Therapeutic Effects of Evasin-1, a Chemokine Binding Protein, in Bleomycin-Induced Pulmonary Fibrosis

Therapeutic Effects of Evasin-1, a Chemokine BindingProtein, in Bleomycin-Induced Pulmonary Fibrosis

Remo C. Russo1*, Ana L. Alessandri1*, Cristiana C. Garcia1, Barbara F. Cordeiro1, Vanessa Pinho2,Geovanni D. Cassali3, Amanda E. I. Proudfoot4, and Mauro M. Teixeira1

1Laboratorio de Imunofarmacologia, Departamento de Bioquımica e Imunologia, 2Departamento de Morfologia, Instituto de Ciencias Biologicas,

and 3Departamento de Patologia Geral, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte,Minas Gerais, Brazil; and 4Merck Serono Geneva Research Centre, Geneva, Switzerland

CC chemokines play an important role in the pathogenesis ofidiopathic pulmonary fibrosis. Few studies have evaluated theefficacy of therapeutically targeting CC chemokines and their re-ceptors during interstitial lung diseases. In the present study, thetherapeutic effects of Evasin-1, a tick-derived chemokine-bindingprotein that has high affinity for CCL3/microphage inflammatoryprotein (MIP)-1a, was investigated in a murine model of bleomycin-induced lung fibrosis. CCL3/MIP-1a concentrations in lung homog-enates increased significantly with time after bleomycin challenge,and this was accompanied by increased number of leukocytes andelevated levels of CCL2/monocyte chemoattractant protein (MCP)-1,CCL5/regulated upon activation, normal T cell expressed and se-creted, TNF-a and transforming growth factor–b1, and pulmonaryfibrosis. Administration of evasin-1 on a preventive (from the day ofbleomycin administration) or therapeutic (from Day 8 after bleomy-cin) schedule decreased number of leukocytes in the lung, reducedlevels of TNF-a and transforming growth factor–b1, and attenuatedlung fibrosis. These protective effects were similar to those observedin CCL3/MIP-1a–deficient mice. In conclusion, targeting CCL3/MIP-1a by treatment with evasin-1 is beneficial in the context of bleomy-cin-induced lung injury, even when treatment is started after thefibrogenic insult. Mechanistically, evasin-1 treatment was associatedwith decreased recruitment of leukocytes and production of fibro-genic cytokines. Modulation of CCL3/MIP-1a function by evasin-1could be useful for the treatment of idiopathic pulmonary fibrosis.

Keywords: chemokine; lung inflammation; pulmonary fibrosis;

bleomycin; evasin-1

Bleomycins are members of the glycopeptide antibiotics, clin-ically used as antineoplasic drugs for a variety of human cancers(1). An important side effect of these compounds is theinduction of lung fibrosis. Because of these effects, bleomycin-induced pulmonary injury and fibrosis in mice is an animalmodel widely used to investigate the biochemical, cellular, andmorphological mechanisms associated with pathogenesis of lung

inflammation and repair, as it resembles some aspects of humanidiopathic pulmonary fibrosis (IPF) (2). IPF is a progressive–degenerative, lethal disease that comprises a group of conditionscharacterized by chronic lung inflammation, disruption of alve-olar structure, interstitial fibroblast proliferation, and excessiveextracellular matrix synthesis and deposition (3). Althoughcauses of pulmonary fibrosis remain unclear and controversial(4, 5), available evidence suggests that leukocyte infiltration playsan important role during exacerbation of pulmonary injury, andalso regulates subsequent lung fibrosis by secretion of suchproinflammatory and profibrogenic factors as TNF-a and trans-forming growth factor (TGF)–b1, respectively (6). The coordi-nated recruitment of neutrophils, macrophages, lymphocytes,and fibroblasts into injured lungs is correlated with differentpatterns of chemokine and cytokine expression during eachphase of lung fibrosis, which are classically described as acuteand chronic lung inflammation, tissue repair, and scarring (7).There is no effective treatment for IPF.

Chemokines are small molecules that signal through seventransmembrane, G protein–coupled receptors, which areexpressed on both hematopoietic and nonhematopoietic cells.The functions of chemokines are diverse, and include chemo-attraction and proliferative effects on diverse cell types (8, 9).Clinical studies and animal models of pulmonary fibrosis haveshown a correlation between certain chemokines with lunginflammation and fibrosis (5–7, 10). For example, CCL2/mono-cyte chemoattractant protein (MCP)-1 production is required forlung fibrosis, and acts on the chemokine receptor, CCR2,expressed by macrophages and fibroblasts (11). It has also beenshown that CCL3/macrophage inflammatory protein (MIP)-1a

and CCL5/RANTES (regulated upon activation, normal T cellexpressed and secreted) contribute to an exacerbated lung injury(12, 13). More recently, Ishida and colleagues (13) demonstratedthat the CCL3/MIP-1a–CCR5 axis is involved in recruitment ofbone marrow–derived fibrocytes, TGF-b1–producing fibroblastprecursors, in bleomycin-induced pulmonary fibrosis.

Chemokine binding-proteins are pathogen-derived factorsthat may be used by viruses, helminthes, and arthropods toevade the host immune response (9, 14). We have recently clonedand characterized a new family of proteins, evasins, isolatedfrom the saliva of the tick, Rhipicephalus sanguineus (15).Evasins have potent chemokine-binding activity in vitro, whichreflects in potent anti-inflammatory effects in vivo (15–20).

CLINICAL RELEVANCE

Chemokines regulate key aspects of pulmonary inflamma-tion and fibrosis. Preventive or therapeutic administrationof evasin-1, a CCL3-binding protein, reduced bleomycin-induced pulmonary inflammation and fibrosis in mice.Evasin-1 decreased of profibrogenic molecules, suggestingthat it may be useful for the treatment of pulmonary fibrosis.

(Received in original form November 6, 2009 and in final form August 11, 2010)

*These authors contributed equally to this work.

This work was supported by the Coordenacxao de Aperfeicxoamento de Pessoal de

Nıvel Superior (Brazil), Fundacxao de Amparo a Pesquisa do Estado de Minas Gerais

(Brazil), Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (Brazil)

and the European Union’s Sixth Framework Program (Innovative Chemokine-

Based Therapeutic Strategies for Autoimmunity and Chronic Inflammation project

grant LSHBCT2005518167).

Correspondence and requests for reprints should be addressed to Mauro Martins

Teixeira, M.D., Ph.D., Laboratorio de Imunofarmacologia, Departamento de

Bioquımica e Imunologia, Instituto de Ciencias Biologicas—Universidade Federal

de Minas Gerais, Avenida Antonio Carlos, 6627, CEP 30882-650–Pampulha, Belo

Horizonte, MG, Brazil. E-mail: [email protected]

This article has an online supplement, which is accessible from this issue’s table of

contents at www.atsjournals.org

Am J Respir Cell Mol Biol Vol 45. pp 72–80, 2011

Originally Published in Press as DOI: 10.1165/rcmb.2009-0406OC on September 10, 2010

Internet address: www.atsjournals.org

An important characteristic of evasins is their fairly restrictedchemokine-binding activity that, nonetheless, results in inhibi-tion of inflammation in experimental models of arthritis (17),graft-versus-host disease (16), colitis (20), and myocardialischemia and reperfusion (19). One of these proteins, evasin-1, has higher affinity for CCL3/MIP-1a, and may also bind toCCL4/MIP-1b and CCL18/pulmonary and activation-regulatedchemokine (PARC), but with lower affinity (15, 17, 18). Thisselectivity for CCL3/MIP-1a over other chemokines, and effectsin various experimental systems, makes evasin-1 a useful phar-macological tool to study these chemokines in vivo. Treatmentof mice with evasin-1 from the time of bleomycin administrationsignificantly decreased lethality, pulmonary inflammation, andfibrosis caused by lethal doses of bleomycin (17). Using twodistinct protocols of bleomycin-induced lung fibrosis, the presentstudy substantially extends the previous observation, suggestingthe possible mechanisms. We demonstrate that evasin-1 de-creases lung fibrosis by preventing local infiltration of leuko-cytes, including T cells, neutrophils, and macrophages, and localproduction of proinflammatory and profibrogenic cytokines,including TNF-a and TGF-b1. The effects of evasin-1 weresimilar to those observed in CCL3/MIP-1a–deficient (CCL3/MIP-1a2/2) mice. Moreover, we demonstrate that therapeuticadministration of evasin-1 (i.e., 8 days after challenge) preservesmost of the beneficial effects of preventive administration.These results highlight the relevance of CCL3/MIP-1a in thecontext of bleomycin-induced fibrosis, and suggest that block-ade of this chemokine is useful in a therapeutically relevantmanner.

MATERIAL AND METHODS

For detailed MATERIAL AND METHODS, see the online supplement.

Bleomycin-Induced Lung Injury and Fibrosis in Mice

Male C57Bl/6J and CCL3/MIP-1a2/2 mice (8–10 wk old) (21)were used in the bleomycin-induced lung injury and fibrosismodel, performed as previously described (22). Briefly, a single25-ml injection containing 0.0625 U or 0.125 U of bleomycindiluted in PBS or PBS only was instilled intratracheally usingHamilton syringes. All experiments were conducted underconditions previously approved by the local animal ethicscommittee, Comite de Etica em Experimentacxao Animal/Uni-versidade Federal de Minas Gerais (protocol no. 146/06).Evasin-1 was produced as previously described (15).

Evasin-1 Treatment Schedules

Bleomycin-instilled mice received twice-daily treatment withvehicle or evasin-1 (10 mg/animal) by subcutaneous injectiondiluted in 100 ml of PBS. In the preventive schedule (Figure1A), evasin-1 was given 45 minutes before bleomycin injectionand then twice daily from Day 0 to Day 25. In the therapeuticschedule, administration of evasin-1 was from Day 8 to Day 25(Figure 1B) after bleomycin challenge.

Bronchoalveolar Lavage and FACS Analysis of

Airway Leukocytes

Bronchoalveolar lavage (BAL) was performed as previouslydescribed (22). Leukocytes recovered from BAL were stainedwith fluorescent-labeled monoclonal antibodies, CD3, CD4,CD8, and CD11b. Stained cells were acquired in FACScancytometer (Becton Dickinson, Franklin Lakes, NJ) and ana-lyzed in FlowJo software (Tree Star Inc., Ashland, OR).Frequencies of analyzed populations are shown as percentagesof total cell counts.

Quantification of Neutrophil and Macrophage by

Myeloperoxidase and N-Acetylglucosaminidase Activity

The extent of neutrophil accumulation in lung tissue was inferredby assaying myeloperoxidase (MPO) activity, as previously de-scribed (22). Macrophage accumulation in tissues was inferred, aspreviously described (23), by assaying N-acetylglucosaminidase(NAG) activity in homogenized lung samples.

Assessment of Cytokine and Chemokine Concentrations

Lung tissues were processed as previously described (22). Theconcentrations of TNF-a, active TGF-b1, CCL2/MCP-1, CCL3/MIP-1a, CCL4/MIP-1b, and CCL5/RANTES were measured inlung tissue homogenates. CXCL1/keratinocyte cytokine (KC) andCXCL2/MIP-2 were also measured in BAL fluid by ELISA, aspreviously described (22).

Assessment of Lung Fibrosis

Lung collagen content was determined by hydroxyprolinequantification, as previous described (22). The lung was re-moved and sections stained with Gomori’s trichrome. Quanti-tative analysis and Ashcroft fibrosis score were performed aspreviously described (22, 24).

Quantification of Anti–Evasin-1 Antibodies

Evasin-1 was diluted in 0.01 M carbonate buffer (pH 9.6) ina concentration of 4 mg per well and incubated overnight. Plateswere washed and incubated for 2 hours with goat anti-mouseIgG or IgG1. After another wash step, samples were incubatedwith peroxidase-labeled rabbit anti-goat followed by substrate(ortho-phenylenediamine). Reactions were stopped with 1 MH2SO4 and absorbance measured at 492 nm. Serum from micesensitized twice with 150 mg of evasin-1 in 2% Alum was used aspositive control. Results are presented as optical density.

Statistical Analysis

All data are presented as mean (6SEM) and were analyzed byone-way ANOVA, followed by Student-Newman-Keuls post hoctest, using GraphPad Prism 4.0 software (GraphPad SoftwareInc., San Diego, CA). Differences were considered significant atP , 0.05.

Figure 1. Evasin (Ev)-1 treatment schedules. Animals were givena single intratracheal challenge of bleomycin (Bleo) (0.0625U) or PBS

and evaluated 4, 8, and 25 days after challenge. Bronchoalveolar

lavage (BAL), lung leukocyte influx, and lung cytokine and chemokinelevels were evaluated at Days 4, 8, and 25. Fibrosis was accessed only at

Day 25 after bleomycin. (A) Preventive schedule: 10 mg/animal of

evasin-1 was given subcutaneously 45 minutes before bleomycin

injection, then twice a day from Day 0 to 25. (B) Therapeutic schedule:10 mg/animal of evasin-1 was given subcutaneously twice a day from

Day 8 to 25. BID, twice daily.

Russo, Alessandri, Garcia, et al.: Evasin-1 Reduces Pulmonary Fibrosis 73

RESULTS

Effects of Evasin-1 on Bleomycin-Induced

Pulmonary Inflammation

Using a severe disease protocol, our preliminary studies con-firmed that CCL3/MIP-1a was produced rapidly after bleomy-cin administration (0.125 U/animal), and that levels of CCL3/MIP-1a were high at least until Day 25 (Figure 2A). We havepreviously shown that evasin-1 inhibited CCL3/MIP-1a–induced granulocyte recruitment into the peritoneal cavity withmaximal inhibition at 10 mg/mice (17). Treatment with evasin-1(10 mg/mice twice daily) using a preventive schedule (Figure1A) reduced CCL3/MIP-1a levels in lungs of wild-type mice at8 days after bleomycin instillation (0.125 U/animal; Figure 2B).There was also decreased neutrophil influx into the airways andreduced production of active TGF-b1 in evasin-1–treated mice(Figures 2C and 2D, respectively). In agreement with a previousstudy (13), there was reduced neutrophil numbers in BAL andlow levels of active TGF-b1 (Figures 2C and 2D, respectively) inCCL3/MIP-1a2/2 mice exposed to bleomycin.

Thereafter, to evaluate the effects of chronic evasin-1administration, we used a milder disease protocol of bleomycin-induced lung fibrosis (0.0625 U/animal) on subsequent exper-iments. Daily treatment with evasin-1 (10 mg/mice twice daily)from the day of instillation (preventive schedule; Figure 1A)led to a significant reduction of total leukocyte number in BAL(Figure 3A), with inhibition of neutrophil recruitment at Days4 and 8 (Figure 3B), and reduced mononuclear cell numbers atDays 8 and 25 (Figure 3C). FACS analysis of BAL samplesshowed that evasin-1 given in a preventive treatment led tosignificant reduction in number of CD41 and CD81 T lym-phocyte in airways (Figure 3D). Evasin-1 also inhibited re-cruitment of CD11b1 cells in the airways 8 days afterbleomycin instillation (Figure 3D). Preventive treatment withevasin-1 also decreased neutrophil accumulation in lung tissuesat Days 8 and 25, as evaluated by MPO assay (Figure 4A).NAG levels, an index of macrophage accumulation in thelungs, were decreased at Day 8, but not at Day 24, afterbleomycin instillation (Figure 4B).

Treatment with evasin-1 from Day 8 after bleomycin in-stillation (Therapeutic schedule, Figure 1B) significantly de-creased the total cell influx in the BAL fluid at Day 25 (Figure3A), mostly due to inhibition of the influx of mononuclear cells(Figure 3C). The therapeutic treatment with evasin-1 failed todecrease MPO or NAG levels in the lung at Day 25 (Figure 4).

Effects of Evasin-1 on Bleomycin-Induced Pulmonary Levels of

Cytokines and Chemokines

Levels of the proinflammatory cytokine, TNF-a, were signifi-cantly increased in lungs of vehicle-treated mice after bleomy-cin administration. In contrast, treatment with evasin-1 in apreventive or therapeutic manner induced a marked reductionof TNF-a levels at all time points evaluated (Figure 5A). Therewas a significant increase in lung levels of active TGF-b1 atDays 8 and 25, but not at Day 4, after bleomycin instillation(Figure 5B). Preventive treatment with evasin-1 did not affectthe levels of this profibrogenic cytokine at Day 8, but bothpreventive and therapeutic administration of evasin-1 decreasedthe active TGF-b1 levels at Day 25 (Figure 5B).

Levels of CCL2/MCP-1 and CCL3/MIP-1a were increasedfrom Day 4 until Day 25 in lungs of vehicle-treated mice afterbleomycin instillation. Treatment with evasin-1 partially de-creased pulmonary levels of CCL2/MCP-1 at Days 4 and 25after bleomycin instillation when the drug was given in a pre-ventive manner (Figure 6A). Therapeutic administration ofevasin-1 failed to modify pulmonary levels of CCL2/MCP-1 at

Figure 2. Comparison of the effects of preventive treatment with

evasin-1 and results in CCL3-deficient mice instilled with bleomycin. (A)Kinetics of CCL3 production in the lungs in response to bleomycin

challenge, (B) pulmonary levels of CCL3, (C ) neutrophil numbers in

BAL fluid, and (D) active transforming growth factor (TGF)–b1 levels in

lungs of wild-type mice, CCL32/2 mice, and mice treated with evasin-1(10 mg/animal) on a preventive schedule. Animals were given a single

intratracheal challenge of bleomycin (0.125U) (white and gray columns)

or PBS (control, black columns), and parameters were evaluated 8 daysafter challenge. Results are representative of two experiments, and

are shown as the mean (6SEM) of seven to eight animals in each

group. *P , 0.05, **P , 0.01, and ***P , 0.001 compared to vehicle-

treated, bleomycin-challenged mice, or to PBS-instilled mice, as in-dicated. N.D., not determined; WT, wild-type.

74 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 45 2011

Day 25 (Figure 6A). Both regimens of evasin-1 administrationdrastically reduced levels of CCL3/MIP-1a, the specific target ofevasin-1 (Figure 6B). CCL4/MIP-1b levels were increased only

at Day 25 after bleomycin challenge, and both protocols ofevasin-1 administration failed to decrease these levels (Figure6C). The production of CCL5/RANTES was greater inbleomycin-instilled mice only at Day 25 after challenge.Whereas preventive treatment with evasin-1 decreased pulmo-nary levels of CCL5/RANTES levels, the therapeutic schedulehad no effect on these parameters. (Figure 6D). Levels ofCXCL1/KC and CXCL2/MIP-2, chemokines that are notrecognized by evasin-1, were accessed in BAL fluid. CXCL1/KCand CXCL2/MIP-2 were increased in BAL fluid of bleomycin-challenged mice at Days 4 and 8, and returned to basal levels atDay 25 (Table 1). Preventive treatment with evasin-1 partiallydecreased CXCL1/KC levels, but not CXCL2/MIP-2 levels, atthese time points in BAL fluid samples (Table 1).

Effects of Evasin-1 on Bleomycin-Induced Pulmonary Fibrosis

Pulmonary fibrosis was assessed by hydroxyproline lung contentand morphometry of Gomori’s Trichrome–stained lung sec-tions. Bleomycin administration caused chronic increase in totallung hydroxyproline levels and total lung wet weight at Day 25after intratracheal instillation (Figures 7A and 7B). Treatmentwith evasin-1 significantly diminished collagen deposition andtotal lung weight in the preventive and the therapeutic regimens(Figures 7A and 7B). We also assessed the effect of evasin-1 onfibrogenesis by morphometry of collagen stained with Gomori’sTrichrome. There was an increase in collagen area (green areas

Figure 3. Effects of preventive and therapeutic treatment with evasin-1

on bleomycin-induced leukocyte influx into the airway space. The

following parameters were evaluated in BAL: (A) total number of

leukocytes; (B) number of neutrophils; and (C) number of mononuclearcells (mononuclears). Animals were given a single intratracheal chal-

lenge of bleomycin (0.0625U) (white and gray columns) or PBS (control,

black columns), and leukocytes were evaluated 4, 8, and 25 days afterchallenge. FACS analysis of BAL fluid at Day 8 after bleomycin in-

stillation is presented (D), showing CD41 and CD81 lymphocytes and

total CD11b1-expressing BAL leukocytes. Results are representative of

two experiments, and are shown as the mean (6SEM) of seven to eightanimals in each group. *P , 0.05, **P , 0.01, and ***P , 0.001

compared to vehicle-treated, bleomycin-challenged mice, or to PBS-

instilled mice, as indicated. Veh, vehicle.

Figure 4. Effects of preventive and therapeutic treatment with evasin-1on bleomycin-induced lung neutrophil (A) and macrophage (B) accu-

mulation. Lung neutrophils and macrophages were evaluated by

determining of myeloperoxidase and N-acetylglucosaminidase activi-

ties, respectively, in lung homogenates. Animals were given a singleintratracheal challenge of bleomycin (0.0625U) (white and gray col-

umns) or PBS (control, black columns) and lung tissue evaluated 4, 8,

and 25 days after challenge. Results are representative of two exper-iments, and are shown as the mean (6SEM) of seven to eight animals

in each group. *P , 0.05, **P , 0.01, and ***P , 0.001 compared to

vehicle-treated, bleomycin-challenged mice, or to PBS-instilled mice, as

indicated. PMN, polymorphonuclear.


and shown as measured area [mm2]) at Day 25 after bleomycininstillation (Figure 7C). Preventive or therapeutic treatmentwith evasin-1 reduced total area of collagen deposition, asassessed by morphometry (Figure 7C).

The Ashcroft scale is a pathological score for the evaluationof bleomycin-induced lung fibrosis in small rodents, employedby visual assessment and analysis of Gomori’s trichrome–stained samples (24). We adopted this score of lung fibrosis toevaluate the effects of evasin-1 in bleomycin-challenged mice.Evasin-1 (preventively or therapeutically administered) led toamelioration of lung pathology at Day 25 after bleomycinchallenge (Figure 7D). Histopathological analysis of lungs atDay 25 after bleomycin challenge revealed loss of pulmonaryarchitecture, with pronounced lung damage, destruction ofalveolar septa, and large, contiguous, fibrotic masses (.50%of microscopic field). Fibrosis was diffuse and dense (Figure7F), contrasting with the preserved lung architecture in controlmice (Figure 7E). In bleomycin-instilled mice treated withevasin-1 in a preventive manner, there was moderate wallthickening with preserved lung architecture, and alveoli werepartly enlarged and rarefied. There was reduced damage andareas with attenuated and single fibrotic masses (<10% ofmicroscopic field) (Figure 7G). Similarly, therapeutic adminis-tration of evasin-1 also reduced pulmonary damage, wallthickening (septum .33 thicker than normal) with singlefibrotic masses (<15% of microscopic field), and preservationof lung structure after bleomycin instillation (Figure 7H).

Subsequently, we evaluated whether chronic subcutaneousinjection of evasin-1 was associated with the presence of anti–evasin-1 antibodies in mice subjected to bleomycin-induced

Figure 5. Effects of preventive and therapeutic treatment with evasin-1on bleomycin-induced pulmonary levels of TNF-a and TGF-b1. Animals

were given a single intratracheal challenge of bleomycin (0.0625U)

(white and gray columns) or PBS (control, black columns), and pulmo-

nary levels of (A) TNF-a and (B) active TGF-b1 evaluated at 4, 8, and 25days after challenge. Results are representative of two experiments, and

are shown as the mean (6SEM) of seven to eight animals in each

group. *P , 0.05, **P , 0.01, and ***P , 0.001 compared to vehicle-

treated, bleomycin-challenged mice, or to PBS-instilled mice, asindicated.

Figure 6. Effects of preventive and therapeutic treatment with evasin-1on bleomycin-induced lung chemokine production. The following

soluble mediators were evaluated by ELISA in lung tissue: (A) CCL2/

monocyte chemoattractant protein-1 (MCP-1) (B) CCL3/macrophageinflammatory protein (MIP)-1a; (C ) CCL4/MIP-1b; and (D) CCL5/

RANTES (regulated upon activation, normal T cell expressed and

secreted). Animals were given a single intratracheal challenge of

bleomycin (0.0625U) (white and gray columns) or PBS (control, blackcolumns), and chemokines were evaluated 4, 8, and 25 days after

challenge. Results are representative of two experiments, and are

shown as the mean (6SEM) of seven to eight animals in each group.

*P , 0.05, **P , 0.01, and ***P , 0.001 compared to vehicle-treated,bleomycin-challenged mice, or to PBS-instilled mice, as indicated.


lung fibrosis. Results show that subcutaneous administrationof evasin-1 for 25 days failed to increase the titer of anti–evasin-1 IgG (Figure 8A) and IgG1 (Figure 8B). In contrast,detectable antibody levels were observed in serum of animalsimmunized with evasin-1 adsorbed to alum (Figures 8A and 8B).

DISCUSSION

In the present study, we significantly broaden our previousobservation that evasin-1 protected against pulmonary inflam-

mation and fibrosis induced by bleomycin (17). The therapeuticand preventive anti-inflammatory effects of evasin-1 adminis-tration are now described, and possible mechanisms involved inprotection are shown. The major findings can be summarized asfollows: (1) preventive treatment with evasin-1 reduced neutro-phil, lymphocyte, and mononuclear migration into the lungs,reduced chemokine, TNF-a, and active TGF-b1 production, andreduced pulmonary fibrosis; (2) therapeutic treatment withevasin-1 from Day 8, at the peak of neutrophil influx, signifi-cantly reduced subsequent mononuclear cell migration, TNF-aand active TGF-b1 production, and reduced pulmonary fibrosis.Chronic subcutaneous administration of evasin-1 in both pro-tocols did not induce an antibody response against evasin-1.Overall, administration of evasin-1 mimicked the findingsobserved in CCL3/MIP-1a2/2 mice exposed to bleomycin,including reduced neutrophil influx into the airways and re-duction of pulmonary levels of active TGF-b1.

Evasin-1 is exquisitely selective for CCL3/MIP-1a andCCL4/MIP-1b over other chemokines (15). Its binding affinityfor CCL3/MIP-1a is some fivefold greater than for CCL4/MIP-1b,and some 20-fold greater than for CCL18/PARC (15), andresults in CCL3-deficient mice shown here and by Ishida andcolleagues (13) suggest that CCL3/MIP-1a, but not CCL4/MIP-1b,is relevant in bleomycin-induced lung injury. Moreover, theparadoxical role of CCL18/PARC in the context of bleomycin isdifficult to interpret, as CCL18/PARC overexpression enhanceslung inflammation, but reduces collagen deposition (25). There-fore, the beneficial effects of evasin-1 administration duringbleomycin-induced pulmonary fibrosis in mice may be ascribedmostly to blockade of CCL3/MIP-1a function.

Several studies have now shown that chemokines, includingCCL2/MCP-1, CCL3/MIP-1a, CCL5/RANTES, CXCL1/KC,CXCL2/MIP-2, CXCL9/monokine induced by gamma interferon(MIG), CXCL10/interferon gamma induced protein 10kDa(IP-10), and their receptors, contribute to the pathogenesis of

TABLE 1. EFFECTS OF EVASIN-1 TREATMENT ON CHEMOKINEPRODUCTION IN BRONCHOALVEOLAR LAVAGE FLUID DURINGBLEOMYCIN-INDUCED LUNG INJURY

Chemokine/Group Day 4 Day 8 Day 25

CXCL1/KC, pg/ml of BALF

Control 32.5 6 11.7

Bleo 1 vehicle 156.2 6 38.8* 117.5 6 31.4† 57.0 6 13.7

Bleo 1 evasin-1 89.1 6 33.9‡x 63.5 6 16.7x 50.8 6 12.2

CXCL2/MIP-2, pg/ml of BALF

Control 52.6 6 13.5

Bleo 1 vehicle 116.0 6 13.2† 103.7 6 16.8‡ 68.2 6 18.1

Bleo 1 evasin-1 110.4 6 11.1† 92.1 6 12.8‡ 87.9 6 28.5

Definition of abbreviations: BALF, bronchoalveolar lavage fluid; Bleo, bleomycin;

KC, keratinocyte cytokine; MIP, macrophage inflammatory protein.

Animals were given a single intratracheal dose of bleomycin (0.0625U) or PBS

(control) and evaluated at Days 4, 8, and 25 after challenge. Evasin-1 was given

subcutaneously twice a day at the dose of 10 mg/animal. The chemokines,

CXCL1 and CXCL2, were evaluated by ELISA assay in BALF. Chemokine levels

were expressed as picograms per milliliter of BALF. Results are representative of

two experiments, and data shown are mean 6 SEM of seven to eight animals in

each group.

* P , 0.001 when comparing all groups with control group.† P , 0.01 when comparing all groups with control group.‡ P , 0.05 when comparing all groups with control group.x P , 0.05 when comparing vehicle-given bleomycin-challenged and evasin-

1–treated, bleomycin-challenged mice.

Figure 7. Effects of preventive

and therapeutic treatment

with evasin-1 on bleomycin-

induced lung fibrosis. Animalswere given a single intratra-

cheal challenge of bleomycin

(0.0625U), and fibrosis wasevaluated 25 days after chal-

lenge. (A) Lung collagen de-

position was assessed by

hidroxyproline quantification.(B ) Total lung weight. (C ) Mor-

phometric analysis of Gomori’s

trichrome–stained areas. (D)

Ashcroft score of lung fibrosis.Bleomycin-challenged (white

and gray columns) or PBS in-

stilled (control, black columns)

mice are shown. Representa-tive sections of Gomori’s tri-

chrome–stained lungs after

PBS administration are shownin (E ). Bleomycin-challenged

animals are also shown (F–H ).

Note the large areas of colla-

gen deposition (green-stainedareas) at Day 25 (F ) after bleo-

mycin instillation. Preventive (G) or therapeutic (H) treatment with evasin-1 decreased the quantity and modified the pattern of collagen deposition

at Day 25 after challenge. Scale bars, 200 mm. Results are representative of two experiments, and are shown as mean (6SEM) of seven to eightanimals in each group. *P , 0.05, **P , 0.01, and ***P , 0.001 compared to vehicle-treated, bleomycin-challenged mice, or to PBS-instilled mice,

as indicated.


chronic inflammation and pulmonary fibrosis induced by theinstillation of bleomycin in the murine lungs (5, 6, 11–13, 22,26). For example, it has been shown that anti–CCL3/MIP-1a

antibodies reduced leukocyte accumulation and subsequentpulmonary fibrosis in bleomycin-instilled mice (27). Similarly,and in accordance with the literature (13), our studies usingCCL3/MIP-1a2/2 mice demonstrate a role for this chemokinein mediating inflammation and fibrosis (data not shown) afterbleomycin instillation. Consistent with these studies in animalmodels, the analysis of bronchoalveolar fluid and biopsies ofpatients with IPF have demonstrated higher levels of CCL3/MIP-1a than in samples from healthy volunteers (28). Inaddition, increased levels of CCL2/MCP-1 and CCL3/MIP-1a

were found in BAL fluid obtained from patients with IPF whencompared with healthy volunteers (29, 30), and in other relatedlung-fibrogenic disease induced by mustard gas inhalation (31).In the present study, we evaluated the therapeutic effects ofa chemokine-binding protein, evasin-1, and show that it reducedbleomycin-induced pulmonary inflammation and fibrosis inmice. Importantly, we show that treatment with evasin-1 waseffective even when started at Day 8 after bleomycin instilla-tion, a time when neutrophil influx was maximal.

The acute inflammatory process in response to bleomycininstillation is characterized by the initial presence of neutrophilsin lung interstitium and airway space, followed by the influx ofmononuclear cells (22, 32), especially lymphocytes and macro-phages. The latter cell types characterize the chronicity of thislung-inflammatory process (32), and were found to be decreasedfor preventive treatment with evasin-1. Neutrophils are thoughtto be major effector leukocytes in the context of IPF, and aremostly found in BAL fluid from sick patients (33). Experimen-tally, the blockade of neutrophil recruitment in the airway byblocking CXCR2 decreases bleomycin-induced lung injury (22,34). Studies in matrix metalloproteinase 8–deficient mice sug-gest that CCL3/MIP-1a is relevant for neutrophil influx ina model of acute lung injury, including that induced bybleomycin (35). The increased CCL3/MIP-1a and lymphocytenumbers in BAL samples from patients with IPF are alsorelated to lung fibrogenic disorders (28, 29, 31). As preventivetreatment started at Day 8, at the time when neutrophilaccumulation is maximal (22), there was no effect on totalMPO activity in the lungs at Day 25. We also observed thatpreventive treatment with evasin-1 reduced CXCL1/KC in BALfluid, but not CXCL2/MIP-2, which may contribute to reducedneutrophil influx into the airways after bleomycin instillation.Altogether, these results suggest that, although inhibition ofneutrophil influx is relevant for the development of fibrosis (13,22, 36), it is unlikely that an inhibitory effect of evasin-1 onneutrophil influx could account entirely for the inhibition offibrosis after bleomycin instillation.

Preventive treatment with evasin-1 also reduced lymphocyteand macrophage cell numbers in the airway space, but hada minor effect on total NAG activity, an index of macrophageinflux, in the lung. A similar effect was observed when the drugwas given from Day 8 after bleomycin instillation. In bothsituations, and in agreement with its mechanism of action(CCL3/MIP-1a binding), evasin-1 greatly decreased CCL3/MIP-1a levels in lungs. These results would suggest that binding to andinhibition of CCL3/MIP-1a function is important for the influx ofmononuclear cells, especially macrophages to the alveolar spaces,but less important to the arrival of these cells in lung parenchyma.In addition, we also observed some inhibition of CCL2/MCP-1and CCL5/RANTES production, especially when the drug wasgiven as a preventive treatment. Inhibition of CCL2/MCP-1 andCCL5/RANTES may be secondary to the inhibition of themigration and activation of both macrophages and neutrophils,which are potential sources of these chemokines. In addition, it ispossible that CCL3/MIP-1a can act on epithelial cells to promotechemokine release (13, 37). Regardless of the mechanism bywhich evasin-1 leads to an indirect inhibition of CCL2/MCP-1and CCL5/RANTES, it is of note that evasin-1 given from Day 8prevented lung fibrosis without affecting CCL2/MCP-1 andCCL5/RANTES levels significantly. Therefore, it appears thata major effect of evasin-1 in our model is to decrease the levelsand function of CCL3/MIP-1a.

TNF-a is expressed by lung macrophages, and is suggested toplay an important role in bleomycin-induced pulmonary fibrosis(38). In our study, preventive and therapeutic treatment withevasin-1 greatly reduced TNF-a levels induced by bleomycininstillation. Smith and colleagues (39) have suggested thatepithelial cells and resident alveolar macrophages are majorsources of IL-6 and TNF-a after bleomycin instillation. Thesecytokines act synergistically to stimulate CCL3/MIP-1a expres-sion by alveolar cells (39). CCL3/MIP-1a is produced by avariety of cells, including neutrophils, lymphocytes, monocytes/macrophages, mast cells, basophils, epithelial cells, and fibro-blasts, and it binds to CCR1 and CCR5 with high affinity toexert its biological effects (9, 40–44). Upon bleomycin exposure,

Figure 8. Effects of preventive and therapeutic treatment with evasin-1on anti–evasin-1 antibody production. Animals were given a single

intratracheal challenge of bleomycin (0.0625U) and titers of anti–

evasin-1 total IgG (A) and anti–evasin-1 IgG1 (B) in serum from mice

challenged with bleomycin and treated with vehicle or each evasin-1administration protocol. Animals immunized twice with evasin-1

adsorbed to alum were used as positive control. Results are presented

as optical density (O.D.) in serum diluted 50-fold. Results are shown as

means (6SEM) of three to nine animals in each group. *P , 0.05, **P ,

0.01, and ***P , 0.001.


there was greater CCL3/MIP-1a staining in alveolar macro-phages by in situ hybridization, suggesting that this resident celltype is the most important source of CCL3/MIP-1a proteinin vivo (39). The released CCL3/MIP-1a can then recruitneutrophils and inflammatory macrophages that can releasefurther TNF-a, CCL3/MIP-1a, and other chemokines, includingCCL2/MCP-1 (39, 43, 45). CCL3/MIP-1a levels could also beregulated by TNF-a that interferes with the state of leukocyteactivation and expression of markers of activation, such asCD11b (46). Together, these cytokines and chemokines cooper-ate to activate macrophages and lymphocytes in the lungs andcontribute to tissue injury. Administration of evasin-1 blocks theaction of CCL3/MIP-1a, hence preventing the positive feedbackcycle among alveolar macrophages and leukocytes, CCL3/MIP-1a, and TNF-a, which culminate in lung injury and fibrosis.

In addition to TNF-a, bleomycin exposure results in up-regulated expression of TGF-b1 mRNA, and TGF-b1 expres-sion appears to be dependent on TNF-a receptor activation(38). Anti–TGF-b1 antibodies have been shown to reduce, butnot to abrogate, bleomycin-induced pulmonary fibrosis (47). Inour study, preventive or therapeutic treatment with evasin-1greatly reduced bleomycin-induced TGF-b1 levels at Day 25,but there was TGF-b1 at Day 8. Moreover, the ability of evasin-1 to prevent CCL3/MIP-1a action leads to a decreased leuko-cyte influx and reduced TNF-a expression. Therefore, theeffects of evasin-1 on TGF-b1 may contribute to its inhibitoryeffects on fibrosis. This is consistent with the study by Ishida andcolleagues (13), which showed that CCL3/MIP-1a2/2 andCCR5-deficient mice had reduced macrophage and fibrocyteinflux, and lower TGF-b1 expression (13).

Neutralizing antibodies induced by natural immunizationcan have a dramatic effect on the potency and efficacy ofproteins used as therapeutics. The administration of foreignproteins could lead to antibody production against this peptideand rejection by the immune system (48). We evaluatedwhether long-term subcutaneous administration of evasin-1could induce an antibody response against evasin-1. Chronicsubcutaneous administration of evasin-1, twice a day for 25 days,did not induce an antibody response against evasin-1, suggestingthat the route of evasin-1 administration in the present treatmentprotocol was not immunogenic for mice.

In conclusion, our results show, for the first time, thattherapeutic administration of evasin-1 is efficient in reducingprogression of pulmonary injury and fibrosis induced by bleo-mycin in mice. The effects of evasin-1 treatment are similar tothose observed in CCL3/MIP-1a2/2 mice. Mechanistically,treatment with evasin-1 is associated with decreased recruit-ment of leukocytes and production of fibrogenic cytokines,including TGF-b1 and TNF-a. Therefore, targeting CCL3/MIP-1a by evasin-1 administration may be useful, therapeuti-cally, for the treatment of certain inflammatory and fibrogeniclung diseases, such as IPF.

Author Disclosure: A.E.I.P. has patents pending for chemokine-binding proteinsfrom tick salivary glands and modified chemokines with anti-inflammatoryactivities. M.M.T. received sponsored grants from MerckSerono for $5,001–$10,000, and Dompe S.Pa, Italy, for $10,001–$50,000. None of the otherauthors has a financial relationship with a commercial entity that has an interestin the subject of this manuscript.

Acknowledgments: The authors thank Valdineria Borges and Ilma Marcxal fortechnical assistance, and are grateful to Denise Carmona Cara and Maria de LourdesM. Noviello from the Departamento de Morfologia, Instituto de Ciencias Biologicas -Universidade Federal de Minas Gerais, for help with the anti–evasin-1 detection.

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Therapeutic Effects of Evasin-1, a Chemokine Binding Protein, in Bleomycin-Induced Pulmonary Fibrosis

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