIa, Fc␥RIIIb, or heterotypic XL of both Downloaded from receptors resulted in significant release of ␣-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-). This release of ␣-defensins, as well as of other constituents (ANCA targets anti-proteinase 3 and and ), was significantly greater in donors homozygous for the NA1 allele of Fc␥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␥R. Based on their chemotactic properties,

␣-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma http://www.jimmunol.org/ formation. The greater activity of the Fc␥RIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele. The Journal of Immunology, 2003, 171: 6090–6096.

uman neutrophils contain many distinct granule popula- and various acid hydrolases. More recently, it has become clear that tions that can be mobilized on inflammatory stimulation among the subpopulations of azurophilic granules expressed in neu- H (1, 2). The release of these granules is a highly regulated trophils (1), there is a subpopulation of granules that contain the ␣-de- process with a gradient of responsiveness to stimulation. For ex- fensin antibacterial/chemotactic peptides (6, 7). The receptor-medi- ample, extracellular release of azurophilic granules requires a ated regulation of ␣- release has not been explored. by guest on September 24, 2021 higher threshold of activation than release of specific or secretory ␣-Defensins are a family of small (3.5- to 4.5-kDa) cationic vesicles (3Ð5). This is in keeping with a primary role for azuro- antimicrobial peptides with three to four intramolecular cysteine philic granule enzymes in intracellular degradation of phagocy- disulfide bonds and are widely distributed in mammals, insects, tosed particles. and plants (8). In addition to their antimicrobial properties, some The control of release is important in lim- of these peptides are potent chemotaxins for mononuclear cells (9), iting neutrophil-mediated tissue destruction at sites of inflamma- including dendritic cells and CD45RAϩ and CD8 T lymphocytes tion. These granules contain key enzymatic mediators of inflam- (10, 11)). Based on the pattern of their cysteine residues and di- 4 mation such as myeloperoxidase (MPO), elastase, collagenase, sulfide connections, six ␣ and two ␤ forms have been characterized in humans. Human ␣-defensins 1, 2, 3, and 4 are found in azuro-

*Division of Clinical Immunology and Rheumatology, Department of Medicine and philic granules in human neutrophils and thus are termed human †Department of Microbiology, University of Alabama, Birmingham, AL 35294; and neutrophil peptides (HNP) (12), whereas human ␣-defensins 5 and XEPTAGEN SpA, Pozzuoli, Italy 6 (HNP 5 and 6) are generated by small intestine Paneth cells (13). Received for publication February 19, 2003. Accepted for publication September Based on their chemotactic activity, human neutrophil ␣-defensins 22, 2003. contribute to modification of acquired immune responses by mo- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance bilization of mononuclear cells, T cells, and dendritic cells (9Ð11). with 18 U.S.C. Section 1734 solely to indicate this fact. Activation of human neutrophils by antineutrophil cytoplasmic 1 This work was supported by National Institutes of Health Grants RO1-AR42476 and Abs (ANCA) is at least partially dependent on engagement of RO1-AR33062. The University of Alabama Arthritis and Musculoskeletal Center Fc␥Rs on the surface of neutrophils. We hypothesized that en- Flow Cytometry Core Facility is supported by National Institutes of Health Grant P60-AR20614. gagement of Fc␥R on neutrophils would mobilize the ␣-defensin- 2 Current address: Department of Internal Medicine, Kitasato University School of positive azurophilic granule compartment and provide a link be- Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa, 228-8555, Japan. tween ANCA-induced neutrophil activation in ANCA-associated 3 Address correspondence and reprint requests to Dr. Jeffrey C. Edberg, Division of vasculitis (in particular Wegener’s granulomatosis (WG)) and Clinical Immunology and Rheumatology, Department of Medicine, University of Al- granuloma formation. Human neutrophils express two structurally abama, 433 Tinsley Harrison Tower, 1900 University Boulevard, Birmingham, AL 35294. E-mail address: [email protected] distinct Fc␥R, Fc␥RIIa and Fc␥RIIIb (14). Both of these receptors 4 Abbreviations used in this paper: MPO, myeloperoxidase; HNP, human neutrophil are functionally polymorphic with the H131/R131 single-nucle- peptide; ANCA, anti-neutrophil cytoplasmic Ab; WG, Wegener’s granulomatosis; ␥ otide polymorphism of Fc RIIa altering the binding of hIgG2 (15) G␣M, goat anti-mouse IgG; mIgG1, mouse IgG1; RT, room temperature; MFI, mean fluorescence intensity; PR3, proteinase 3; cANCA, cytoplasmic staining ANCA; and mIgG1 (16) and the NA1/NA2 single-nucleotide polymor- pANCA, perinuclear staining ANCA. phisms of Fc␥RIIIb altering the quantitative functional capacity of

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 6091 the receptor (17Ð19). ANCA have been shown to engage both ELISA for ␣-defensin and elastase ␥ neutrophil Fc R upon binding to cell-associated ANCA-target Ag The concentration of ␣-defensins (HNP 1Ð3) in diluted cell-free superna- with preferential engagement of Fc␥RIIIb presumably due to its tant was measured with the HNP 1Ð3 ELISA Kit (Cell Sciences, Norwood, numeric predominance over Fc␥RIIa (20Ð22). MA) according to the manufacturer’s instructions. This assay quantitates In this study, we show that cross-linking of neutrophil Fc␥R the three principal ␣-defensins, HNP 1Ð3, that are unique to neutrophils Ͼ results in significant release of ␣-defensins. The magnitude is in- and account for 99% of the total defensin content of these cells. The concentration of elastase in diluted cell-free supernatants was also mea- fluenced by the host receptor genetics, with donors homozygous sured by ELISA. Microtiter plates were coated with 100 ␮l of a rabbit for the NA1 allele of Fc␥RIIIb displaying significantly greater re- polyclonal anti-elastase (Biodesign, Saco, ME) (51 ␮g/ml) for2hatRT, lease. This release is induced by both monoclonal and human cy- and the wells were blocked with 0.1% BSA in PBS (PBS/0.1%BSA). The toplasmic staining ANCA (cANCA) (anti-proteinase 3 (PR3)) 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 from patients with WG, and importantly, its blockade by the Fc- incubated for2hatRTfollowed by four washes with PBS, 0.1% BSA. specific TG19320 peptide suggests an intervention strategy that Biotinylated rabbit polyclonal anti-elastase Ab prepared with the Biotin can be carried to the in vivo setting. Protein Labeling Kit (Roche Diagnostics, Mannheim, Germany) was added at 100 ␮g/ml in PBS, 0.1% BSA and incubated for2hatRT.After four washes, plates were incubated for 30 min at RT with avidin-alkaline phos- Materials and Methods phatase (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 ␮l Blood donors of 1 mg/ml p-nitrophenyl phosphate (Sigma-Aldrich) in 10% diethanol- amine (Sigma-Aldrich). After 30 min at RT, the reaction was stopped with Whole blood anticoagulated with EDTA was obtained from healthy vol- ␮ unteers. All donors were genotyped for both neutrophil Fc␥Rs (Fc␥RIIa 50 l of 0.1% EDTA and OD405 was determined (VMAX; Molecular Downloaded from and Fc␥RIIIb) by allele-specific PCR (23, 24). In some studies, neutrophils Devices, Sunnyvale, CA). were isolated by centrifugation through a discontinuous Ficoll-Hypaque density gradient as previously described (25). The protocol for phlebotomy Functional MPO activity assay was approved by the Institutional Committee on Human Rights in Research. Functional MPO activity was performed as previously described (28). Briefly, isolated neutrophils (2 ϫ 106/ml) were added to wells coated with IgG (100 ␮g/ml) and incubated at 37¡Cfor1htoheterotypically cross-link Antibodies with Fc␥RIIa and Fc␥RIIIb. After stimulation, cell-free supernatants were http://www.jimmunol.org/ collected and stored at Ϫ20¡C. Released MPO was quantitated by mixing ␥ Ј ␥ Anti-Fc RIIIb mAb 3G8 F(ab )2 or IgG, anti-Fc RII mAb IV.3 Fab and 300 ␮l of dilutions of cell-free supernatants with 250 ␮lof80mM FITC-conjugated 3G8 (mIgG1) and IV.3 (mIgG2b) were purchased from ␮ Ј Ј NaH2Po4 buffer, pH 5.4, and 80 lof20mM3,3,5,5 -tetramethylbenzi- Medarex (Annandale, NJ). F(abЈ) fragments of F(abЈ) -specific goat anti- ␮ 2 2 dine in dimethylformamide. Then 100 l of 1.0 mM H2O2 were added, and mouse IgG (G␣M) for anti-Fc␥R mAb cross-linking, Fc-specific PE-con- ⌬ the OD655 was recorded at 15-s intervals at 25¡C for 1 min (29). Released jugated G␣M F(abЈ) for detection of primary Abs, and murine F(abЈ) ⌬ 2 2 functional MPO activity is represented as OD655/min during the liner were obtained from Jackson ImmunoResearch Laboratories (West Grove, phase of the reaction. PA). The anti-PR3 mAb CLB 12.8 (mIgG1) was from Research Diagnos- tics (Flanders, NJ), and the anti-MPO mAb MPO-7 (mIgG1) was from Dako (Carpinteria, CA). Anti-CD66b mAb CLB-B13.9-FITC (mouse IgG1 cANCA-induced neutrophil activation by guest on September 24, 2021 (mIgG1)) and FITC- or PE-conjugated isotype-matched controls were pur- ANCA (mAb or human) stimulation of neutrophils in washed whole blood chased from Caltag (San Francisco, CA). Human cANCA (anti-PR3) or was performed as described above except that the washed cells were pre- perinuclear staining ANCA (pANCA) (anti-MPO) plasmas were obtained incubated for 40 min at 37¡C to induce cell surface expression of protein- from five patients with active WG. IgG from the plasmas was purified as ase 3 (PR3) ANCA target. Preliminary studies indicated that near maximal previously described (21, 22). surface PR3 was detected at this time point. Aliquots were then incubated with or without anti-PR3 mAb CLB 12.8 (10 ␮g/ml) or diluted cANCA- Washed whole blood assay positive plasma for 45 min. After stimulation, supernatants were harvested as described above, and cells in washed whole blood were analyzed for Neutrophils in washed whole blood, which eliminates isolation-induced CD66b expression by flow cytometry as described above (30). neutrophil activation (26), were stimulated as described previously (27). In some studies, we used the IgG-Fc region-binding peptide (TG19320) Briefly, EDTA-anticoagulated blood was chilled to 4¡C, washed twice in (31) or a scrambled TG19320 peptide to block ANCA-induced neutrophil modified PBS (125 mM sodium chloride, 10 mM phosphate, 5 mM po- activation. In these studies, aliquots of washed whole blood were incubated tassium chloride, 5 mM glucose, pH 7.35), and then resuspended in the with anti-PR3 mAb or human ANCA pretreated with various concentration original volume. Washed whole blood was keep at 4¡C, and all washes of peptides at 37¡C at 45 min. were performed with modified PBS at 4¡C. For neutrophil stimulation, aliquots (300 ␮l) of washed whole blood were incubated with saturating Flow cytometry concentrations of the anti-Fc␥RII- and/or anti-Fc␥RIII-specific mAb frag- ␮ ␮ Ј ments (1 g/ml IV.3 Fab and/or 2 g/ml 3G8 F(ab )2) for 15 min at 4¡C, Data were collected using a BD Biosciences FACSCalibur. The instrument washed twice, and resuspended in buffer with 1.09 mM CaCl2 and 1.62 was routinely calibrated using fluorescent beads (Rainbow Calibration Par- Ј ␮ mM MgCl2. Cell-bound mAb fragments were cross-linked with F(ab )2 ticles, 3.5 m; Spherotech, Libertyville, IL). Neutrophils in whole blood G␣M (35 ␮g/ml) at 37¡C for 15 min. Alternately, cells were stimulated were identified by characteristic light scatter properties and confirmed by with PMA (Sigma-Aldrich, St. Louis, MO; 10 ␮g/ml) as a positive control Fc␥R characteristic expression (14) using FITC-conjugated IV.3 and 3G8. Ј ␣ or with F(ab )2 G M as a negative control. Endotoxin levels in all reagents As previously reported, cell surface expression of PR3 displayed a bimodal were below detection limits (Limulus ameobocyte assay; Sigma-Aldrich) pattern (32, 33). as described (22, 27). In some studies, cells were pretreated with 50 ng/ml Analysis of flow cytometry listmode date was done using CellQuest (BD rTNF-␣ (R&D, Minneapolis, MN) for 20 min at 37¡C before mAb cross- Biosciences). The results are expressed as mean fluorescence intensity linking. After stimulation, cell-free supernatants were then collected and (MFI) of the histogram data. stored at Ϫ20¡C. After two washes, the remaining G␣M binding sites on Ј ␮ the cells were blocked with murine F(ab )2 (88 g/ml). Aliquots of stim- Data analysis ulated cells were incubated with primary mAb for flow cytometry for 30 min at 4¡C. Unconjugated mAb were detected with PE-conjugated G␣M For quantitation of azurophilic granule release in donors homozygous for Ј ␥ ␥ F(ab )2 for 30 min. Fc R expression on neutrophil in washed whole blood Fc RIIIb alleles, experiments were performed in a matched paired donor was quantitated with FITC-conjugated anti-Fc␥RIIIb mAb 3G8 or anti- design. The effect of activating stimuli on Fc␥R-mediated Fc␥RII mAb IV.3. All samples were then treated with FACS Lysing So- are presented as percent increase relative to control incubations. Data are lution (BD Biosciences, San Jose, CA) for 10 min at room temperature displayed as mean Ϯ SEM. The effects of stimuli were compared using the (RT), washed once, and analyzed by flow cytometry (FACSCalibur; BD t test. p ϭ 0.05 was used to reject the null hypothesis that there is no Biosciences). difference between the groups or conditions. 6092 ANCA-INDUCED ␣-DEFENSIN RELEASE

Results Neutrophil Fc␥R-mediated release of ␣-defensin To study the release of ␣-defensins in response to Fc␥R cross- linking, we used ELISA to quantitate the concentration of HNP 1Ð3 in cell-free supernatants after targeted Fc␥R cross-linking on neutrophils in washed whole blood. No significant release of HNP 1Ð3 above baseline was detected in response to homotypic cross- linking of either Fc␥RIIa or Fc␥RIIIb. However, HNP 1Ð3 were released from neutrophils after co-cross-linking of both Fc␥RIIa and Fc␥RIIIb together and after stimulation with 10 ␮g/ml PMA (Fig. 1A). Because TNF-␣ can prime neutrophils for enhanced FIGURE 2. Greater release of ␣-defensins from TNF-␣-primed neutro- stimulus-mediated degranulation (28, 34, 35), we examined the phils from donors homozygous for the NA1 allele of Fc␥RIIIb (NA1/1) ␣ ␥ ␣ release of -defensins induced by Fc R cross-linking in TNF- - relative to donors homozygous for the NA2 allele of Fc␥RIIIb (NA2/2) in primed neutrophils to enhance release of azurophilic granules. response to heterotypic cross-linking of Fc␥RIIa and Fc␥RIIIb. Neutro- TNF-␣ (50 ng/ml) alone induced significant release of HNP 1Ð3 phils in washed whole blood from normal donors homozygous for the NA1 and markedly enhanced the Fc␥R-mediated release of HNP 1Ð3 or NA2 alleles were studied in a matched paired experimental design (n ϭ (Fig. 1B). In the presence of TNF-␣, homotypic cross-linking of 7 pairs). Released ␣-defensins (HNP 1Ð3) were measured as described in Fc␥RIIa or Fc␥RIIIb and heterotypic cross-linking of Fc␥RIIa and Materials and Methods. Data are presented as percent release relative to Downloaded from Fc␥RIIIb induced significant release of HNP 1Ð3 relative to con- control incubated samples (mean Ϯ SEM). CTRL, Control incubation of cells with mouse F(abЈ) . Statistical analysis of the data was performed trol incubation. These results indicated that cross-linking of Fc␥Rs 2 could induce the release of the ␣-defensin subpopulation of azuro- 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 ϭ 0.004). philic granules in both unprimed and primed neutrophils.

Fc␥R-mediated release of ␣-defensins from neutrophils is

␥ http://www.jimmunol.org/ Fc RIIIb allele dependent allele of Fc␥RIIIb showed significantly greater release of HNP 1Ð3 Donors homozygous for the NA1 allele of Fc␥RIIIb display a (Fig. 2). quantitatively greater Fc␥R-dependent phagocytic capacity (17) There was no difference in the concentration of released HNP and greater Fc␥RIIIb enhancement of Fc␥RIIa-specific phagocy- 1Ð3 in washed whole blood supernatants after incubation with tosis relative to NA2-homozygous donors (19). To test whether the TNF-␣ alone in these donors (HNP1Ð3 (ng/ml): 198.7 Ϯ 26.42 vs NA1 allele might have a greater capacity to mobilize azurophilic 196.1 Ϯ 38.07 for NA1/1 vs NA2/2, p ϭ 0.4340; seven pairs). The granules relative to the NA2 allele, we assessed the influence of surface expression of Fc␥RIIa and Fc␥RIIIb on neutrophils was Fc␥RIIIb-NA1/NA2 polymorphism on the quantitative magnitude identical in the two groups (Fig. 3), with the mean MFI for ␥ Ϯ Ϯ ϭ of release of ␣-defensins in response to heterotypic cross-linking Fc RIIa being 56.3 3.2 vs 55.1 2.7 ( p 0.571; seven pairs) by guest on September 24, 2021 of Fc␥RIIa and Fc␥RIIIb on TNF-␣-primed neutrophils. Indeed, and the mean MFI for Fc␥RIIIb being 366.4 Ϯ 47.4 vs 377.6 Ϯ heterotypic cross-linking from donors homozygous for the NA1 49.4 ( p ϭ 0.688; seven pairs) for NA1/1 and NA2/2 donors, re- spectively. Fc␥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␥RIIa-mediated functions (36, 37). Because we have used Fab

FIGURE 1. Fc␥R cross-linking induces release of ␣-defensins. A, Het- erotypic cross-linking but not homotypic cross-linking of Fc␥RIIa and Fc␥RIIIb on unprimed neutrophils induced significant release of ␣-de- fensins (HNP 1Ð3). B, Both heterotypic and homotypic cross-linking of Fc␥RIIa and/or Fc␥RIIIb on TNF-␣-primed neutrophils in washed whole blood induced significant release of ␣-defensins (HNP 1Ð3). Unprimed or TNF-␣-primed neutrophils in aliquots (300 ␮l) of a washed whole blood were stimulated with specific cross-linking of Fc␥RIIa with IV.3 Fab (1 ␮ ␥ Ј ␮ ␣ Ј ␥ ␥ g/ml) and/or Fc RIIIb with 3G8 F(ab )2 (2 g/ml) by G M F(ab )2 (30 FIGURE 3. Expression of Fc RIIa and Fc RIIIb on the surface of neu- ␮g/ml) for 15 min at 37¡C. The concentrations of HNP 1Ð3 were measured trophils in washed whole blood from donors homozygous for the NA1 by ELISA (n ϭ 2Ð4). Bars, SEM. CTRL, Control incubation of cells with allele (NA1/1) (black line) or the NA2 allele (NA2/2) (gray line). Histo- Ј ␣ ␣ ␥ mouse F(ab )2. TNF- significantly enhanced release of -defensins in all grams of a representative experimental pair are shown. Expression of Fc R -p Ͻ 0.001 was measured with FITC-conjugated anti-Fc␥RIIa mAb IV.3 or anti ,ءء ,p Ͻ 0.05, relative to control incubation ,ء .(cases (p Ͻ 0.03 relative to control incubation. Fc␥RIIIb mAb 3G8 using flow cytometry. Dotted lines, Isotype controls. The Journal of Immunology 6093

FIGURE 4. Release of azurophilic granule constituents in response to Fc␥R cross-linking in the presence of TNF-␣. Neutrophils in aliquots (300 ␮l) of washed whole blood were primed with TNF-␣ (50 ng/ml) for 20 min at 37¡C and then stimulated with specific cross-linking of Fc␥RIIa with IV.3 Fab (1 ␮g/ml) and/or ␥ Ј ␮ ␣ Fc RIIIb with 3G8 F(ab )2 (2 g/ml) by G M Ј ␮ F(ab )2 (30 g/ml) for 15 min at 37¡C. Released elastase was measured by ELISA (A), and cell surface-associated MPO (B) and PR3 (C) were ,ء .quantified by flow cytometry. CTRL, Control p Ͻ 0.05; n ϭ 5.

Ј ␥ ␥ ␣ or F(ab )2 for engagement of Fc Rs on neutrophils, Fc RIIa al- philic granules, including the -defensin subpopulation, was quan- leles should not influence the magnitude of ␣-defensin release in titatively greater in donors homozygous for the NA1 allele relative this system. Indeed, there was no difference in the release of HNP to donors homozygous for the NA2 allele of Fc␥RIIIb. 1Ð3 in donors homozygous for the H131 allele and the R131 allele Downloaded from of Fc␥RIIa when donors were matched for the Fc␥RIIIb NA ge- ANCA-induced release of ␣-defensins notype (data not shown). ANCA bind neutrophil-associated ANCA target and engage Fc␥R ␥ Fc␥RIIIb allele-dependent release of other azurophilic granule with preferential binding to Fc RIIIb (20Ð22). Although resting constituents neutrophils in washed whole blood expressed little to no PR3 on the cell surface, incubation at 37¡C for 40 min induced detectable http://www.jimmunol.org/ There was little evidence of elastase release or display of MPO and cell surface PR3 expression as measured by the anti-PR3 mAb PR3 on the cell surface after Fc␥R cross-linking on unprimed neu- CLB12.8 (33). Incubation of cells with CLB12.8 for an additional trophils in washed whole blood. However, Fc␥R induced release 45 min at 37¡C induced further degranulation as assessed by an of elastase was strikingly enhanced by TNF-␣ (Fig. 4A). Likewise, increase in CD66b expression (22), the magnitude of which was cell surface expression of MPO and PR3, both ANCA target Ags dependent on the density of PR3 expression (Fig. 6A). This acti- found in azurophilic granules, was significantly up-regulated in vation also showed Fc␥RIIIb allele sensitivity (Fig. 6B). response to Fc␥R cross-linking after TNF-␣ priming (Fig. 4, B and Anti-PR3 mAb also induced release of ␣-defensins (HNP 1Ð3) C). Expression of MPO after receptor-specific stimulation was also (Fig. 7A). ␣-Defensin release by cells incubated alone (44.1 Ϯ 8.8 significantly greater in NA1/1 donors relative to NA2/2 donors ng/ml; Fig. 7A) was not different from cell incubated with an iso- by guest on September 24, 2021 (Fig. 5), as was Fc␥R-stimulated release of soluble MPO, mea- type control mAb (45.7 Ϯ 7.3 ng/ml). Similarly, purified IgG from sured functionally in the medium (Table I). In contrast to MPO, cANCA- and pANCA-containing plasmas, but not purified IgG PR3 remains mainly membrane bound and is released only in from control donors that lack ANCA activity, resulted in signifi- minute amounts into the extracellular medium (38). Together, our cant release of HNP 1Ð3 (Fig. 8), and the magnitude of release was results demonstrate that Fc␥R-mediated degranulation of azuro- 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-␣ (Fig. 8). To demonstrate conclusively that the ␣-defensin-releasing ac- tivity in ANCA-containing plasma is indeed due to IgG-Fc region engagement of Fc␥R, we used the Fc-region binding peptide TG19320, a peptide previously shown to block IgG-Fc region binding to Fc␥R both in vitro and in vivo (31). ␣-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 nonfunc- tional 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 de- rived plasma are responsible for inducing ␣-defensin release. Thus, ANCA-mediated neutrophil activation, initiated by mono- clonal or polyclonal cANCA, induces significant release of the FIGURE 5. The increase in cell-associated MPO in heterotypic Fc␥R- ␣-defensins HNP1Ð3 and is completely dependent on Fc␥R stimulated TNF-␣-primed neutrophils is greater in donors homozygous for interactions. the NA1 allele (NA1/1) relative to donors homozygous for the NA2 allele of Fc␥RIIIb (NA2/2). Histograms from a representative experiment are shown: black thick line, NA1/1 donor; gray thick line, NA2/2 donor. MPO Discussion expressions on TNF-␣-primed neutrophil (thin lines) and matched isotype ANCA-mediated cross-linking of neutrophil Fc␥R induces extra- control (IgG1) (dotted lines) after incubation were identical in both donors. cellular release of ␣-defensins capable of recruiting mononuclear, 6094 ANCA-INDUCED ␣-DEFENSIN RELEASE

Table I. Azurophilic granule release from primed neutrophils

Granule Protein Releasea

Azurophilic Granule Constituents NA1/1 NA2/2 pb

HNP 1Ð3 (soluble) (%) 253.8 Ϯ 37.7 209.7 Ϯ 37.2 0.004 (n ϭ 7) Elastase (soluble) (%) 570.2 Ϯ 117.0 379.8 Ϯ 77.1 0.043 (n ϭ 6) MPO (cell associated) (%) 221.4 Ϯ 16.1 170.7 Ϯ 13.5 0.002 (n ϭ 6) PR3 (cell associated) (%) 337.7 Ϯ 29.7 295.0 Ϯ 23.3 0.005 (n ϭ 6) MPO (functional) (⌬OD/min)c 0.354 Ϯ 0.033 0.195 Ϯ 0.051 0.010 (n ϭ 6)

a Azurophilic granule marker release from primed neutrophils induced by heterotypic cross-linking of Fc␥RIIa and Fc␥RIIIb is greater in donors homozygous for the NA1 allele (NA1/1) relative to the NA2 allele (NA2/2) of Fc␥RIIIb. Numbers represent percent increase relative to control incubation (mean Ϯ SEM). Release of ␣-defensins (HNP 1Ð3) and elastase in the supernatant was quantitated by ELISA, and quantitation of cell-associated MPO and PR3 was assessed by flow cytometry. b p values were analyzed by one-tailed paired t test in six or seven pairs. c Isolated neutrophils were activated with immobilized IgG, and released functional MPO activity was measured as described in Materials and Methods. Numbers represent difference from control incubation (mean Ϯ SEM). dendritic, and T cells to ANCA-induced granuloma. The magni- ther on the cell surface or in cell-free supernatant as markers for tude of release is affected by the Fc␥RIIIb genotype, which may release of azurophilic granules. Display of cell-associated PR3 oc- Downloaded from explain the association of Fc␥RIIIb genotype with disease pheno- curred more readily than other azurophilic markers, in part because type (39). The ability of the TG19320 Fc-blocking peptide, which PR3 is contained in secretory vesicles as well as in azurophilic has been used effectively in some models of autoimmunity (31) to granules and in part because very little is released into supernatant block this release, reveals a potential novel therapeutic approach to (38). Because myeloperoxidase is also released from monocytes, ANCA-related diseases. we used isolated neutrophils for measurement of both cell associ- Because isolation procedures can induce neutrophil activation ated and soluble MPO in supernatants. Our results clearly show (26, 33), we used a washed whole blood assay system, which that Fc␥R cross-linking is able to induce the release of azurophilic http://www.jimmunol.org/ avoids density gradient centrifugation and hypotonic lysis. We also granules. Interestingly, up-regulation of CD63, a commonly used avoided cytochalasin B, which enhances release of neutrophil membrane marker for release of azurophilic granules, was less granule constituents but which is clearly nonphysiological. Previ- sensitive than other markers (data not shown). TNF-␣ was able to ously, we have reported that Fc␥R-specific stimulation induces enhance release of all azurophilic constituents induced with Fc␥R exocytosis of secretory vesicles in isolated neutrophils (27). Our cross-linking. current data demonstrate that Fc␥R engagement initiated release of The quantitative difference in release of ␣-defensins induced by the ␣-defensin subpopulation of azurophilic granules, an effect that the NA1 and NA2 alleles of Fc␥RIIIb is not due to differential ␣ Ј ␥ was enhanced by TNF- . This effect was also sensitive to the binding of 3G8 F(ab )2 to Fc RIIIb alleles (41) or to quantitative by guest on September 24, 2021 functional NA1/NA2 polymorphism of Fc␥RIIIb (Table I), as has differences in Fc␥RIIa or Fc␥RIIIb expression. Furthermore, the been seen with specific granules (30). ␣-Defensins HNP1Ð3 are Fc␥RIIa-H131/R131 polymorphism does not influence quantita- specific for neutrophils, and elastase, although found in monocytes tive magnitude of release of ␣-defensins in response to receptor- and , predominates in neutrophils (40). Thus, measure- specific cross-linking. Thus, the differences in granule release seen ment of release of both ␣-defensins and elastase into the superna- between NA1- and NA2-homozygous donors relates directly to dif- tant was feasible in the washed whole blood paradigm. Of course, ferences in the function of these two different alleles. The same Fc␥RIIIb is expressed by neutrophils and not by monocytes, pro- relationship is seen with ANCA-induced release. viding another parameter of cell type specificity. In the washed whole blood paradigm, quantitative differences in Because subpopulations of azurophilic granules are found in cell surface PR3 expression correlate with quantitative degranula- neutrophils (1) and the dynamics of azurophilic constituent release tion induced by anti-PR3 (Fig. 6). We found heterogeneous levels vary (38), we measured several other azurophilic constituents ei- of cell surface PR3 expression on neutrophils among our donors

FIGURE 6. Monoclonal anti (␣)-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 ϭ 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␥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 37oC for 40 min (MFI ϭ 40.7 and 39.9 for the NA1/1 and NA2/2 donors, respectively). The Journal of Immunology 6095

FIGURE 8. IgG purified from a cANCA-positive WG plasma (anti- PR3ϩ), a pANCA-positive WG plasma (anti-MPOϩ) but not from an ANCA-negative plasma (ANCAϪ) from a disease-free control donor in- duce significant release of ␣-defensins. Neutrophils in washed whole blood were incubated at 37¡C for 40 min in the absence (Ⅺ) or presence of TNF-␣ (f), as described in Materials and Methods, followed by incubation for 45 min with the IgG fractions (diluted 1/87 to final IgG concentrations Downloaded from ranging from 113 to 141 ␮g/ml). Control cells were incubated for the same periods of time in the absence of any IgG addition. Released ␣-defensins (HNP 1Ð3) in supernatant were measured by ELISA. Data represent four determi- nations using each IgG preparation. TNF-␣ treatment resulted in a significant increase in HNP 1Ð3 secretion (p Ͻ 0.05) in the presence of the IgG prepa- rations from PR3- and MPO-containing plasmas but not IgG from a control

/p Ͻ 0.005 http://www.jimmunol.org ,ءء ;p Ͻ 0.05 ,ء .plasma or in the nonstimulated control

FIGURE 7. ANCA-induced whole blood neutrophil activation can in- Fc␥R and ANCA target, eliciting functional responses. The ability ␣ ␥ duce -defensins release in an Fc R-dependent manner. Neutrophils in of monoclonal anti-PR3 (so called monoclonal cANCA) to also washed whole blood were incubated at 37¡C for 40 min, followed by in- ␮ directly stimulate neutrophils suggests that there are preformed cubation for 45 min with anti-PR3 mAb CLB12.8 (10 g/ml) (A) or human ␥ cANCA (anti-PR3)-containing plasma (diluted 1/87 to final IgG concen- clusters of PR3 (ANCA target) that facilitate Fc R cross-linking or trations ranging from 113 to 141 ␮g/ml; B) in the presence or absence of possibly that there are small preformed mAb aggregates. It is also the IgG-Fc region-binding peptide (TG19320) (1000 ␮g/ml). Released possible that the direct cross-linking of Fc␥R-PR3 results in func- ␣-defensins (HNP 1Ð3) in supernatant were measured by ELISA. p values tional responses. It is also interesting to speculate on the role that

Data represent 11 determinations other Fc␥R might play in ANCA-induced cell activation. ANCA by guest on September 24, 2021 ,ء .were analyzed with one-tailed t test using 4 PR3-ANCA-positive plasmas from 3 patients with WG. CTRL, have been demonstrated to activate monocytes in an Fc␥R-depen- Control. dent manner (48), implicating Fc␥RIIa but also Fc␥RIa. Likewise, activated neutrophils exposed to IFN-␥ or IL-10 will also express Fc␥RIa, making this an additional potential neutrophil Fc␥R tar- that appeared to be a stable individual characteristic, as reported by geted by ANCA. The recent description of the expression of the others (32, 33). Therefore, to establish the role of Fc␥RIIIb genet- inhibitory Fc␥R, Fc␥RIIb, on monocytes (49) raises the intriguing ics in anti-PR3-induced activation, we had to control for the level suggestion that the magnitude of ANCA-induced cell activation of PR3 expression among Fc␥RIIIb-homozygous donors to dem- could be modulated by changes in the balance of expression of the onstrate the predicted difference in degranulation (Fig. 6). activating and inhibitory Fc␥R. The expression of Fc␥RIIb on hu- Although the contribution of Fc␥RIIIb alleles to ANCA-induced man neutrophils is under active investigation. activation support an important role for Fc␥R, we also explored the Our results documenting ANCA-induced ␣-defensin release ability of an IgG-Fc region blocking peptide, TG19320, which from neutrophils provides a mechanism to link ANCA-Fc␥R in- blocks IgG binding to Fc␥R (31), to alter ANCA-induced activa- teractions with the recruitment of key elements of the acquired tion. Strikingly, TG19320 peptide, but not its corresponding immune response to ANCA-induced granuloma. The Fc␥RIIIb al- scrambled peptide, completely blocked cANCA-induced release of lele sensitivity of this is consistent with previous observations that ␣-defensins. The same results were shown using multiple cANCA- Fc␥R alleles are significantly associated with disease activity in containing plasmas and from isolated IgG preparations from pa- patients with WG (39, 50, 51). Furthermore, the ability of a small tients with WG (Fig. 7B). stable and soluble peptide inhibitor (TG19320) of IgG-Fc␥R bind- ANCA are typically observed in the circulation of patients with ing presents the possibility of a new strategy in the treatment of WG, microscopic polyangiitis, and Churg-Strauss syndrome. The WG and also suggests possible approaches to the treatment and two major ANCA targets are PR3 and MPO, and cANCA (anti- study of Ig-mediated diseases. PR3) occurs in 80Ð90% of patients with WG (42) whereas other less common ANCA specificities have been reported (43, 44). Acknowledgments ANCA can directly activate neutrophils (20Ð22, 45), and some We thank Dr. Richard Jones (University of Alabama Capstone Medical data indicate that ANCA titers may parallel disease activity (46). Center, Tuscaloosa, AL) for kindly providing WG plasmas and clinical The Fab components of the ANCA IgG molecule ligate ANCA data and Debbie McDuffie for expert technical assistance with the MPO functional assay and the ELISAs. targets. Bound ANCA IgG triggers neutrophils through Fc␥RIIIb and Fc␥RIIa; Fc␥R-independent pathway(s) of activation may also References play a role (47). It is likely that the polyclonal nature of the ANCA 1. Borregaard, N., and J. B. Cowland. 1997. Granules of the human neutrophilic autoantibody repertoire results in the direct clustering of multiple polymorphonuclear leukocyte. Blood 89:3503. 6096 ANCA-INDUCED ␣-DEFENSIN RELEASE

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