Eosinophil Ribonucleases and Their Cutaneous Lesion-Forming Activity Douglas A. Plager, Mark D. P. Davis, Amy G. Andrews, Michael J. Coenen, Terry J. George, Gerald J. Gleich and This information is current as Kristin M. Leiferman of September 26, 2021. J Immunol 2009; 183:4013-4020; Prepublished online 28 August 2009; doi: 10.4049/jimmunol.0900055 http://www.jimmunol.org/content/183/6/4013 Downloaded from References This article cites 46 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/183/6/4013.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 26, 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Eosinophil Ribonucleases and Their Cutaneous Lesion-Forming Activity1 Douglas A. Plager,*2 Mark D. P. Davis,* Amy G. Andrews,† Michael J. Coenen,* Terry J. George,* Gerald J. Gleich,‡§ and Kristin M. Leiferman3* Eosinophil granule proteins are deposited in cutaneous lesions in many human diseases, but how these proteins contribute to patho- physiology is obscure. We injected eosinophil cationic protein (ECP or RNase 3), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil peroxidase (EPO), and major basic protein-1 (MBP1) intradermally into guinea pig and rabbit skin. ECP and EDN each induced distinct skin lesions at >2.5 ␮M that began at 2 days, peaking at ϳ7 days and persisting up to 6 wk. These lesions were ulcerated (ECP) or crusted (EDN) with marked cellular infiltration. EPO and MBP1 (10 ␮M) each produced perceptible induration and erythema with moderate cellular infiltration resolving within 2 wk. ECP and EDN localized to dermal cells within 2 days, whereas EPO and MBP1 remained extracellular. Overall, cellular localization and RNase activity of ECP and EDN were critical for lesion formation; differential Downloaded from glycosylation, net cationic charge, or RNase activity alone did not account for lesion formation. Ulcerated lesions from patients with the hypereosinophilic syndrome showed ECP and EDN deposition comparable to that in guinea pig skin. In conclusion, ECP and EDN disrupt skin integrity and cause inflammation. Their presence in ulcerative skin lesions may explain certain findings in human eosi- nophil-associated diseases. The Journal of Immunology, 2009, 183: 4013–4020. econdary eosinophil granules contain several highly cat- nicity of the granule proteins (pIs of ϳ11 for ECP, EPO, and http://www.jimmunol.org/ ionic proteins, including eosinophil cationic protein MBP1 and a pI of 8.7 for EDN) (2, 11, 12). Additionally, ECP and S (ECP,4 RNase 3)4 eosinophil-derived neurotoxin (EDN, EDN possess RNase activity, and EPO has peroxidase activity, RNase 2), eosinophil peroxidase (EPO), and major basic protein-1 whereas MBP1 is a C-type lectin (13). ECP and EDN are unique (MBP1) (1). EDN, EPO, and MBP1 are among the most abun- among the granule proteins in their ability to induce neurotoxicity, dantly transcribed genes in developing eosinophils (2) and are cor- referred to as the Gordon phenomenon (14, 15), and their RNase respondingly abundant in isolated peripheral blood eosinophils (3). activity appears critical for this ability (15). MBP1 activates sev- These granule proteins are extensively deposited in skin in several eral human cell types, such as mast cells, basophils, and eosino- dermatoses, such as atopic dermatitis, bullous pemphigoid, and phils (16–18). Thus, eosinophil granule proteins have broad po- urticaria, and in the hypereosinophilic syndrome (HES) (4). In tential to impact cellular and tissue functions. by guest on September 26, 2021 atopic dermatitis, eosinophil granule proteins are deposited at rel- Few studies have examined the in vivo effects of eosinophil granule atively high local concentrations (Ͼ1 ␮M) (5) without apparent proteins in skin. They induce cutaneous wheal and flare reactions (19, cutaneous eosinophilia, evidently as a result of cytolytic degranu- 20) and increase cutaneous vasopermeability (5). Overall, the impor- lation (6–8). tance of eosinophils in the pathophysiology of atopic disease, includ- Numerous in vitro studies have reported cytotoxic properties of ing atopic dermatitis, is controversial (21–24). Nonetheless, in dis- eosinophil granule proteins against mammalian cells, as well as eases with extensive cutaneous eosinophil granule protein deposition, pathogenic organisms, such as helminthes, bacteria, and viruses (1, such as in HES and severe atopic dermatitis, cutaneous lesions are 9, 10). Part of this cytotoxicity appears attributable to the catio- common. To define the in vivo effects of eosinophil granule protein deposition, we injected them intradermally into guinea pig skin and monitored cutaneous lesion formation. Subsequently, we examined *Department of Dermatology, †Department of Comparative Medicine, ‡Department the mechanism(s) of lesion formation, particularly with ECP and of Immunology, and §Department of Medicine, Mayo Clinic, Rochester, MN 55905 EDN, and compared immunohistologic staining of eosinophil granule Received for publication January 9, 2009. Accepted for publication July 11, 2009. proteins to that observed in erosive and ulcerative lesions in the hy- The costs of publication of this article were defrayed in part by the payment of page pereosinophilic syndrome. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants AI 11483, AI Materials and Methods 09728, AI 07047 (Training Grant), AI 34577, and AI 50494; the American Academy Human eosinophil granule proteins of Allergy, Asthma and Immunology Women Physicians in Allergy Grant (to K.M.L.); and by The Mayo Foundation, the Kieckhefer Foundation, and the Dr. Smith After approval by the Mayo Clinic Institutional Review Board, eosinophils H. and Lucille Gibson Postdoctoral Research Fellowship in Dermatology (to D.A.P.). from patients with marked eosinophilia (up to 84%) were obtained by cyta- 2 Address correspondence and reprint requests to Dr. Douglas A. Plager, Department pheresis (25). The methods for purifying eosinophil granules and granule pro- of Dermatology, Mayo Clinic, Guggenheim 4-94, 200 First Street SW, Rochester, teins have been described (14, 26–28). Distilled and deionized water was used MN 55905. E-mail address: [email protected] throughout all eosinophil granule protein purifications. Briefly, after cell lysis 3 Current address: Department of Dermatology, University of Utah Health Sciences and granule isolation, granules were solubilized in 0.01 M HCl (pH 3.0) with Center, Salt Lake City, UT 84132. sonication. After centrifugation (40,000 ϫ g, 20 min), the supernatant was ϫ 4 Abbreviations used in this paper: ECP, eosinophil cationic protein; Ang, pyroglu- fractionated at 4°C on a 1.2 100-cm Sephadex G-50 column equilibrated angiogenin; CM, carboxymethyl; EDN, eosinophil-derived neurotoxin; EPO, eosin- with 0.025 M sodium acetate, 0.15 M NaCl (pH 4.2). ophil peroxidase; HES, hypereosinophilic syndrome; MBP1, major basic protein-1. Fractions containing EPO were pooled, dialyzed against 0.05 M Na2HPO4,0.05MKH2PO4 (pH 8.0), centrifuged, applied to a carboxy- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 methyl (CM)-Sepharose column, and eluted with a linear NaCl gradient www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900055 4014 SKIN LESIONS INDUCED BY EOSINOPHIL RIBONUCLEASES (0.15–1.5 M). Fractions rich in EPO activity were pooled and dialyzed Injection sites were examined at least twice weekly. Lesions were as- against PBS (8.1 mM Na2PO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM sessed for appearance using the grading scale: 0, no visible or palpable KCl (pH 7.40)) and concentrated. The concentrated solution was centri- lesion; 0.5, trace visible erythema, edema, and/or palpable lesion; 1, visible fuged, and the supernatant was removed and frozen at Ϫ70°C. All prepa- erythema, edema with a palpable lesion; 2, erythematous, edematous, in- rations of EPO had absorbance ratios (absorbance at 412 nm/absorbance at durated palpable lesion; 3, palpable lesion with induration and mainly non- 280 nm) between 0.95 and 1.04. erosive epidermal changes (epidermal disruption with Ͻ50% of lesion Fractions from the Sephadex G-50 column containing ECP and EDN eroded or ulcerated); 4, induration with crusting or ulceration (Ͼ50% of were pooled, dialyzed against one-half concentration PBS (pH 7.4), con- lesion). For skin biopsy collection, animals were killed by intracardiac centrated, and fractionated on a heparin-Sepharose CL-6B column (1.2 ϫ injection of pentobarbital (Nembutal), and biopsies were immediately ob- 8 cm) equilibrated with one-half concentration PBS. Proteins bound to the tained by gently lifting the skin and using curved scissors to cut around column were eluted with a linear NaCl gradient (0.07–1.5 M NaCl). EDN the injection sites. Twenty-two guinea pigs and two rabbits were used in the eluted at a low salt concentration (ϳ0.2 M NaCl), whereas ECP eluted at studies; not all were tested with the same injections at the same time. The ϳ0.4 M NaCl (14). Extinction coefficients at 280 nm of 1.64 (mg/ number tested is listed in each table for the respective experiments. ml)Ϫ1cmϪ1 for EDN and 1.55 (mg/ml)Ϫ1cmϪ1 for ECP were used to de- termine protein concentrations (14).