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Antibody-Induced Release of Chemotaxins FcγRIIIb Allele-Sensitive Release of α -Defensins: Anti-Neutrophil 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 References This article cites 51 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/171/11/6090.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 neutrophils 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 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 ␣-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-proteinase 3). This release of ␣-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␥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 ␣-defensin 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 (3–5). 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 azurophilic granule 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 (9–11). 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 (17–19). 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 1–3) in diluted cell-free superna- with preferential engagement of Fc␥RIIIb presumably due to its tant was measured with the HNP 1–3 ELISA Kit (Cell Sciences, Norwood, numeric predominance over Fc␥RIIa (20–22). 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 1–3, 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.
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