Cutaneous Lesion-Forming Activity Eosinophil Ribonucleases and Their

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

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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 © 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 pathophysiology 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 eosinophil-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 determine protein concentrations (14). To obtain ECP1 and ECP2 (14), two Indirect immunofluorescence and histologic staining forms of differentially glycosylated ECP, ECP fractions pooled from the Human ECP, EDN, EPO, and MBP were detected in formalin-fixed, par- initial heparin-Sepharose column were subjected to a second heparin- affin-embedded biopsy specimens of cutaneous injection sites by indirect Sepharose chromatography. These latter fractions were analyzed by SDS- immunofluorescence. Briefly, serial tissue sections (5 ␮m) were mounted PAGE followed by colloidal Coomassie blue staining (GelCode Blue; on positively charged microscope slides, deparaffinized, and incubated in Pierce), and ECP1 and ECP2 were pooled separately. 0.1% trypsin for1hat37°C.Theslides were incubated overnight in 10% Fractions containing MBP1 (2, 29) from the Sephadex G-50 column normal goat serum at 4°C. The next day, sections were washed and over- were pooled and stored at Ϫ70°C in 0.025 M sodium acetate, 0.15 M NaCl

laid with either control rabbit Ab (normal rabbit IgG or rabbit preimmu- Downloaded from (pH 4.2) to inhibit polymerization. MBP1 concentrations were determined Ϫ Ϫ nization serum) or granule protein-specific Ab (affinity chromatography- using an extinction coefficient at 277 nm of 2.63 (mg/ml) 1cm 1 (26). Use Ϫ Ϫ purified rabbit anti-human EDN and MBP or rabbit antiserum against ECP of a more recently determined extinction coefficient (3.67 (mg/ml) 1cm 1) and EPO, all produced in our laboratory). Slides were washed and treated would have resulted in 28% lower calculated MBP1 concentrations (2). with 1% chromotrope 2R (J. T. Baker) to eliminate nonspecific eosinophil The original calculations were used throughout the study for consistency staining (30). Subsequently, slides were overlaid with FITC-conjugated with other granule protein concentrations. All eosinophil granule protein goat anti-rabbit IgG (SouthernBiotech). After a final wash, sections were preparations were pure as assessed by Coomassie blue staining following mounted in a glycerol solution containing p-phenylenediamine to prevent SDS-PAGE. fading (31). Representative photomicrographs were taken with a Zeiss Ax- http://www.jimmunol.org/ iophot microscope equipped with excitor barrier filter set: blue BP450/490- RNase activity and lesion formation LP520/560 (N 487910). Histologic examination with H&E stains was also performed to examine cellular infiltration. To test the influence of RNase activity on ECP and EDN lesion formation, injectable-grade RNasin, an RNase inhibitor that effectively inhibits the RNase activity measurement RNase activity of EDN (15), was mixed with ECP and EDN for a final concentration of 3.3 ␮M protein and 17 ␮M RNasin (64 ␮gof78U/␮g Mouse liver RNA was isolated using RNA-STAT protocol (Tel-Test) and RNasin in a final volume of 75 ␮l) just before intradermal injections of quantitated by absorbance at 260 nm. RNA was stored in 1ϫ Tris-acetate- each mixture into two guinea pigs. For comparison, ECP or EDN mixed EDTA buffer (40 mM Tris-acetate, 1 mM EDTA (pH 8.3) in diethylpyro- with sterile PBS in place of the RNasin was also injected. carbonate-treated sterile H2O). To assay for RNase activity (32), stock

To test further the effect of RNase activity, 1 mg of purified ECP or YOYO-1 dye (1 mM; Invitrogen) was added to 10 ␮g/ml mouse liver RNA by guest on September 26, 2021 EDN was diluted in 200 mM MES, 167 mM NaCl (pH 5.5) and carboxy- in Tris-acetate-EDTA buffer to a final YOYO-1 concentration of 2 ␮M, methylated by incubating with 75 mM sodium iodoacetate (Sigma-Aldrich) and this solution was heated at 65oC for 10 min. This RNA/YOYO-1 at 37°C for5hinthedark (15). As controls, a “sham” carboxymethylation solution (100 ␮l) was added to the wells of a polystyrene flat-bottom 96- procedure without addition of sodium iodoacetate was performed with pu- well plate and measured using a Cytofluor plate reader series 4000 (Per- rified ECP or EDN, and these products are referred to as unmodified-ECP Septive Biosystems) with excitation at 485 nm and emission at 530 nm. and unmodified-EDN. After incubation, the samples were diluted 10-fold The RNA/YOYO-1 complex was allowed to equilibrate for 1 h before with 8.1 mM Na2PO4, 1.5 mM KH2PO4 (pH 7.4) (“no salt” PBS) and addition of protein (EDN, ECP, RNase A, or Ang) to duplicate wells, and loaded onto a heparin Sepharose CL-6B column (Amersham Biosciences) fluorescence readings were recorded. Initial RNase velocity values were equilibrated with no salt PBS. The column was washed with no salt PBS calculated from the change in arbitrary fluorescence units occurring from 2 and a gradient from 0 to 430 mM NaCl in PBS was initiated. Absorbance to 4 min after the addition of protein and normalized per mass of protein at 280 nm of collected fractions (to estimate protein content) and RNase added. activity of selected peak fractions (see “RNase activity measurement” below) were measured. This procedure resulted in carboxymethylated ECP Results (CM-ECP) and CM-EDN that eluted from the heparin-Sepharose column at somewhat lower salt concentrations and that had RNase activity dimin- Cutaneous lesion formation ished by 80–90%, based on the change in initial RNase velocity. Reducing SDS-PAGE was used to assess the purity of the eosinophil granule protein preparations (Fig. 1). Fig. 2 shows represen- Intradermal injections and their assessment tative lesions following eosinophil granule protein injection, with The Institutional Animal Use and Care Committee approved the studies. robust lesions from ECP and EDN, and less intense or no lesions Hartley guinea pigs, 400–600 g, were anesthetized with 100 mg/ml ket- from EPO, MBP1, and bovine RNase A. In this experiment, gran- amine hydrochloride containing 1% xylazine (Ketaset; Fort Dodge Labo- ule proteins at 10 ␮M and 2.5 ␮M and their storage buffer solu- ratories) by i.m. injections of 0.5 ml per kilogram body mass. Fifty or 90 ␮l of ECP, EDN, EPO, and MBP1 at concentrations ranging from 0.5 to 50 tions were injected intradermally, and injection sites were ob- ␮M prepared in sterile buffers using LPS-free water (sterile water for ir- served for up to 4 wk; Table I shows the lesion grade for each rigation (USP); Baxter Healthcare) was injected intradermally into shaved granule protein. Overall, ECP induced the most severe lesions, back skin. Sterile acetate buffer (0.025 M sodium acetate, 0.15 M NaCl (pH followed by EDN; both ECP and EDN had greater lesion-forming 4.2); MBP1 storage buffer), phosphate buffer with 1 M NaCl (elution and activity than did EPO or MBP1. ECP lesions were distinctly and storage buffer for ECP, EDN, and EPO with a NaCl concentration greater than or equal to that needed to elute each of these proteins from their consistently ulcerative, and EDN lesions typically showed white, respective cation exchange columns during purification), and sterile PBS dry crusts (Fig. 2). ECP and EDN lesion formation began within 2 were also injected. Additional intradermal injections included poly-L-argi- days and peaked ϳ7 days after injection (Table I). Injection sites nine hydrochloride (5–15 kDa), bovine RNase A, and LPS (from Esche- of identical samples on duplicate guinea pigs were similar in mag- richia coli J5) from Sigma-Aldrich. Injectable grade RNasin was from ␮ Promega. Pyroglu-angiogenin (Ang) was provided by Dr. R. Shapiro (Har- nitude and appearance, while the overall severity of the 2.5 M vard Medical School, Boston, MA). To determine that the injection-site injection sites was diminished relative to the 10 ␮M sites (Table I). reactions were not species-restricted, rabbits were also tested. Bovine RNase A, sterile PBS, and phosphate buffer with 1 M NaCl The Journal of Immunology 4015

tions, three approaches were taken. First, puriﬁed LPS at concentrations up to 100 ␮g/ml was intradermally injected into guinea pigs. Lesion-forming activity by endotoxin was minimal, never exceeding a trace lesion (0.5 score), even at 100 ␮g/ml, from 2 to 14 days after injection. Second, to avoid bacterial contamination of injected protein samples, all samples were centrifuged at 12,000 ϫ g for 10 min before injection, and only the supernatants were injected. A supernatant sample of ECP was tested for bacterial contamination by culture at the Mayo Clinic Microbiology Laboratory, and no microorganisms were detected after 5 days of culture. Finally, to rule out a pathogenic bacterial infection, a representative lesion induced by ECP was biopsied, and a portion was cultured. Testing was FIGURE 1. Purity of eosinophil granule proteins. A, An excess of each performed on both agar plates and in liquid broth; coagulase- of the four eosinophil granule proteins was assessed using SDS-PAGE negative Staphylococcus and Streptococcus viridans, likely under reducing conditions. EPO is composed of both heavy (ϳ50 kDa) and commensal organisms, were cultured from the biopsy. light (ϳ10 kDa) chains, and the arrowhead points to the most prominent, more highly glycosylated forms of EDN and ECP in their respective lanes

(14). B, CM-ECP and unmodified ECP (unmod-ECP) after elution from a Mechanism of ECP- and EDN-induced cutaneous lesion Downloaded from second heparin-Sepharose CL-6B column was analyzed by SDS-PAGE formation under reducing conditions. Note that a greater proportion of the lower ECP-2 band is likely present in the unmod-ECP lane because the peak of Cellular localization of ECP and EDN and cellular infiltration. unmodified ECP, which was from exactly the same ECP sample as that Within 24 h after ECP injection, heterophil and lymphomono- used to generate the CM-ECP, eluted across two main fractions, and it was nuclear subdermal cell infiltrates were prominent in the dermis, the latter of these fractions (no. 16; i.e., f16) that was used for this including near cutaneous trunci muscles, and these infiltrates re- SDS-PAGE. mained substantial 2, 3, and 4 days after injection (Figs. 3, A and http://www.jimmunol.org/ B). Cellular infiltration increased in the upper dermis during this time and peaked at approximately day 7; cell infiltrates began to injection sites did not cause lesions after injection (Fig. 2). Undi- decrease at approximately day 14. Similar patterns of cellular in- luted acetate buffer caused a trace lesion (0.5 score) that returned to normal after 1 wk. However, for control purposes, this was an filtration occurred for the other three granule proteins, but with extreme acetate buffer concentration because the stock MBP1 in intensities proportional to the lesion-forming activities of the gran- acetate buffer was diluted Ͼ20-fold with sterile PBS before injec- ule proteins (i.e., less cellular infiltration with decreased lesion tion. Intradermal injections of the granule proteins at 25 ␮M were formation). Likewise, intradermal injection of PBS and RNase A, also performed in two rabbits. Similar lesion formation occurred neither of which induced a skin lesion, resulted in minimal or no by guest on September 26, 2021 except that lesions from EPO were relatively more intense and detectable cellular infiltration during the first week. approached the severity of lesions from EDN (Table I). Immunofluorescence staining showed that most of the intrader- Additional experiments were performed with a wider range of mally injected ECP and EDN, diffusely distributed in tissues ini- ECP and EDN concentrations. Lesions were consistently elicited tially after injection (Fig. 3C), were associated with cells in the with Ն2.5 ␮M ECP or EDN, and visible lesions frequently formed dermis within 48 h (Fig. 3D–F). In contrast, EPO and MBP1 re- even at 1 ␮M ECP or EDN. Lesions remained detectable for Ͼ2 mained primarily extracellular and appeared to bind to dermal ma- wk with Ն2.5 ␮M ECP or EDN (Table I). Alopecia persisted at trix fibers (Fig. 3, G and H) (5). least 4 wk at 10 EDN injection sites (two 9 ␮M and eight 10 ␮M) Differential glycosylation. Glycosylation influences protein rec- on four guinea pigs. The 10 ␮M EDN injection sites on one guinea ognition and activity, and because of heterogeneous glycosylation, pig were monitored up to 6 wk after injection and showed hair ECP exists in two predominant forms, that is, ECP1 and ECP2 regrowth at this time. Alopecia and crusting persisted longer than (14). Therefore, a sample of ECP containing a mixture of ECP1 4 wk at three ECP injection sites (all 10 ␮M) on each of two and ECP2 was subjected to a second heparin-Sepharose chromato- guinea pigs. graphic separation with NaCl gradient elution. SDS-PAGE analy- To rule out a potential contribution from bacterial or LPS ses showed fractions containing predominantly the higher molec- contamination in purified eosinophil granule protein prepara- ular mass ECP1 or the lower molecular mass ECP2 that eluted

Table I. Cutaneous lesion severity gradea and kinetics following intradermal injection of eosinophil granule proteins

ECP EDN EPO MBP1

Guinea pigs Rabbits Guinea pigs Rabbits Guinea pigs Rabbits Guinea pigs Rabbits Observation Time 2.5 ␮M10␮M25␮M 2.5 ␮M10␮M25␮M 2.5 ␮M10␮M25␮M 2.5 ␮M10␮M25␮M

Day 1 1 1 1 0.75 1 1 0 0.25 1 0 0.5 0 Day 3 2 1.75 3 1.25 1.75 0.75 0 0.25 0.75 0 0.25 0 Day 7 3.5 4 3.5 2 2.5 2.5 0.25 0.75 2 0.25 1 1 Day 14 3 3.25 NDb 2 2 ND 0.5 0.5 ND 0.5 0.5 ND

a Average lesion grade for single 50-␮l intradermal injections of respective concentrations, 2.5 and 10 ␮M (into two guinea pigs for each concentration at each time point) and 25 ␮M (into two rabbits for each concentration at each time point); lesion grading scale from 0 to 4, see Materials and Methods for lesion score description. b ND, not done. 4016 SKIN LESIONS INDUCED BY EOSINOPHIL RIBONUCLEASES

protein net cationic charge on lesion formation was also examined by injecting RNases of different charges. Ninety microliters of ECP (10 ␮M), EDN (10 ␮M), Ang (10 ␮M), and RNase A (15 ␮M) was each injected intradermally in duplicate into two guinea pigs. Table II shows the course of lesion formation; lesion scores for duplicate injections on the same guinea pig and on different guinea pigs, as graded by two investigators, varied by Յ0.5. Lesion severity was not directly proportional to the net cationic charge among these RNases because, although ECP (ϩ14 charge) and RNase A (ϩ4 charge) caused the most and least severe lesions, respectively, the more highly cationic Ang (ϩ10 charge) did not induce more severe lesions than those of EDN (ϩ7 charge). RNase activity. To test the influence of RNase activity on ECP and EDN lesion formation, RNasin was mixed with ECP and EDN before intradermal injections of each mixture. For comparison, ECP or EDN mixed with sterile PBS was also injected. The ECP plus RNasin lesion was diminished in both size and intensity by FIGURE 2. Cutaneous lesions induced by eosinophil granule proteins in guinea pig skin. Fifty microliters of proteins, each at 10 ␮M, was in- 50–70%; however, the EDN plus RNasin lesion was unaffected. Downloaded from jected intradermally. Proteins were injected to the right of the numeric Immunofluorescence staining of sites biopsied 4 days after injec- labels and included MBP1 (1), ECP (2), EDN (3), EPO (4), and bovine tion detected minimal ECP in the ECP plus RNasin injection site; RNase A (5). Seven days after injections, the guinea pig was anesthetized in contrast, staining of ECP and EDN in the remaining three sites, and photographed as shown. that is, ECP without RNasin and EDN with and without RNasin, was comparable to untreated injection sites. To address further the role of RNase activity in lesion for- from the heparin-Sepharose column at lower and higher salt con- mation, an active site histidine of ECP and EDN was carboxym- http://www.jimmunol.org/ centrations, respectively, and that displayed an ϳ2 kDa difference ethylated. We first confirmed the relative RNase activities of in SDS-PAGE mobility. Intradermal injections of unseparated ECP, EDN, Ang, and RNase A (Fig. 4A). Following sham treat- ECP, ECP1, and ECP2 at 10 and 2.5 ␮M resulted in lesions of ment or treatment with 75 mM iodoacetate, samples of the un- similar severity among the 10 and 2.5 ␮M injections, respectively. modified (sham-treated) and CM-ECP or EDN were reisolated These injection sites were biopsied 1 wk later and stained by by heparin-Sepharose chromatography. Peak fractions of un- immunofluorescence for ECP. For all three ECP preparations, modified-ECP, unmodified-EDN, CM-ECP, and CM-EDN were specific staining was detected, and the majority of staining ap- tested for RNase activity; carboxymethylation reduced the peared localized to cells. RNase activity of ECP and EDN by ϳ80 and 87% (Fig. 4B), by guest on September 26, 2021 Net cationic charge. The limited lesion-forming activity of MBP1 respectively. Carboxymethylation of ECP and EDN strikingly (13.8 kDa, ϩ16 net positive charge) suggested that strong cationic reduced lesion-forming activities by two to three grades both on charge is not sufficient to induce cutaneous lesions as intense as those days 3 and 7 (Table III). Immunofluorescence staining for ECP caused by ECP or EDN. Similarly, intradermally injected poly-L-ar- at injection sites of unmodified- and CM-ECP at day 3 ginine at concentrations up to 25 ␮M showed a maximum of a grade demonstrated that carboxymethylation did not alter the cellular 1 lesion that subsided to a trace lesion after 1 wk. The influence of localization or the overall immunofluorescence distribution of

FIGURE 3. Immunofluorescence localization of ECP and EDN following intradermal injection. H&E staining of the epidermis and upper dermis (A) and the subdermal area surrounding the trunci muscle (B) 3 days after injection of 10 ␮M ECP (magnification, ϫ200) shows prominent cellular infiltration in the dermis. Immunofluorescence staining for EDN in the dermis less than1h(C), 2 days (D), and 4 days (E) after intradermal injection at 10 ␮M (magnification, ϫ400) shows generally diffuse deposition on cells and tissues (C) at 1 h, whereas the later injection sites (D and E) show an increased proportion of cell-localized staining. Immunofluorescence staining for ECP (F), EPO (G), and MBP1 (H) 4 days after intradermal injection at 10 ␮M (magnification, ϫ400) shows relatively greater cellular staining for ECP, similar to EDN, compared with the predominantly extracellular staining for EPO and MBP1. The Journal of Immunology 4017

Table II. Cutaneous lesion severity gradea and kinetics following intradermal injection of various RNases

ECP, ϩ14b EDN, ϩ7 Ang, ϩ10 RNase A, ϩ4 (10 ␮M) (10 ␮M) (10 ␮M) (15 ␮M)

Observation Guinea Guinea Guinea Guinea Guinea Guinea Guinea Guinea Time pig 1 pig 2 pig 1 pig 2 pig 1 pig 2 pig 1 pig 2

Day 1 0.75 1 1 0.75 0.5 0.5 0 0 Day 3 3 3.5 3 3 1.5 1.5 0 0 Day 7 3.5 NDc 3ND1ND0ND Day 14 3 ND 3 ND 1 ND 0 ND

a Average of lesion grades for duplicate 90-␮l injections on each of two guinea pigs; lesion grading scale from 0 to 4, see Materials and Methods for lesion score description. b Excess number of basic amino acids for the given RNase. c ND, not done. the protein (Fig. 5); immunofluorescence staining patterns were (Fig. 6, D and F), similar to that observed in guinea pig skin at similar for unmodified-EDN and CM-EDN. different time points following intradermal protein injections (Fig. 3), with the exception that the human tissue showed stain- Relationship to human skin disease ing of intact eosinophils. The MBP1 cellular staining, as rep- Downloaded from Morphologically, the lesions resulting from ECP and EDN injections resented by brightly fluorescent ovals, reflects the relative num- into guinea pig skin showed similarity to erosive and ulcerative le- bers of intact eosinophils (Fig. 6E). Both EDN (Fig. 6F) and sions in patients with HES (Fig. 6A–C) (33). Immunofluorescence especially ECP (Fig. 6D) staining shows more extensive cellu- staining of lesional tissue from four such patients demonstrated both lar localization than accountable by MBP1-associated eosino- cellular localization and extracellular deposition of ECP and EDN phil staining, suggesting their localization to cells other than

eosinophils. http://www.jimmunol.org/ Discussion Eosinophil granule protein deposition occurs in a variety of dermatoses (4). Several of the granule proteins induce a cutaneous wheal- and-flare reaction and increase cutaneous vasopermeability in vivo (5, 19, 20). The in vitro cytotoxicity of eosinophil granule proteins is also well documented (1). Additionally, the hypereosinophilic syndromes are often associated with cutaneous lesions (34), and atopic dermatitis shows strong association with eosinophil activity (35, 36). Even so, by guest on September 26, 2021 other reports suggest that eosinophils have a limited impact on asthma and atopic dermatitis inflammation (21–24). To help clarify these is- sues, we investigated the effects of eosinophil granule proteins on guinea pig and rabbit skin through injections into the dermis, a site to which the granule proteins localize in cutaneous diseases. Intradermal injection of micromolar concentrations of four eosinophil granule proteins, ECP, EDN, EPO, and MBP1, produced cutaneous lesions with cellular infiltration. The ECP- and EDN-induced lesions were substantially more pronounced than the EPO- and MBP1-induced lesions. This was unexpected for EDN because of its limited in vitro cytotoxicity toward K562 cells relative to that of ECP, EPO, and MBP1 (2). The lesion-forming activity of ECP and EDN was associated with their localization in dermal cells. Furthermore, RNase activity appears important to their lesion-forming activities, and their respective net positive charges also may have modulated lesion formation. However, neither RNase activity nor high net positive charge alone was sufficient to account for full lesion formation. FIGURE 4. RNase activity of unmodified and carboxymethylated The marked lesion-forming activities of ECP and EDN focused our RNases. A, RNase activity, reflected by a decrease in fluorescence, after 60 attention on these proteins (Fig. 2). An initial concern was whether a min equilibration, is shown for PBS (F), Ang (E), ECP (Œ), EDN (‚), and contaminant or an infectious component might have contributed to bovine RNase A (f). Each protein was added at 1 ␮g, and data are plotted lesion formation. Because lesion-forming activity varied among the as the percentage of maximum fluorescence. B, An equal volume of the different granule proteins and was consistent for a given granule pro- heparin-Sepharose CL-6B column buffer (F), the peak fraction of CM- tein, we think that no systematic factor or contaminant was substan- E EDN (absorbance at 280 nm of 0.205; ), and the peak fraction of un- tially contributing to EDN- and ECP-induced lesions. Several ap- modified EDN (absorbance at 280 nm of 0.271; Œ) were each added to proaches, addressing potential endotoxin or bacterial contamination of separate wells in duplicate after the 60-min equilibration. Initial RNase velocities (the change in fluorescence over the 2-min interval beginning 2 the protein preparations and postinjection infection, showed that bac- min after protein addition and normalized per unit of absorbance at 280 terial or endotoxin contamination did not explain EDN- and ECP- nm) for CM-EDN (911/2/0.205 ϭ 2222) and for unmodified EDN (9121/ induced lesions. Finally, the ability of RNasin, a specific RNase in- 2/0.275 ϭ 16,584) indicated an 87% reduction of RNase activity following hibitor, to diminish an ECP-induced lesion by ϳ60% and the ability carboxymethylation of EDN. of carboxymethylation to diminish ECP- and EDN-induced lesions 4018 SKIN LESIONS INDUCED BY EOSINOPHIL RIBONUCLEASES

Table III. Cutaneous lesion severity gradea and kinetics following intradermal injection of carboxymethylated-ECP and -EDN

Unmodiﬁed-ECP (10 ␮M) CM-ECPb (10 ␮M) Unmodiﬁed-EDN (9 ␮M) CM-EDNb (8.3 ␮M)

Observation Guinea Guinea Guinea Guinea Guinea Guinea Guinea Guinea Guinea Guinea Guinea Guinea Time pig 1 pig 2 pig 3 pig 1 pig 2 pig 3 pig 1 pig 2 pig 3 pig 1 pig 2 pig 3

Day 3 3 3 3 0.5 0.5 1 3 3 3 0.5 0.5 0 Day 7 3 3.5 NDc 1 1 ND 3 2.5 ND 0 0.5 ND

a Lesion grades for single injections on three separate guinea pigs; lesion grading scale from 0 to 4, see Materials and Methods for lesion score description. b Initial RNase velocity of CM-ECP was 20% of unmodiﬁed ECP, and CM-EDN was 13% of unmodiﬁed EDN. c ND, not done.

(Table III) support the speciﬁc lesion-forming activities of ECP and were as follows: RNase A was more active than EDN, and these EDN. Notably, RNasin (pI of 4.7) probably did not inhibit an EDN- were substantially more active than ECP, which was substantially induced lesion because of the relatively short half-life of the EDN- more active than Ang (Fig. 4). These relative activities are ex- RNasin complex (EDN, ϩ7 net charge) compared with that of an- pected from previous reports. However, the relative lesion-forming giogenin (ϩ10 net charge) (15), and presumably to that of ECP (ϩ14 activities of ECP and EDN were substantially greater than that of net charge). A short EDN-RNasin half-life compared with that of Ang, which was substantially greater than that of RNase A (Table Downloaded from ECP-RNasin is also consistent with the immunoﬂuorescence detec- II). Therefore, lesion formation does not depend solely and directly tion of intradermally injected EDN, but not ECP, when mixed with on RNase activity. Second, we tested carboxymethylated ECP and RNasin. Overall, it is unlikely that the cutaneous lesions caused by EDN, and this treatment strikingly inhibited lesion formation (Ta- intradermal injections of ECP or EDN are attributable to a contami- ble III). Thus, at least for ECP and EDN, RNase activity is required nating substance or infectious component. for maximal lesion formation. Because carboxymethylation inserts

The ECP- and EDN-induced lesions developed over 1 wk, oc- one negative charge per carboxymethyl group added, these http://www.jimmunol.org/ curred at concentrations as low as 1 ␮M, and occurred in both guinea changed molecular charges of the carboxymethylated ECP and pig and rabbit skin (Fig. 2 and Table I). ECP and EDN concentrations EDN could be confounding factors (see below). Also, factors other Ͼ1 ␮M are likely deposited in human skin disease (5). Given the than RNase activity appear to be involved, because while the pronounced lesion-forming activities of ECP and EDN, we consid- RNase activity of ECP is substantially less than that of EDN, ECP ered distinctive properties of ECP and EDN compared with the less produces a more prominent lesion. active lesion-forming EPO and MBP1. Two such properties are neu- Cationicity is often associated with cytotoxicity (45). Thus, net rotoxicity and RNase activity. A third property, distinguishing ECP positive charge, as suggested by the cytotoxic activity of the non- and EDN from EPO and MBP1, is cellular localization after intrader- RNase MBP1 (ϩ16, molecular mass of 13.8 kDa) (2), may con- mal injection (Fig. 3) (5). Cellular localization of ECP and EDN ap- tribute to lesion formation. For example, a more highly cationic by guest on September 26, 2021 pears consistent with previous observations for another RNase, onco- protein may have an increased partitioning to the cell’s surface or nase (37), and with our staining for ECP and EDN compared with increased direct cytotoxicity or both. The protein cationicity of the MBP1 in HES skin lesions (Fig. 6). Interestingly, cellular internaliza- RNases decreases in the following order: ECP, Ang, EDN, and tion can increase the neurotoxicity of an RNase by 1000-fold (38). RNase A (Table II). Because the more highly charged Ang does Thus, internalization of ECP and EDN, as suggested by the appear- not produce a more pronounced lesion than that caused by EDN, ance of the immunofluorescent cells within the dermis containing nor does MBP1 or poly-L-arginine cause such pronounced lesions, ECP and EDN (Fig. 3), might account for the unexpectedly high a direct relationship between net positive charge and lesion-form- lesion-forming activity of EDN. Unfortunately, due to the paucity of ing activity does not exist. Glycosylation can affect a protein’s net guinea pig-specific reagents, the identity of the cells accumulating charge, and both ECP and EDN are glycosylated, while Ang and ECP and EDN remains unknown. However, ECP has been observed RNase A are not. A recent study showed that the N-linked carbo- in macrophage-like dermal cells after house dust mite patch testing of hydrates on ECP are in part made up of sialic acid, galactose, and humans (39), and EDN has recently been reported as an endogenous acetylglucosamine (46). These individual carbohydrates are sug- TLR2-dependent “alarmin” acting on dendritic cells (40). If EDN gestive of “complex type” N-linked glycosylation, which typically binds directly to TLR2, a precedent for it being internalized after involves noncharged carbohydrates and negatively charged sialic ligation exists (41). Additionally, EDN is chemotactic for dendritic acid. This form of glycosylation would only diminish ECP’s cells and stimulates release of inflammatory mediators, such as IL-8 cationicity and presumably any associated cation-dependent cyto- and TNF-␣ (40, 42). IL-8 and TNF-␣ can lead to further cell recruit- toxicity. Alternatively, such glycosylation could confer other prop- ment, the latter, at least in part, via increased endothelial cell adhesion erties to ECP and EDN that contribute to their distinct lesion- molecule expression (43), and TNF-␣ induces an inflammatory lesion forming capabilities compared with Ang and RNase A, even when injected into rabbit skin (44). Thus, ECP and EDN appear to be though differential glycosylation of ECP1 and ECP2 did not no- internalized by resident dermal cells and, directly or indirectly via ticeably alter their lesion-forming activities. induction of other inflammatory mediators, cause additional cellular Thirteen members of the human RNase family have been identified infiltration and cutaneous disruption. (47). Among these 13 genes, ECP and EDN form a distinct clad with Given the potential importance of RNase activity, we examined substantial homology. Thus, unidentified molecular characteristics its influence in ECP- and EDN-induced lesion formation, and two specific to ECP and EDN may be critical to their cutaneous lesion- approaches, in addition to the use of RNasin (discussed above), forming activity. For instance, after intradermal injection, it is un- were taken. First, we compared the lesion-forming activities of known whether Ang and RNase A localize to dermal cells like ECP ECP and EDN to two other RNases, Ang and bovine RNase A. and EDN. However, similar to their difference in lesion-forming po- The relative RNase activities for these four RNases against the tency reported herein, the alarmin adjuvant effect of EDN reported by RNA substrate used in this study (i.e., mouse liver total RNA) Yang et al. was not recapitulated by human angiogenin (40). The The Journal of Immunology 4019

sions in an HES variant (Fig. 6A–C) that, until recently, was associated with a grave prognosis (33). HES with ulcerative lesions appears to be a presentation of myeloproliferative HES (associated with a deletion on chromosome 4 resulting in a fusion gene product, FIP1L1- PDGFRA, and yielding a novel kinase sensitive to imatinib mesylate therapy with long-term disease control) (49, 50). The biopsy specimens from 4 HES patients showed both eosinophil infiltration and eosinophil granule protein deposition; the staining of cell-localized ECP and EDN was extensive, out of proportion to the number of infiltrating eosinophils identified by MBP1 staining, and similar to that observed in guinea pig skin at later time points following injection of ECP and EDN. Ongoing deposition of ECP and EDN in human tissue, unlike single injections of ECP or EDN into guinea pig skin, likely contributed to the relatively greater extracellular staining of ECP and EDN in human tissue compared with later guinea pig intradermal injection sites (Ն 2 days postinjection). Because of ethical considerations, it is not possible directly to study the effects of eosin-

ophil granule proteins injected into human skin; however, these anal- Downloaded from ogous ﬁndings by formation of lesions and by immunostaining support the conclusion that ECP and EDN cause cutaneous lesions in human disease. In summary, all four eosinophil granule proteins, ECP, EDN, EPO, and MBP1, induce a cutaneous lesion after intradermal

injection into guinea pig and rabbit skin at pathophysiologically http://www.jimmunol.org/ relevant concentrations. The ECP- and EDN-induced lesions are more intense than lesions induced by EPO and MBP1, and FIGURE 5. Immunofluorescence staining for intradermally injected un- this difference appears to be closely associated with cellular modified-ECP and CM-ECP. Injection site biopsies were taken from one of internalization and RNase activity of ECP and EDN. Net cat- three guinea pigs 3 days after injecting unmodified ECP (A) and CM-ECP ionic charge may also modulate lesion-forming activity. How- (B). Polyclonal rabbit anti-ECP was used to stain for ECP in these speci- ever, neither cationicity or RNase activity directly correlates mens, and a representative area of the dermis in each shows comparable with lesion-forming activity, suggesting the potential impor- cellular and extracellular staining (magnification, ϫ160).

tance of other properties speciﬁc to ECP and EDN. Overall, by guest on September 26, 2021 these data provide further direct evidence that deposition of the relatively speciﬁc lesion-inducing activities of ECP and EDN com- eosinophil granule proteins, particularly ECP and EDN, at mi- pared with other RNases are also reminiscent of their unique neuro- cromolar concentrations, can severely effect cutaneous structure toxic and antiviral activities (48). and function in diseases in which granule proteins are deposited Morphologically, the lesions resulting from ECP or EDN injection including in the hypereosinophilic syndrome associated with into guinea pig skin showed similarity to erosive and ulcerative le- mucocutaneous ulcerations (Fig. 6) (33, 34, 50).

FIGURE 6. Immunohistologic staining similarities to ulcerated human tissue in HES lesion. A, Lesion on guinea pig ﬂank skin from ECP injection shows similar appearance to eroded and ulcerated lesions in an HES patient. B, Ulcerations and erosions in patient with HES. C, Oral lesions in HES with central epithelial ulceration and surrounding erythema from which a biopsy specimen was obtained and stained for eosinophil granule proteins. Photomicrographs (magniﬁcations, ϫ160) of serial sections of lesional mucosa (C) from a patient with HES stained with anti-ECP (D), anti-MBP1 (E), anti-EDN (F), and H&E (G). 4020 SKIN LESIONS INDUCED BY EOSINOPHIL RIBONUCLEASES

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