of April 3, 2018.This information is current as Mice

Does Not Modify Graft-versus-Leukemia inProtects from Graft-versus-Host Disease but

Binding Protein Evasin-1−αProtein-1The CCL3/Macrophage Inflammatory

Vanessa PinhoSilva, Amanda E. I. Proudfoot, Mauro M. Teixeira and Sousa, Rosa M. E. Arantes, Danielle G. Souza, Tarcília A.Resende, Ana L. Alessandri, Caio T. Fagundes, Lirlândia P. Marina G. M. Castor, Bárbara Rezende, Carolina B.

http://www.jimmunol.org/content/184/5/2646doi: 10.4049/jimmunol.0902614January 2010;

2010; 184:2646-2654; Prepublished online 25J Immunol 

Referenceshttp://www.jimmunol.org/content/184/5/2646.full#ref-list-1

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The Journal of Immunology

The CCL3/Macrophage Inflammatory Protein-1a–BindingProtein Evasin-1 Protects from Graft-versus-Host Disease butDoes Not Modify Graft-versus-Leukemia in Mice

Marina G. M. Castor,*,† Barbara Rezende,* Carolina B. Resende,* Ana L. Alessandri,*

Caio T. Fagundes,* Lirlandia P. Sousa,* Rosa M. E. Arantes,‡ Danielle G. Souza,*,x

Tarcılia A. Silva,{ Amanda E. I. Proudfoot,‖ Mauro M. Teixeira,* and Vanessa Pinho*,#

CCL3 is a protein of the CC chemokine family known to be important for T cell recruitment in inflammatory diseases. The aim of the

current study was to evaluate the effects and putative mechanism of action of evasin-1, a novel CCL3-binding protein, in the path-

ogenesis of acute graft-versus-host disease (GVHD). GVHD was induced by the transplantation of splenocytes from C57BL/6J to

B6D2F1 mice. Treatment of recipient mice with evasin-1 prevented mortality associated with GVHD. This was correlated with

reduced weight loss and clinical disease severity. Analysis of the small intestine showed that evasin-1 treatment reduced the his-

topathological score and decreased levels of IFN-g and CCL5. Mechanistically, evasin-1 treatment reduced the number of CD4+

and CD8+ T cells infiltrating the small intestine, as assessed by immunohistochemistry, and the adhesion of leukocytes to intestinal

venules of recipient mice, as assessed by intravital microscopy. Evasin-1 was also able to decrease liver damage, as seen by

reduction of inflammatory infiltrate and IFN-g levels. Treatment with evasin-1 did not interfere with graft-versus-leukemia.

Altogether, our studies demonstrate that CCL3 plays a major role in mediating GVHD, but not graft-versus-leukemia in mice

and suggest that blockade of CCL3 with evasin-1 has potential therapeutic application in patients undergoing bone marrow

transplantation. The Journal of Immunology, 2010, 184: 2646–2654.

Graft-versus-host disease (GVHD) is a major complicationof allogeneic bone marrow transplantation (BMT),leading to significant morbidity and mortality in humans

(1). Allogeneic BMT is a transplantation between two geneticallynonidentical individuals (2). This therapy is currently indicated fortreatment of a number of malignant and nonmalignant diseases,including acute and chronic leukemia, myelomas, lymphomas,aplastic anemia, solid tumors, and severe immunodeficiency (1, 2).Acute and chronic GVHD occurs when transplanted donor-derived T cells recognize and react to histo-incompatible recipientAgs and cells (3). Acute GVHD occurs within 100 d and isa rapidly progressive syndrome characterized by profound wast-

ing, immunosuppression, and tissue injury in a number of organs,including the intestines, spleen, skin, liver, and lung (1, 4–6).Mononuclear phagocytes and other leukocytes are thought to be

responsible for both initiation of graft-versus-host reaction and forthe subsequent injury to host tissues after complex interactions withcytokines and chemokines (1, 2, 7–9). Three phases have beendescribed in experimental GVHD. During the first phase, the con-ditioning regimen (irradiation and/or chemotherapy) leads to dam-age, activation of host tissues, including intestinal mucosa, andinduces the secretion of inflammatory cytokines (8–11). The secondphase consists of donor T cell activation, proliferation and differ-entiation into effector T cells (3, 8, 9). Activated donor T cellsrecognize allogeneic Ags presented by host APC and are furtheractivated by costimulation expressed by APC (3, 8, 9). At the finalphase, reactive effector T cells are recruited by chemokines, in-cluding CCL3/MIP-1 a, into GVHD target organs, resulting in hosttissue injury (2, 3, 8–13). CCL3 is a member of the CC chemokinesubfamily that has been shown to be expressed in GVHD and toaffect T cell recruitment and proliferation in GVHD (8, 9, 12, 13).A number of distinct chemokine-binding proteins (CBP) have

been isolated from virus and other parasites, including ticks, that

inhibit chemokine activity both in vitro and in vivo and play an

important role in immune evasion (14–20). CBP derived from

ticks, named evasins, appear to have unusually stringent chemo-

kine selectivity, differentiating them from broader spectrum viral

CBPs. In vitro, evasin-1 binds and inactivates CCL3 (17) and,

in vivo, treatment with evasin-1 decreased rolling, adhesion, and

transmigration of leukocytes induced by CCL3 and reduced

bleomycin-induced lung fibrosis and death (18). The present

study examined the effect of the treatment with evasin-1 in the

pathogenesis of acute GVHD in mice and compared findings with

results obtained in CCL3-deficient (CCL32/2) mice or mice

treated with dexamethasone. We also evaluated whether CCL3

*Laboratorio de Imunofarmacologia, Departamento de Bioquımica e Imunologia; †De-partamento de Fisiologia e Biofısica; ‡Laboratorio de Neuro-Imunopatologia Experimen-tal, Departamento de Patologia; xDepartamento de Microbiologia; {Departamento deClınica, Patologia, e Cirurgia Odontologicas, Faculdade de Odontologia; #Departamentode Morfologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais,BeloHorizonte,Brazil; and ‖MerckSerono,GenevaResearchCenter,Geneva, Switzerland

Received for publication August 10, 2009. Accepted for publication December 29,2009.

This work was supported by grants from Coordenacao de Aperfeicoamento de Pes-soal do Ensino Superior, Conselho Nacional de Desenvolvimento Cientifico e Tec-nologico, Fundacao de Amparo a Pesquisa do Estado de Minas Gerais, and theEuropean Union’s Sixth Framework Programme (Innovative Chemokine-BasedTherapeutic Strategies for Autoimmunity and Chronic Inflammation, LSHB-CT-2005-518167).

Address correspondence and reprint requests to Dr. Mauro M. Teixeira, Departa-mento de Bioquimica e Imunologia, Instituto de Ciencias Biologicas, UniversidadeFederal de Minas Gerais, Av. Antonio Carlos, 6627-Pampulha 31270-901, BeloHorizonte, MG, Brazil. E-mail address: mmtex@icb.ufmg.br

Abbreviations used in this paper: BMT, bone marrow transplantation; CBP, chemo-kine-binding protein; GVHD, graft-versus-host disease; GVL, graft-versus-leukemia;NAG, N-acetyl glucosaminidase; WT, wild type.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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would affect the ability of the engrafted murine host to deal with

challenge from a leukemic cell line.

Materials and MethodsMice

Eight- to twelve-week-old mice were housed under standard conditions ina temperature-controlled room (23 6 1˚C) on an automatic 12-h light/darkcycle and had free access to commercial chow and water. All proceduresdescribed in this study were approved by the Ethics Committee on AnimalExperimentation of the Universidade Federal de Minas Gerais (number:077/08). Male C57BL/6J and (C57BL/6J 3 DBA/2) F1 were obtainedfrom the Bioscience unit of Universidade Federal de Minas Gerais., CCL3-deficient mice (21) were purchased from The Jackson Laboratory (BarHarbor, ME), and they were maintained in the animal facilities of theUniversidade Federal de Minas. Transgenic mice expressing enhancedGFP (22) were kindly donated by Professor Okabe (Osaka University,Osaka, Japan) and maintained in the animal facilities of on Animal theUniversidade Federal de Minas.

Induction of GVHD and treatment protocols

For GVHD induction, 33 107 splenocytes from either syngeneic (B6D2F1)or semiallogeneic (C57BL/6J) donors were injected into recipient B6D2F1mice that had been irradiated with 4 Gy total body irradiation (source: 60CO)2 d prior to transplantation. B6D2F1 mice, which received splenocytes fromB6D2F1 mice (B6D2F1 → B6D2F1), did not develop disease and wereconsidered the control group. The GVHD group received splenocytes fromC57BL/6J mice (C57BL/6J → B6D2F1) and developed classic disease.Animals in this group were treated with PBS (the vehicle for drugs used) s.c.in 200 ml twice per day. The evasin-1 group received splenocytes fromC57BL/6J (C57BL/6J → B6D2F1) and 10 mg evasin-1 s.c. in 200 ml PBS,30 min before transplantation and then twice per day during the entire du-ration of the experiments. The dexamethasone group received splenocytesfrom C57BL/6J (C57BL/6J → B6D2F1) and 25 mg dexamethasone (Hipo-labor Pharmaceuticals, Sao Paulo, Brazil) s.c. in 200 ml PBS once a day. Agroup of B6D2F1 mice (CCL32/2 group) received C57BL/6J splenocytesfrom CCL3-deficient mice (C57BL/6J CCL32/2→ B6D2F1). Evasin-1 wasproduced in HEK293 cells and purified by Merck Serono (Geneva, Swit-zerland) according to a previously published study (17).

Mortality rate and assessment of GVHD clinical score

A group of mice was monitored daily for survival posttransplantation andassessed clinically by a standard scoring system that generates a compositeGVHD score comprising individual scores for weight loss, posture(hunching), activity, fur texture, skin integrity, diarrhea (11, 23), and occultblood in feces. Each parameter was evaluated every 2 d and graded 0–2

according to occurrence and degree of intensity. A clinical index wassubsequently generated by summation of scores of the seven criteria(maximum index = 14).

Histopathology

A set of experiments were conducted for quantification of histopathologicalparameters in the intestine and liver, important target organs in GVHD. Theintestinal rolls and liver were fixed in buffered formaldehyde (10% in PBS) for24 h and routinely processed for paraffin embedding. Sections 5mm-thickwereobtained and stainedwith H&E. The entire extension of the jejunum and ileumwall and liver was examined under an optical microscope (Olympus BX51,Tokyo, Japan) using a 103 objective. The images were captured under the

FIGURE 1. Kinetics of the expression of CCL3 after GVHD induction.

GVHD was induced by the transfer of splenocytes from allogeneic (WT

C57BL/6J or C57BL/6J-CCL32/2 mice) donors to B6D2F1 mice. Mice

that received splenocytes from syngeneic (B6D2F1) mice did not develop

disease and were considered the control group. Intestinal samples were

collected at the indicated time points after GVD induction, and CCL3 was

detected by ELISA. Results are presented as the mean 6 SEM (n = 6).

pp , 0.01 compared with control; #p , 0.01 compared with GVHD.

FIGURE 2. Evasin-1 treatment prevented mortality and clinical GVHD.

GVHD was induced by the transfer of splenocytes from allogeneic (WT

C57BL/6J or C57BL/6J-CCL32/2 mice) donors to B6D2F1 mice. Mice that

received splenocytes from syngeneic (B6D2F1) mice did not develop disease

and were considered the control group. Evasin-1 (10 mg in 200 ml of PBS, s.c.)

was given to WT mice 30 min before transplantation and twice per day during

the entire duration of the experiments. After induction of GVHD, mice were

monitored every 2 d for survival (A), body weight (B), and GVHD clinical

scores (C). Control group (n), n = six mice/group; GVHD group (:), n =

eight mice/group; evasin-1 group (3), n = 9 mice/group; and CCL32/2 group

(▼), n = 7 mice/group. pp, 0.05 when compared with the control group; #p

, 0.05 when compared with the GVHD group.

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microscope with a resolution of 720 3 480 pixels (Cool SNAP-Procf Color,Media Cybernetics, Bethesda, MD) and evaluated using the program ImageProExpress version 4.0 for Windows (Media Cybernetics).

Histopathological scores were determined according to the criteriapreviously established (11, 24, 25). Samples were obtained from mice atdays 3, 10, and 20 posttransplantation, which correspond to the followingphases of clinical disease in mice: latency, onset of clinical disease, andmortality, respectively. Some mice had intestines removed for histopath-ological analysis 24 h postirradiation and before the transplantation tocontrol for the consequences of irradiation to the intestine.

Cytokine and chemokine quantification

Cytokines and chemokines were measured according to the proceduressupplied by the manufacturer (R&D Systems, Minneapolis, MN). Briefly,100 mg duodenum or liver was homogenized in 1 ml PBS (0.4 mol/l NaCland 10 mmol/l NaPO4) containing antiproteases (0.1 mmol/l phenylmethylsulfonyl fluoride, 0.1 mmol/l benzethonium chloride, 10 mmol/l EDTA,and 20 KI aprotinin A) and 0.05% Tween 20. The samples were thencentrifuged for 10 min at 30003 g, and the supernatants immediately usedfor ELISA assays at a 1:4 dilution in PBS.

Intravital microscopy

GVHD was induced in B6D2F1 mice, using GFP+C57BL/6J mice sple-nocytes. At day 20 posttransplantation, mice were anesthetized, and thejejunum-ileum was exposed in a perfusion system containing warmbicarbonate-buffered saline (pH 7.4). An intravital microscope (OlympusBX50F4) with a 103 objective lens was used to examine the mesentericmicrocirculation. A video camera (5100 HS; Panasonic, Osaka, Japan) wasused to project the images onto a monitor, and images were recorded forplayback analysis, using a conventional videocassette recorder.

Intestinal venules (20–40 mm) were selected and the number of rollingand adherent GFP+leukocytes determined offline during video playbackanalysis. Rolling leukocytes were defined as those cells moving at a velocityless than that of erythrocytes within a given vessel. The flux of rolling cellswas measured as the number of rolling cells passing by a given point in thevenule per minute. A leukocyte was considered adherent if it remainedstationary for at least 30 s, and total leukocyte adhesion was quantified as thenumber of cells in the intravascular space within an area of 100 mm.

Evasin-1 was given on two separate treatment schedules. In the firstgroup of experiments, evasin-1 (10 mg/mouse) was administered just beforeGVHD induction and then every 12 h until the intravital microscopyprocedure. In the second group, mice were treated with evasin-1 (10 mg/mouse) 30 min before the intravital procedure; that is, in the latter group,

animals only received the compound just before evaluation of leukocyteand endothelial cell interactions.

Immunohistochemistry of intestine

Paraffin-embedded jejunum-ileum obtained from mice at day 20 post-transplantation were sectioned (3 mm), placed on glass slides coated with 2%3-aminopropyltriethylsilane (Sigma-Aldrich, St. Louis, MO), deparaffinized,and incubated with 3% hydrogen peroxide diluted in methanol (1:1) for 20min. Sections were then immersed in citrate buffer (pH 6.0) for 20 min at95˚C for Ag retrieval. The material was blocked by incubation with 3%normal goat serum diluted, at room temperature, for 20 min. The slides werethen incubated with anti-mouse CD8 or CD4 monoclonal Abs diluted at1:1000, at 4˚C overnight in a humidified chamber. After washing in TBS, thesections were revealed using the LSAB+ System-HRP (DakoCytomation,Carpinteria, CA) in accordance with instructions given by the supplier.Sections were counterstained with Mayer´s hematoxylin. Negative controlswere obtained by the omission of primary Abs, which were substituted with1% PBS-BSA and with mouse serum (X501-1, Dako).

The number of positive cells was counted in 10 alternate high-powermicroscopic fields (3400) under an optical microscope, Olympus BX50 Q(Melville, NY), using an integration reticule (4740680000000-Netzmikr-ometer 12.53, Carl Zeiss, Gottingen, Germany) with the aid of Q-CapturePro software (QImaging, Surrey, British Columbia, Canada).

Quantification of macrophage infiltration

The relative number of macrophages infiltrating the intestine and liver werequantified by measuring N-acetyl glucosaminidase (NAG) activity at day20 posttransplantation. A portion of 100 mg of the small intestine wasresuspended in saline 0.9% (4˚C) containing 0.15 v/v Triton X-100 (MerckSharp & Dohme, Sao Paulo, Brazil), homogenized, and centrifuged at 4˚for 10 min at 1500 rpm. The supernatants were collected and immediatelyassayed for NAG using a 1:10 dilution, as described previously (26).

Graft-versus-leukemia induction

P815, a mouse mastocytoma cell line (H-2d, American Type CultureCollection, Rockville, MD) transduced with a lentiviral vector (EF1aGFP),was kindly provided by A.C. Leal and M. Bonamino from Divisao deMedicina Experimental, Instituto Nacional de Cancer (Rio de Janeiro,Brazil). These cells were used for graft-versus-leukemia (GVL) experi-ments and were maintained in RPMI 1640/10% FCS at 37˚C, 5% CO2. Thesame protocols for irradiation and GVHD induction were used in theseexperiments. A total of 6000 GFP+ P815 cells were injected i.v. in B6D2F1

FIGURE 3. Evasin-1 treatment reduced intestinal damage in mice subjected to GVHD. GVHD was induced by the transfer of splenocytes from allo-

geneic (C57BL/6J) donors to B6D2F1 mice. Mice that received splenocytes from syngeneic (B6D2F1) mice did not develop disease and were considered

the control group. Evasin-1 (10 mg in 200 ml of PBS s.c.) was given 30 min before transplantation and twice per day during the entire duration of the

experiments. After induction of GVHD, mice were killed and the jejunum-ileum sampled for histopathological analysis and scored at 3 (A), 10 (B), and 20

(C) days posttransplantation. Results are presented as the mean 6 SEM (n = 6). pp , 0.05 compared with control; #p , 0.05 compared with GVHD. D–F,

Histological aspects of small intestine (jejunum-ileum) of control, GVHD, and evasin-1–treated mice, respectively, at day 20 posttransplantation. Scale

bar, 50 mm for all panels.

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recipients on day 0 just after the splenocyte transplantation. At day 10posttransplantation, mice were sacrificed and lymph nodes prepared forFACS analysis. Erythrocytes were lysed and cells centrifuged for 5 min at350 g. Cells (1 3 107) were resuspended in buffer containing 5% BSA and0.01% sodium azide and fixed in a solution containing 2% formaldehyde inPBS. Flow cytometry was performed using a FACScan (BD Biosciences,San Jose, CA), and 20,000 events per sample were evaluated. The presenceof tumor cells was determined by assessing the frequency of GFP+ cells,using the program FlowJo (Tree Star, Ashland, OR). The frequency ofGFP+ P815 cells was analyzed in tumor-bearing mice that received or didnot receive evasin-1 treatment (10 mg/mouse, s.c., twice per day).

Statistical analysis

Data in text are given as mean 6 SEM. Comparison between groups wascarried out by performing ANOVA followed by Student-Newman-Keulspost hoc analysis. A log rank test was used to compare the relevant survivalcurve. Analyses were carried out using the software SPSS v.15.0. A p ,0.05 was considered significant.

ResultsInitial experiments were performed to evaluate the kinetics of CCL3production in our model (Fig. 1). The transplantation of splenocytesfrom wild type (WT) C57BL/6J mice to WT B6D2F1 mice (GVHDgroup) induced an increase in levels of CCL3 in intestine whencompared with B6D2F1 mice that received transplants of isogeniccells (control group). When WT B6D2F1 mice received parentalsplenocytes of C57BL/6J CCL32/2 (CCL32/2 group), the levels ofCCL3 in intestine were reduced at 10 and 20 d, but not at day 3,posttransplantation (Fig. 1). The latter results indicate that residentcells (which are CCL3+) contribute to the initial production of CCL3.All B6D2F1 mice that received splenocytes from C57BL/6J

mice (GVHD group) died before day 45, as shown in Fig. 2A. Incontrast, the control group did not develop GVHD and were allalive by the end of the experiment. The transplantation of sple-nocytes from C57BL/6J CCL32/2 mice to WT B6D2F1 mice(CCL32/2 group) resulted in marked protection (88% survivalrate), confirming the role of CCL3 in GVHD (Fig. 2A). Theevaluation of weight (Fig. 2B) and clinical score (Fig. 2C) of miceconcurred with the mortality data. Indeed, whereas there wasa marked weight loss in mice subjected to GVHD, weight loss andclinical score were greatly reduced in CCL32/2 mice (Fig. 2B,2C), indicating the role of CCL3 in GVHD development.

Reduced clinical disease and mortality in evasin-1–treatedmice subjected to GVHD

Once the role of CCL32/2 was determined in our model, we testedwhether treatment with evasin-1 was efficient to ameliorate GVHD.Evasin-1 treatment, 30 min before transplantation and every 12 hthereafter, prevented mortality and provided partial alleviation of theGVHD clinical presentation (Fig. 2). Overall, the protection affordedby evasin-1 was similar to that observed in CCL32/2 mice (Fig. 2).For comparison, experiments conducted with the glucocorticosteroiddexamethasone showed that 75% of mice were alive at day 45 afterGVHD induction and therewas decrease of clinical disease similar tothat observed with evasin-1 treatment (data not shown). Evasin-1treatment was able to decrease CCL3 levels in the gut at 10 d afterGVHD induction (pg/100 mg of tissues: control mice, 63 [69];vehicle-treated GVHD mice, 2988 [6165]; evasin-1–treated mice,2281 [6233]; n = 6 and p , 0.05) and 20 d after GVHD induction(pg/100 mg of tissues: vehicle-treated GVHD mice. 1518 [6123];evasin-1–treated mice: 756 [649]; n = 6 and p , 0.05).

Reduced intestinal damage in evasin-1–treated mice subjectedto GVHD

Intestinal sections were evaluated at 3, 10, and 20 d post-transplantation. The dose of irradiation given to the animalsappeared not to cause significant changes in the intestine of mice

when they were evaluated 24 h postirradiation (data not shown). Incontrast, the grafted transplant caused significant changes in theintestine that were already apparent at day 3 and evolved to muchmore significant disease at day 20 posttransplantation (Fig. 3). Atday 3, mild edema and congestion of the lamina propria werepresent, and this condition was associated with focal areas ofvillous enlargement (data not shown). These initial changes werenot modified significantly by treatment with evasin-1 or in ex-periments in CCL32/2 mice (Fig. 3A).At day 10 after disease induction, there was increased cellularity

in the lamina propria and the inflammatory process reached themuscular layer, interposing between degenerated muscle cells.Peyer patches were increased in volume and exhibited signs ofreactivity; phagocytosis events were frequent, and swelling andincreased vascularization were observed in the surrounding mucosa(data not shown). Treatment with evasin-1 induced a significantpreservation of the muscular and serous layer, resulting in a re-duction in overall score (Fig. 3B). The effects of the treatmentwere qualitatively and quantitatively similar to results obtained inCCL32/2 mice (Fig. 3B).At day 20 posttransplantation, epithelial changes were signifi-

cant, including crypt cell hyperplasia, increased cellularity of thelamina propria, edema, and congestion. There were also de-generative changes and necrosis of the muscular and serous layersin the GVHD group (Fig. 3C–E). Treatment with evasin-1 causeda significant amelioration of the overall pathological score. Pro-tection of the mucosa was observed; it showed only superficial andrare erosions, with regenerative cell changes restricted to the top

FIGURE 4. Evasin-1 treatment reduced concentration of CCL5 and

IFN-g in the small intestine of mice subjected to GVHD. GVHD was

induced by the transfer of splenocytes from allogeneic (C57BL/6J) donors

to B6D2F1 mice. Mice that received splenocytes from syngeneic

(B6D2F1) mice did not develop disease and were considered the control

group. Evasin-1 (10 mg in 200 ml of PBS s.c.) was given 30 min before

transplantation and twice per day during the entire duration of the ex-

periments. At day 20 after induction of GVHD, mice were killed and the

concentrations of CCL5 (A) and IFN-g (B) evaluated by ELISA. Results

are presented as the mean 6 SEM (n = 6). pp , 0.05 compared with

control; #p , 0.05 compared with GVHD.

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of the villous layer. Moreover, cellularity in the lamina propriawas discreet, and the muscular and serous layers were preserved(Fig. 3C, 3F). As differences were very striking at day 20, weconcentrated at this time point on further evaluating the effects ofevasin-1 on GVHD.

Reduced production of CCL5 and IFN-g and recruitment ofCD4+ and CD8+ cells in the intestine of evasin-1–treated micesubjected to GVHD

The induction of GVHD resulted in a significant increase in thelevels of CCL5 and IFN-g in the jejunum-ileum at day 20 after celltransplantation (Fig. 4). The treatment with evasin-1 showed therewas a significant reduction of GVHD-associated increase in thelevels of CCL5 and IFN-g (Fig. 4). We also observed an increasein TNF levels after GVHD induction, but treatment with evasin-1failed to alter TNF levels (pg TNF/100 mg of tissues: controlmice, not detected; vehicle-treated GVHD mice, 136 [631];evasin-1–treated mice, 125 [626]; n = 6 and p . 0.05).Infiltration of lymphocytes in the intestine is thought to be an

important contributor to intestinal injury in GVHD (9, 25, 27, 28).

Thus, we evaluated whether the treatment with evasin-1 wouldmodify the recruitment of CD8+ and CD4+ cells into the intestineof mice subjected to GVHD. As seen in Fig. 5, there was a markedinfiltration of CD8+ and CD4+ cells in the lamina propria of thesmall intestine of GVHD mice, and treatment with evasin-1greatly decreased CD8+ and CD4+ cell recruitment.Histological analysis of samples showed an overall decrease in

inflammatory cell influx in the intestine of evasin-1–treated micesubjected to GVHD when compared with vehicle-treated mice(Fig. 3). Moreover, a significant decrease in NAG activity (asurrogate marker for macrophage influx) (26) was noted in theintestine of evasin-1–treated mice (Relative number of macro-phages/mg of tissue: control mice, 5.6 3 107 [60.2]; vehicle-treated GVHD mice, 8.43 107 [60.2]; evasin-1–treated mice, 6.03107 [60.1]; n = 6 and p , 0.05).The experiments above showed inhibition of the total re-

cruitment of CD8+ and CD4+ cells and macrophages in GVHDmice given evasin-1. To examine whether inhibition of chemo-kine-induced leukocyte adhesion and consequent migration wasthe mechanism by which evasin-1 functioned in the system, we

FIGURE 5. Evasin-1 treatment reduced the

number of CD8+ and CD4+ cells in the small

intestine of mice subjected to GVHD. GVHD

was induced by the transfer of splenocytes from

allogeneic (C57BL/6J) donors to B6D2F1 mice.

Mice that received splenocytes from syngeneic

(B6D2F1) mice did not develop disease and

were considered the control group. Evasin-1 (10

mg in 200 ml of PBS s.c.) was given 30 min

before transplantation and twice per day during

the entire duration of the experiments. At day

20 after induction of GVHD, mice were killed

and the number of CD8+ cells identified by

immunohistochemistry. A and B, Numbers of

CD8+ and CD4+ cells, respectively, in control

(n = 4), GVHD, and evasin-1 groups (n = 5).

Results are presented as the mean 6 SEM.

pp , 0.01 compared with control; #p , 0.01

compared with GVHD. H&E-stained tissue

sections from control (C, D), GVHD (E, F) and

evasin-1–treated (G, H) mice are shown for

illustration. Arrows show CD4+ and CD8+ cells

in E and F, respectively. Scale bar, 20 mm.

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performed intravital microscopy of an intestinal venule, post-capillary. Irradiated B6D2F1 or C57BL/6J mice received leuko-cytes from GFP+ C57BL/6J mice. Mice were treated with vehicleor evasin-1 (10 mg/mouse) using two different protocols, and theinteraction of GFP+ cells with endothelium was evaluated on day20 after GVHD induction. As seen in Fig. 6, there was a markedincrease in the number of leukocytes adhering to the intestinalvenules of mice undergoing GVHD. In the first protocol, micewere treated daily with evasin-1 from the onset of GVHD (Fig. 6,top panel). Consistent with its ability to decrease lethality andtissue injury, daily evasin-1 treatment greatly reduced leukocyteadhesion (Fig. 6). Of more interest, administration of evasin-1 justbefore intravital microscopy on day 20 after GVHD inductiongreatly decreased the adhesion of leukocytes to the mesentericvenules (Fig. 6). These results suggest that blockade of chemo-kine-induced leukocyte adhesion is a major mechanism for theprotective action of evasin-1 in the system.

Evasin-1–treated mice subjected to GVHD exhibited reductionof IFN-g production and inflammatory infiltrate in liver

In addition to the effects on the intestine, hepatic damage is animportant feature of GVHD. Liver damage was scored at 3, 10, and20 d posttransplantation. There was no significant liver damage atdays 3 and 10 after induction of GVHD (data not shown). At 20 d,a marked inflammatory infiltrate was noted in periportal andintralobular areas in mice subjected to GVHD. Treatment withevasin-1 decreased leukocyte infiltration in animals subjected toGVHD (Fig. 7A, 7D–F). Consistent with the overall leukocyteinflux observed on histological sections, evasin-1–treated micesubjected to GVHD showed a significant decrease in NAG activity2in the liver at day 20 posttransplantation (Fig. 7B). Levels of IFN-gwere enhanced by GVHD at day 20, and this increase was pre-

vented by treatment with evasin-1 (Fig. 7C). Hepatic levels ofCCL5 and IL-10 did not increase in mice subjected to GVHD at day20 (data not shown).

Evasin-1 treatment maintains GVL effect after GVHDinduction

GFP+ P815 cells were injected at day 0 of splenocyte trans-plantation, and the frequency of these cells in the lymph nodeswas determined by FACS at day 10 posttransplantation. A groupof mice that received splenocyte transplant and did not receivetumor cells served as negative controls to set the gates for GFP+

cells. There was a marked increase in the frequency of GFP+ P815cells in the lymph nodes of mice that were not subjected to GVHD(i.e., did not receive the splenocyte transplant) (Fig. 8). In contrast,transfusion of splenocytes into mice that had the tumor implantedresulted in a significant decrease in the frequency of GFP+ P815tumor cells, suggesting a significant GVL effect (Fig. 8). Treat-ment with evasin-1 had no major effect on the ability of the graftto decrease the frequency of GFP+ P815 tumor cells in the lymphnodes (Fig. 8). Entrapment of effector T cells could have ac-counted for the antitumor effect in evasin-1–treated animals.However, treatment with evasin-1 did not interfere with accumu-lation of activated donor (GFP+) in spleen or lymph nodes(number of GFP+CD8+CD69+ cells/spleen: vehicle-treated GVHDmice, 56.5 3 105 [613.4]; evasin-1–treated mice, 66.3 3 105 [611.8]; number of GFP+CD8+CD69+ cells/lymph node: vehicle-treated GVHD mice, 9.23 105 [64.2]; evasin-1–treated mice, 6.13105 [62.4]; n = 5 and p . 0.05).

DiscussionCCL3 is expressed during experimental GVHD and may be im-portant for the inflammatory response caused by acute GVHD (12,13). The results reported herewith confirm the relevance of CCL3in mediating GVHD in mice. Moreover, we report for the first timethat blockade of CCL3 with a CBP, evasin-1, which preventsCCL3 function (17), ameliorated GVHD and prevented death.Treatment with evasin-1 prevented the influx of leukocytes, es-pecially CD8+, CD4+ cells and macrophages, to the small intestineand decreased tissue damage in the liver. Mechanistically, in-hibition of leukocyte influx to the intestine was due to inhibitionby evasin-1 of the ability of leukocytes to adhere to endothelialcells in affected tissues. The latter results suggest that mediationof leukocyte adherence and subsequent migration is the majormechanism by which CCL3 participates in murine GVHD. Fi-nally, the protective effects of evasin-1 against GVHD did notinterfere with the beneficial effect of the graft against a leukemiccell line, hence showing that GVL was still operative in evasin-1–treated mice.The production of CCL3 peaked at day 10 after GVHD but was

already noticeable at day 3 and remained elevated at day 20,suggesting that the chemokine was generated continuouslythroughout the course of the disease. Studies in which donor cellswere obtained from C57BL/6JCCL32/2 mice showed that initiallevels of CCL3 were mostly derived from cells of recipient mice,whereas a considerable proportion of the production of CCL3derived from donor cells at days 10 (.80%) and 20 (.95%)postinduction. Hence, the cells transferred during grafting becomemajor contributors of CCL3 production as the disease progresses.In agreement with previous studies (12, 13), transfer of CCL3-deficient cells was accompanied by decreased GVHD-associatedclinical score, weight loss, and lethality. Treatment with evasin-1resulted in protective effects that were qualitatively and quanti-tatively similar to those observed in GVHD experiments usingcells from CCL32/2 mice. Indeed, the ability of evasin-1 to

FIGURE 6. Effects of treatment with evasin-1 on the number of cells

adherent to the mesenteric venules of mice subjected to GVHD, as as-

sessed by intravital microscopy. GVHD was induced by the transfer of

splenocytes from allogeneic (C57BL/6J) donors to B6D2F1 mice. Mice

that received splenocytes from syngeneic (B6D2F1) mice did not develop

disease and were considered the control group. In one group (evasin-1

daily), evasin-1 (10 mg in 200 ml of PBS s.c.) was given 30 min before

transplantation and twice per day during the entire duration of the ex-

periments. In another experiment (evasin-1 30 min), evasin-1 was given at

a single dose 30 min before the intravital microscopy. At day 20 after

induction of GVHD, mice were anesthetized, and intestinal venules (20–40

mm) were selected for counting the number of adherent leukocytes, using

intravital microscopy. Results are presented as the mean 6 SEM. pp ,0.01 compared with control; #p , 0.01 compared with GVHD; n = 6.

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decrease intestinal injury associated with GVHD may underlie itsability to prevent clinical deterioration and death. Evasin-1 ischaracterized by its exquisite selectivity for three CC chemokines:CCL3, CCL4, and the closely related chemokine CCL18, with K(D) values of 0.16, 0.81, and 3.21 nm, respectively (17), and hasbeen shown to effectively block the function of CCL3 in vivo (18).It is difficult to determine the contribution of inhibition of CCL4and CCL18 to the beneficial effects of evasin-1 in the system.However, the similarity of effects between evasin-1 treatment andCCL32/2 mice suggests that neutralization of CCL3 is probablythe major mechanism of evasin-1 in murine GVHD. Therefore,our experiments concur with the view that CCL3 is important fortissue injury and consequent clinical disease and lethality asso-ciated with GVHD; they are novel in suggesting that CCL3-basedtherapies are indeed effective in blocking GVHD in mice.A series of experiments were then carried out to understand the

mechanisms by which a blockade of CCL3 with evasin-1 wouldprotect from GVHD. CD8+ and CD4+ cells, as well as macro-phages, are leukocytes known to play an effector role in GVHD (7,25, 27–32). In evasin-1–treated animals, there was significant in-hibition of the influx of these leukocytes to the intestine of micesubjected to GVHD. Inhibition of recruitment was secondary tothe ability of evasin-1 to prevent the adhesion of donor leukocytesto the affected tissues, as assessed by intravital microscopy. Thelatter experiments suggest that CCL3 plays a major role in drivingadhesion and subsequent recruitment of leukocytes in the intestineand liver of mice subjected to GVHD. Moreover, there was also anindirect inhibition of the intestinal levels of the chemokine CCL5.As the interaction of CCL5 with its receptors contributes to therecruitment and proliferation of allospecific T cells in GVHD (33),the effects of evasin-1 on CCL5 levels may also be relevant for theoverall inhibition of intestinal infiltration of leukocytes. In thispaper, we have not investigated the mechanisms by which theinhibition of CCL3 would lead to the inhibition of CCL5, but thedata suggest that CCL3 is involved in the recruitment of cell typesthat are relevant for CCL5 production in the intestine of mice

subjected to GVHD. CCL5 is a known chemoattractant for T cellswith a predominant Th1 profile and may also be released by theselymphocytes (33–36). In agreement with the latter studies, thelocal production of IFN-g was decreased in the intestine and liverof evasin-1–treated mice subjected to GVHD. As IFN-g is shownto play a role in GVHD (37–40), a decrease in the levels of thiscytokine may also contribute to the overall inhibition of GVHD-associated disease and lethality. Moreover, studies have suggesteda role for TNF-a in GVHD (1–3, 8–10). In our studies, we ob-served an increase in TNF-a levels after GVHD induction, buttreatment with evasin-1 fails to alter TNF-a levels. The observa-tion that TNF-a levels were not modified, despite clinical

FIGURE 7. Evasin-1-treatment reduces inflammation and levels of IFN-g in the liver of mice subjected to GVHD. GVHD was induced by the transfer of

splenocytes from allogeneic (C57BL/6J) donors to B6D2F1 mice. Mice that received splenocytes from syngeneic (B6D2F1) mice did not develop disease

and were considered the control group. Evasin-1 (10 mg in 200 ml of PBS s.c.) was given 30 min before transplantation and twice per day during the entire

duration of the experiments. After induction of GVHD, mice were killed and the liver sampled for histopathological analysis and scored at 20 d post-

transplantation (A). The relative numbers of macrophages (B) and IFN-g (C) also were verified. Results are presented as the mean 6 SEM (n = 6). pp ,0.05 compared with control; #p , 0.05 compared with GVHD. D–F, Histological aspects of H&E-stained liver sections in control, GVHD, and evasin-1–

treated mice, respectively, at day 20 posttransplantation. Scale bar, 100 mm for all panels.

FIGURE 8. Evasin-1 treatment does not interfere with GVL in mice

subjected to GVHD.GVHDwas induced by the transfer of splenocytes from

allogeneic (C57BL/6J) donors to B6D2F1 mice. Mice that received sple-

nocytes from syngeneic (B6D2F1) mice did not develop disease and were

considered the control group. Evasin-1 (10 mg in 200 ml of PBS s.c.) was

given 30 min before transplantation and twice per day during the entire

duration of the experiments. GFP+P815 cells were injected i.v. into B6D2F1

recipients on day 0 of transplantation. At day 10 of transplantation, mice

were killed and the frequency of GFP+P815 cells evaluated in inguinal

lymph nodes. Results are presented as the mean6 SEM (n = 6). pp, 0.01

compared with mice that received no tumor cells; #p, 0.01 compared with

mice that received tumor but were not subjected to GVHD.

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protection and decreased death rates, is in line with the ability ofevasin-1 to decrease, but not abolish, disease. Whether thesemaintained levels of TNF-a contribute to effective GVT (seebelow) remains to be determined.BMT has become standard treatment for a number of malignant

and nonmalignant diseases, including acute and chronic leukemia,myelomas, lymphomas, aplastic anemia, solid tumors, and severeimmunodeficiency (1, 2). In these diseases, it is thought that BMTmay help in the elimination or decrease in growth of the tumor(41). The ability of grafted leukocytes to reduce tumor growth isreferred to as the GVL effect (41–46). Unfortunately, allograftedcells become activated against the host and cause GVHD (41–46).Therefore, it is generally accepted that, in the context of hema-topoietic stem cell transplantation, a therapy should decrease orprevent GVHD but ideally should not modify the associated GVL(42–46). In our study, there was significant GVL after the trans-plantation of splenocytes, as assessed by the inhibition of thegrowth of P815-GFP+, a mastocytoma cell line that becomesleukemic. Importantly, treatment with evasin-1 had no effect onGVL against P815-GFP+ cells, demonstrating that it preventeddisease without disrupting the beneficial effect of the host againstthe leukemic cell line.CBPs have arisen as new proteins capable of modulating in-

flammatory responses andmight be explored as potential therapeuticstrategies for inflammatory diseases. In this study, we observeddecidedly beneficial effects of the CCL3-binding protein evasin-1 ina murine model of GVHD. Altogether, these results suggest thatCCL3 blockade by evasin-1may be exploited clinically, a possibilitythat deserves further evaluation.

DisclosuresA.E.I.P. is an employee of Merck Serono, and the company holds the rights

for the development of evasin-1 for the therapy of inflammatory diseases.

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