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Bronchopulmonary Aspergillosisin a Murine Model of Allergic Immunomodulatory Role of C10 Chemokine

LukacsRobert M. Strieter, Steven L. Kunkel and Nicholas W. Cory M. Hogaboam, Chad S. Gallinat, Dennis D. Taub,

http://www.jimmunol.org/content/162/10/60711999; 162:6071-6079; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Immunomodulatory Role of C10 Chemokine in a MurineModel of Allergic Bronchopulmonary Aspergillosis1

Cory M. Hogaboam,2* Chad S. Gallinat,* Dennis D. Taub,‡ Robert M. Strieter,†

Steven L. Kunkel,* and Nicholas W. Lukacs*

The immunomodulatory role of the chemokine C10 was explored in allergic airway responses during experimental allergicbronchopulmonary aspergillosis (ABPA). The intratracheal delivery ofAsperigillus fumigatusAg into A. fumigatus-sensitized miceresulted in significantly increased levels of C10 within the bronchoalveolar lavage, and these levels peaked at 48 h afterA.fumigatuschallenge. In addition, C10 levels in BAL samples were greater than 5-fold higher than levels of other chemokines suchas monocyte-chemoattractant protein-1, eotaxin, and macrophage-inflammatory protein-1a. From in vitro studies, it was evidentthat major pulmonary sources of C10 may have included alveolar macrophages, lung fibroblasts, and vascular smooth musclecells. Experimental ABPA was associated with severe peribronchial eosinophilia, bronchial hyperresponsiveness, and augmentedIL-13 and IgE levels. The immunoneutralization of C10 with polyclonal anti-C10 antiserum 2 h before the intratrachealA.fumigatus challenge significantly reduced the airway inflammation and hyperresponsiveness in this model of ABPA, but had noeffect on IL-10 nor IgE levels. Taken together, these data suggest that C10 has a unique role in the progression of experimentalABPA. The Journal of Immunology,1999, 162: 6071–6079.

A irway allergic responses to the ubiquitous fungal organ-ism Aspergillus fumigatuscan complicate asthma andare often characterized by a Th2-type cytokine response

with varying severity of bronchial hyperresponsiveness, eosino-philia, and elevations in IgE (1, 2). These symptoms follow thedevelopment of hypersensitivity toA. fumigatusAg (3), and col-lectively describe a disease known as allergic bronchopulmonaryaspergillosis (ABPA)3 (4, 5). While the pathogenesis of ABPAstill remains speculative, murine models of ABPA have providedclues regarding the immune events that precipitate this form ofallergic airway disease (6). For example, both IL-4 and IL-5 con-tribute to the elevations in IgE, eosinophilia, and bronchial hyper-responsiveness associated with experimental ABPA (7–9, 10). In-terestingly, lung injury in A. fumigatus Ag-challenged miceappears to require eosinophils and CD41 T cells (10) in the ab-sence of IgE-mediated events. This latter observation is supportedby mutual observations thatA. fumigatusAg exposure induceseosinophilia in mice before the elevation of serum IgE levels (11,8) and that ABPA proceeds normally in IgE knockout mice (12, 9).Other recent studies have revealed that this allergic disease is not

necessarily a Th2-polarized event either, rather a combined Th1-and Th2-type cytokine response is present and these cytokine re-sponses are regulated by endogenous IL-10 (13).

Despite advances in the characterization of immune events inexperimental ABPA, a major deficit in knowledge persists regard-ing the role of chemotactic cytokines in airway allergic responsesto Aspergillusfungus. Clinical observations in asthmatic patientsshow that C-C chemokines such as monocyte-chemoattractant pro-tein-1 (MCP-1), RANTES, eotaxin, and macrophage-inflamma-tory protein-1a (MIP-1a) are elevated in allergic asthmatic pa-tients before and following Ag challenge (14–16, 17). Thesefindings have supplied the impetus for the exploration of the roleof these and other chemokines in experimental allergic airway re-sponses to a number of small protein allergens (18–20). Surpris-ingly, functional redundancy among the chemokines involved inthe allergic airway response appears to be minor due in large partto the orchestrated timing and the tissue-specific localization ofchemokine production (18, 21).

Growing evidence also suggests that certain chemokines possessshadow or modulating roles within immune responses (22). Orig-inally identified in GM-CSF-activated bone marrow cells (23),MIP-related protein 1 or C10 is a chemokine that is postulated tofit this role. C10 has a genomic structure that includes an additionalexon, making it unique from other chemokines (24), and this ad-ditional exon is necessary for a significant portion of the biologicactivity of this molecule (25). C10 is chemotactic for B cellsand CD41 T cells (26), and is highly homologous to human che-mokines such as MIP-1d (27), CCF18 (28), HCC-1, and HCC-2(29). These human chemokines all possess similar affinity for CCchemokine receptor 1 and promote T cell and monocyte chemo-taxis. Previous studies indicated that unlike numerous other che-mokines, C10 is IL-4- but not LPS-inducible in macrophages andrequires de novo protein synthesis that delays its appearance forat least 24 h after cell activation (26). Thus, the present studywas directed at elucidating the role of C10 in the developmentof allergic airway inflammation and hyperresponsiveness toA. fumigatuschallenge.

Departments of *Pathology and†Internal Medicine, Division of Pulmonary and Crit-ical Care, University of Michigan Medical School, Ann Arbor, MI 48109; and‡Na-tional Institute of Aging, National Institutes of Health, Baltimore, MD 21224

Received for publication November 30, 1998. Accepted for publication February22, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants 1P50HL56402,HL35276, HL31963, and AI36302.2 Address correspondence and reprint requests to Dr. Cory M. Hogaboam, Depart-ment of Pathology, University of Michigan Medical School, 1301 Catherine Rd., AnnArbor, MI 48109-0602. E-mail address: hogaboam@path.med.umich.edu3 Abbreviations used in this paper: ABPA, allergic bronchopulmonary aspergillosis;BAL, bronchoalveolar lavage; MCP, monocyte-chemoattractant protein; MIP, macroph-age-inflammatory protein; VSMC, vascular smooth muscle cell.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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Materials and MethodsMurine model of allergic bronchopulmonary aspergillosis

Specific pathogen-free, female CBA/J mice were purchased from The Jack-son Laboratory (Bar Harbor, ME) and were maintained under specificpathogen-free conditions before and during experiments. Sensitization ofmice to solubleA. fumigatusAgs was achieved using a previously de-scribed procedure (30, 31). Briefly, all mice received a total of 10mg of A.fumigatuscrude Ag (Greer Laboratories, Lenoir, NC) dissolved in 0.2 mlof IFA (Sigma, St. Louis, MO). One-half of this preparation was thendeposited in the peritoneal cavity, and the remainder was delivered s.c.Two weeks later, mice received a total of 20mg of A. fumigatusAgsdissolved in normal saline via the intranasal route. Four days after theintranasal challenge, mice received 20mg of A. fumigatusAg dissolved innormal saline via the intratracheal route. In additional groups ofA. fumiga-tus-sensitized mice, 0.5 ml of polyclonal anti-C10 antiserum or normalrabbit serum was delivered into the peritoneal cavity of these mice 2 hbefore the intratracheal Ag challenge. Mouse lung responsiveness to i.v.methacholine administration and a number of additional parameters of al-lergic airway inflammation were examined at various times after theA.fumigatusintratracheal challenge. Prior approval for mouse usage in thesestudies was obtained from University Laboratory Animal Medicine(ULAM) facility at the University of Michigan Medical School (Ann Ar-bor, MI).

Anti-C10 Ab generation

Polyclonal anti-C10 antiserum was generated by the multiple site immu-nization of a New Zealand White rabbit using anEscherichia coli-ex-pressed C10 protein (25, 26). The resulting anti-C10 antiserum was puri-fied over a protein A affinity column, and 10ml of rabbit anti-C10antiserum neutralized approximately 20 ng of C10 in vitro (data notshown). This antiserum was titrated by direct ELISA, and no cross-reac-tivity with the following recombinant murine cytokines and chemokines:IL-1b, TNF-a, IL-4, IFN-g, IL-10, IL-6, MIP-1a, MCP-1, MIP-1b,RANTES, KC, eotaxin, MIP-2, and MARC (MCP-3). For this and othermethods described below, all recombinant murine cytokines and chemo-kines were obtained from R&D Systems (Minneapolis, MN), Genzyme(Cambridge, MA), or Pepro Tech (Rocky Hill, NJ).

Cell isolation and culture

Alveolar macrophages were isolated from bronchoalveolar lavage (BAL)samples taken from nonsensitized CBA/J mice. BAL samples were ob-tained through the multiple intratracheal introduction of 1 ml of PBS con-taining 50 mM EDTA. MC-9 mast cells (ATCC CRL 8306) represent amature, nontransformed mast cell line, and these cells were maintained inculture with IL-3 supplementation and DMEM growth medium containing1% (v/v) antibiotic-antimycotic and 15% (v/v) FBS (DMEM-15). Fibro-blasts and vascular smooth muscle cells (VSMCs) from nonsensitizedCBA/J mice were grown out from lung using techniques previously de-scribed in detail elsewhere (32, 33). Briefly, pulmonary fibroblasts weregrown out from mechanically dispersed whole lungs in 175-ml tissue cul-ture flasks containing DMEM-15. VSMCs were grown from explants oflarge pulmonary vessels in 60-mm tissue culture plates in the presence ofDMEM-15. After a minimum of three passages, homogenous populationsof fibroblasts and VSMCs were transferred to six-well tissue culture plates.Before an experiment, lung fibroblasts and VSMCs were stained foraactin, desmin, anda-naphthyl acetate esterase. After the third passage, lungfibroblasts stained weakly fora actin, suggesting a myofibroblast-type phe-notype, and cultures of these cells were found to be completely free ofa-naphthyl acetate esterase-positive cells such as macrophages (data notshown). In contrast, VSMCs stained strongly fora actin (i.e.,$95% pos-itive), and confluent cultures of these cells exhibited a “hill and valley”appearance that is typical of cultured smooth muscle cells, and these cul-tures of VSMCs were devoid of macrophages. Lung fibroblasts andVSMCs were used in these experiments up to the sixth passage.

Cell culture protocols

Preparations of alveolar macrophages were typically greater than 95%pure, and these cells were suspended in RPMI 1640 containing 10% FBS(RPMI-10) at 13 106 cells/well of a six-well tissue culture plate. Indi-vidual wells were then exposed to RPMI-10 alone or to IL-1b, TNF-a,IL-4, IFN-g, or IL-10 at 10 ng/ml in RPMI-10 for 24 h. MC-9 mast cellsat a density of 23 105 cells/well in six-well tissue culture plates were leftuntreated or received 10mg/ml of compound 48/80, a potent nonspecificmast cell activator (34). Each well in a six-well tissue culture plate wasseeded with approximately 13 106 fibroblasts or VSMCs. Twenty-fourhours later, the DMEM growth medium was replaced with RPMI contain-

ing 10% FBS (RPMI-10) containing IL-1b, TNF-a, IL-4, IFN-g, or IL-10at 10 ng/ml. Cytokine combinations of IL-1b 1 IL-4, IL-1b 1 IFN-g,IL-1b 1 IL-10, TNF-a 1 IL-4, TNF-a 1 IFN-g, or TNF-a 1 IL-10 (10ng/ml of each) were also added to other wells. Twenty-four hours after theaddition of cytokines to cultured fibroblasts and VSMCs, cell-free super-natants were removed for the measurement of C10 levels by ELISA.

Eosinophil chemotaxis

A modified Boyden chamber technique was used to quantify eosinophilchemotactic responses to C10 and eotaxin, a potent and selective eosino-phil chemoattractant (35). The eosinophils used in this experiment wereisolated from mice chronically infected withSchistosoma mansoni(thesemice were obtained from the National Institute of Health, Bethesda, MD).Briefly, 1 ml of thioglycolate broth was delivered by i.p. injection into eachmouse, and 48 h later the peritoneal cavity of these mice was thoroughlylavaged with normal saline. The collected cells were suspended in RPMI1640 containing 5% FBS, 2-ME (10mM), sodium pyruvate (2 mM),L-glutamine (20 mM), penicillin (100 U), and streptomycin (100 mg/ml).Eosinophils were adhesion purified in 175-ml tissue culture flasks for 1 hto yield preparations that contained approximately 85% eosinophilic gran-ulocytes. Contaminating cells were primarily lymphocytes. These cellswere suspended at 13 107 cells/ml of RPMI-10, and a 100-ml aliquot ofthis suspension was placed in individual wells of a 24-well microchemo-taxis chamber. The upper wells were separated from lower wells containing10–100 ng/ml of C10 or eotaxin by a 3-mm-pore-size polycarbonate filter(Nucleopore, Pleasanton, CA). The microchemotaxis chambers were incu-bated for 4 h at 37°C in a 5% CO2 incubator, after which the filters werefixed in 4% paraformaldehyde, stained in hematoxylin and eosin, andmounted onto a glass microscope slide for light microscopy visualization.Migrated eosinophils were subsequently quantified in at least twenty3400fields of view.

Assessment of bronchial hyperresponsiveness

Bronchial hyperresponsiveness was assessed in a Buxco plethysmograph(Buxco, Troy, NY) specifically designed for the low tidal volumes of mice,as described previously (36). Sodium pentobarbital (Butler, Columbus,OH; 0.04 mg/g of mouse body weight)-anesthetized mice were intubatedand constantly ventilated using a Harvard pump ventilator (Harvard Ap-paratus, Reno, NV). The following ventilation parameters were employedfor each mouse: tidal volume5 0.25 ml, breathing frequency5 120/min,and positive end-expiratory pressure> 3 cm H2O. Within the sealed ple-thysmograph mouse chamber, transpulmonary pressure (i.e.,D trachealpressure2 D mouse chamber pressure) and inspiratory volume or flowwere monitored online by an adjacent computer. Airway resistance wascontinuously calculated online by a specialized computer software program(Buxco), and this measurement was the result of the division of thetranspulmonary pressure by the change in inspiratory volume. Following abaseline period, mice received 10mg of methacholine by tail vein injection,and airway responsiveness to this nonselective bronchoconstrictor wasagain calculated online. Nonsensitized mice exhibited little change in air-way resistance following a similar challenge with methacholine. At theconclusion of the assessment of airway responsiveness, a BAL was per-formed on each mouse. The cell-free supernatant from each BAL samplewas frozen at220°C before chemokine and cytokine ELISA. Whole lungswere then dissected from the thoracic cavity, and snap frozen in liquid N2

or prepared for histologic analysis.

Quantification of leukocytes in BAL

Cells suspended in the BAL were pelleted onto glass slides by cytocen-trifugation and subjected to Diff-Quik (Baxter, McGraw Park, IL) staining,and polymorphonuclear and mononuclear cells were then quantified bylight microscopy at3200 magnification.

Chemokine and cytokine ELISA analysis of BAL and whole lung

Murine eotaxin, MCP-1, C10, KC, MIP-2, MARC (mouse MCP-3), MIP-1a, IL-13, and IL-10 were determined in 50-ml supernatant samples fromcell-free BAL samples or whole lung homogenates using a standardizedsandwich ELISA (37). Briefly, Nunc-immuno ELISA plates (MaxiSorp)were coated with the appropriate cytokine capture polyclonal Ab at a di-lution of 1–5mg/ml of coating buffer (0.6 M NaCl; 0.26 M H3BO4; 0.08M NaOH; pH 9.6) for 16 h at 4°C. The rabbit polyclonal Abs directedagainst murine eotaxin, C10, KC, IL-10, and IL-13 were purchased fromR&D Systems. The rabbit polyclonal Abs directed against murine MCP-1,MIP-2, and MARC were purified from the sera of immunized rabbits, aspreviously described (37). These rabbit polyclonal Abs were used for cap-ture and detection in the ELISA system, and the specificity of each was

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confirmed by running a panel of recombinant cytokines and chemokinesthrough each ELISA (see panel listed above). The unbound capture Ab waswashed away, and each plate was blocked with 2% BSA-PBS for 90 minat 37°C. Each ELISA plate was then washed with PBS Tween-20 (0.05%;v/v), and 50-ml samples either undiluted or diluted 1/10 were added toduplicate wells and incubated for 1 h at 37°C. Following the incubationperiod, the ELISA plates were then thoroughly washed and the appropriatebiotinylated polyclonal rabbit anti-cytokine Ab (3.5mg/ml) was added. Thepolyclonal Abs were biotinylated using an EZ-Link system from Pierce(Rockford, IL). After washing the plates 30 min later, streptavidin-perox-idase (Bio-Rad Laboratories, Richmond, CA) was added to each well for30 min, and each plate was thoroughly washed again. Chromagen substrate(Bio-Rad Laboratories) was added, and optical readings at 492 nm wereobtained using an ELISA plate scanner. Recombinant murine cytokinesand chemokines were used to generate the standard curves from which theconcentrations present in the samples were derived. The limit of ELISAdetection for each cytokine was consistently above 50 pg/ml.

Lung histologic analysis

Whole lungs fromA. fumigatus-sensitized mice were fully inflated by in-tratracheal perfusion with 4% paraformaldehyde before dissection from thethoracic cavity and placement in fresh paraformaldehyde for 24 h. Routinehistologic techniques were used to paraffin-embed this tissue, and 5-mmsections of whole lung were counterstained with Mayer’s hematoxylin(Mayer & Myles Laboratories, Coopersburg, PA) for the visualization andidentification of eosinophils. Inflammatory infiltrates and other histologicchanges were monitored around blood vessels and airways using light mi-croscopy at a magnification of3200.

Determination of systemic IgE

Sera fromA. fumigatus-sensitized mice were analyzed for total IgE beforeand following intratrachealA. fumigatuschallenge. Complementary cap-ture and detection Ab pairs for mouse IgE were obtained from PharMingen(San Diego, CA), and the IgE ELISA was performed according to theenclosed directions. Duplicate sera samples were diluted 1/100, IgE levelsin each were calculated from OD readings at 492 nm, and IgE concentra-tions were calculated from a standard curve generated using rIgE (5–2000pg/ml).

Data statistical analysis

All results are expressed as mean6 SEM (SE). All test conditions werecompleted in duplicate wells of a tissue culture plate. A Student’st test wasused to determine statistical significance between the control and anti-C10groups;p , 0.05 was considered statistically significant.

ResultsExperimental allergic bronchopulmonary aspergillosis isassociated with greatly augmented C10 levels in the BAL

Although C-C chemokines have previously been shown to exertsignificant roles during airway allergic responses to a number ofsmall protein Ags, the expression of chemokines during experi-mental ABPA was previously uncharacterized. The results fromELISA analysis of BAL cell-free supernatants from our experi-mental model of ABPA for the presence of C-C chemokines (i.e.,MCP-1, eotaxin, MIP-1a, and MARC), CXC chemokines (i.e.,MIP-2 and KC), and C10 are shown in Fig. 1. Major elevationsabove baseline values (measured in BAL from sensitized micebefore intratrachealA. fumigatuschallenge) were observed in BALlevels of C10, MCP-1, and eotaxin, but C10 was increased thegreatest to approximately 30 ng/ml by 48 h after intratracheal chal-lenge (Fig. 1). By comparison, MCP-1 and eotaxin levels reached5 and 1 ng/ml, respectively, over the same period of time (Fig. 1).At 72 h afterA. fumigatuschallenges, BAL levels of all of thechemokines examined had returned to baseline values or were notdetected by ELISA. These findings suggested that the allergic air-way responses toA. fumigatusAg involved profound changes inC10 generation, particularly in the 48-h period afterA. fumigatusAg challenge.

Alveolar macrophages, pulmonary fibroblasts, and VSMCsgenerate C10

Given the very dramatic elevations in C10 observed in the ABPAmice following A. fumigatus challenge, the putative cellularsources of C10 in the lung were next explored. Consistent withprevious studies showing that C10 is an IL-4-inducible chemokinein isolated peritoneal macrophages (26), isolated alveolar macro-phages from nonsensitized mice were a constitutive source of C10,and IL-4 was the most potent inducer of C10 release by these cells(Table I). Another immune cell, the MC-9 mast cell, was a muchpoorer constitutive source of C10 (0.386 0.02 ng/ml), and 48/80activation of these mast cells did not markedly augment their re-lease of C10 (0.446 0.02 ng/ml) above the constitutive level. Ourprevious studies suggested that structural cells in the lung such aslung fibroblasts and smooth muscle might also generate C10 (38).Thus, the putative contribution of fibroblasts and VSMCs to theoverall C10 production within the lung was also examined. Asshown in Fig. 2, constitutive and cytokine-inducible C10 produc-tion was detected in 24-h cultures of both structural cell types. InVSMCs, only IL-1b, either alone or in combination with IL-4,IFN-g, or IL-10, enhanced C10 levels approximately 3-fold abovelevels detected in untreated (i.e., control) cultures (Fig. 2). Thepresence of either IL-1b, TNF-a, or IL-10 in cultures of pulmo-nary fibroblasts enhanced C10 production at least 2-fold above

FIGURE 1. BAL chemokine levels fromA. fumigatus-sensitized miceat various times after intratracheal challenge withA. fumigatusAg. Imme-diately following the determination of the airway hyperresponsiveness tomethacholine challenge (seeMaterials and Methods), mice were intratra-cheally lavaged with PBS containing 50 mM EDTA. The total volume ofthe lavage obtained from each mouse was 1 ml. The cells contained in theBAL were pelleted onto microscope slides for quantification, and specificELISA analyzed the cell-free supernatants. Data are mean6 SE from aminimum of five mice per timepoint.

Table I. C10 generation by purified alveolar macrophagesa

Cytokine Treatment (allcytokines at 10 ng/ml)

C10 Levels(ng/ml)

Control 116 0.1IL-1b 196 3.0TNF-a 4.26 1.2IL-4 40 6 0.4p

IFN-g 156 1.1IL-10 166 2.0

a Alveolar macrophages were purified from BAL samples of nonsensitized CBA/Jmice. A total of 1.03 106 cells were exposed to cytokines at 10 ng/ml for 24 h priorto the removal of cell-free supernatants for ELISA analysis for C10.

p p # 0.05 compared with control.

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control levels (Fig. 2). C10 levels were also enhanced between 3-and 5-fold (above control level) in cultures of pulmonary fibro-blasts that were incubated with similar combinations of cytokinesfor 24 h. Taken together, these data indicated that alveolar mac-rophages and pulmonary structural cells generated C10 in responseto inflammatory and immune cytokines.

Airway hyperresponsiveness during experimental aspergillosis ismodulated by C10

To evaluate the significance of changes in C10 generation withinthe allergic airways, C10 was immunoneutralized inA. fumigatus-sensitized mice 2 h before intratracheal challenge withA. fumiga-tus Ag. C10 levels in BAL samples from both treatment groupswere markedly greater than levels measured in the whole homog-enates, consistent with the observation noted above that alveolarmacrophages were a major source of C10 particularly followingexposure to IL-4 (see Table I). The successful immunoneutraliza-tion of C10 using an i.p. injection of anti-C10 antiserum was con-firmed in BAL (Fig. 3) and in whole lung homogenates (Fig. 3)removed before and 24 and 48 h after intratrachealA. fumigatusAg challenge. However, in the lung homogenates at 48 h afterA.fumigatuschallenge, C10 levels were not different between the twotreatment groups.

Airway responses to methacholine inA. fumigatus-sensitizedmice immediately before (i.e., att 5 0) intratracheal challengewith A. fumigatusAg were not different from responses measuredin nonsensitized mice (data not shown). However, at 24 and 48 hafter the intratracheal administration ofA. fumigatusAg in sensi-

tized mice, airway hyperresponsiveness to methacholine provoca-tion was significantly elevated approximately 3-fold above airwayresponses measured prior att 5 0 (Fig. 4). Immunoneutralizationof C10 in A. fumigatus-sensitized mice reversed the increase inairway hyperresponsiveness at 24 and 48 h after intratracheal chal-lenge. In addition, airway hyperresponsiveness to methacholine at48 h post-A. fumigatuschallenge was significantly lower than re-sponses measured in allergic mice that received normal rabbit se-rum (Fig. 4).

C10 is an eosinophil chemoattractant

The directed migration or chemotaxis of leukocytes down a che-mokine concentration gradient is a classical hallmark of chemo-kine activation (39). The chemotaxis of eosinophils following che-mokine activation is limited to a select group of C-C chemokinesthat until now did not include C10. However, the present experi-ments showed that eosinophils from the peritoneal cavity of micechronically infected withS. mansoniexhibited marked chemotac-tic responses to 100 ng/ml of C10 (Table II). Nevertheless, C10was not as potent as similar concentrations of eotaxin in the che-motaxis of eosinophils (Table II).

Immunoneutralization of C10 markedly diminishes airwayeosinophilia during experimental ABPA

As depicted in Fig. 5, the presence of eosinophils in the BAL ofallergic mice that received anti-C10 antiserum was significantlyreduced at 24 and 48 h afterA. fumigatuschallenge compared withallergic mice that were pretreated with normal serum. Also at the

FIGURE 2. C10 chemokine synthesis by puri-fied VSMCs and fibroblasts derived from normalmouse lungs. Primary cultures of both cell typeswere left untreated (i.e., control) or exposed to cy-tokines for 24 h, after which cell-free supernatantswere removed for ELISA determination of C10levels. Data are representative of three similarexperiments.

FIGURE 3. Effects of anti-C10 antiserum treatment on pulmonary C10 levels inA. fumigatus-sensitized mice before and at 24 and 48 h after intratrachealA. fumigatusAg challenge. Sensitized mice received 0.5 ml of anti-C10 antiserum or normal rabbit serum (i.e., control) via an i.p. injection 2 h before theAg challenge. C10 was measured in cell-free BAL samples and in whole lung homogenates taken from allergic mice immediately following the deter-mination of bronchial hyperresponsiveness (seeMaterials and Methods). Data are mean6 SE from a minimum of five mice per timepoint in both treatmentgroups.p, p # 0.05 compared with the corresponding control mice that received normal rabbit serum beforeA. fumigatusAg challenge.

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24-h timepoint afterA. fumigatuschallenge, significantly fewerlymphocytes and macrophages were present in cytospins of BAL(Fig. 5). However, macrophage counts in the BAL of anti-C10antiserum-pretreated mice were significantly elevated at 48 h afterAg challenge compared with BAL counts from mice that werepretreated with normal serum. Neutrophil counts were not differentbetween the two treatment groups.

Further confirmation of the role of C10 in the tissue eosinophiliaassociated with experimental ABPA was confirmed from histo-logic samples derived from mice beforeA. fumigatusAg challenge(Fig. 6,A andB), at 24 h (Fig. 6,C andD), and at 48 h (Fig. 6,EandF) after this challenge. Little inflammation was present in ei-ther the normal serum (Fig. 6A) or anti-C10 antiserum (Fig. 6B)treatment groups before the intratracheal challenge of theseAs-pergillus-sensitized mice withA. fumigatusAg. After A. fumigatuschallenge, dramatic histologic changes in the lungs of ABPA miceincluded major inflammatory infiltrates surrounding various sizedairways and blood vessels at 24 h (Fig. 6C) and at 48 h (Fig. 6E)after A. fumigatus Ag challenge. The inflammatory infiltratearound the airways of these mice that received normal serum wascomposed of abundant eosinophils, accompanied by smaller quan-tities of lymphocytes, macrophages, and neutrophils. Conversely, a

FIGURE 4. Effect of anti-C10 antiserum treatment on bronchial hyper-responsiveness inA. fumigatus-sensitized mice before and at 24 and 48 hafter intratrachealA. fumigatusAg challenge. Sensitized mice received 0.5ml of anti-C10 antiserum or normal rabbit serum (i.e., control) via an i.p.injection 2 h before the Ag challenge. Bronchial hyperresponsiveness toi.v. methacholine provocation was conducted as described inMaterials andMethods. Data are mean6 SE from a minimum of five mice per timepointin both treatment groups.p, p # 0.05 compared with mice beforeA. fu-migatusAg challenge (i.e.,t 5 0). t, p 5 0.034 compared with the cor-responding control mice that received normal rabbit serum beforeA. fu-migatusAg challenge.

FIGURE 5. The presence of eosinophils, lymphocytes, neutrophils, and macrophages in BAL fromA. fumigatus-sensitized mice at 24 and 48 h afterintratrachealA. fumigatusAg challenge. Sensitized mice received 0.5 ml of anti-C10 antiserum or normal rabbit serum (i.e., control) via an i.p. injection2 h before the Ag challenge. BAL counts were made from pelleted whole cell preparations from each mouse immediately after i.v. methacholine provocation(seeMaterials and Methods). Only macrophages were present in BAL samples from both groups immediately before the intratrachealA. fumigatuschallenge (data not shown).ppp, p # 0.001;p, p # 0.05 compared with the corresponding control mice that received normal rabbit serum beforeA.fumigatusAg challenge.

Table II. Eosinophil chemotactic responses to C10 and eotaxina

Response(mean6 SE)

Control 576 8C10 (ng/ml)

1 1316 910 1496 4

100 1916 15Eotaxin (ng/ml)

10 1456 15100 2886 6

a Migrated eosinophils were counted on fixed and stained polycarbonate mem-branes by light microscopy at3400. Data are the mean6 SE of a minimum of 20fields of view. Similar findings were obtained in an additional experiment.

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clear paucity of inflammatory cells surrounding large airways wasobserved when ABPA mice received anti-C10 antiserum, most no-tably at the 24-h (Fig. 6D) and 48-h (Fig. 6F) timepoints afterA.fumigatuschallenge. Thus, these data suggested that C10 has amajor effect on the recruitment or movement of eosinophils andother leukocytes into the airways during experimental ABPA.

Immunoneutralization of C10 attenuates eotaxin and MCP-1levels in BAL and whole lung

While the previous data suggested that C10 directly affected therecruitment of leukocytes to the allergic airway, we next examinedthe possibility that changes in recruitment were partly the conse-quence of C10 modulating the generation of other chemotacticcytokines within the lung. To test this postulate, specific ELISAexamined MCP-1 and eotaxin levels in BAL and whole lung ho-mogenates. These C-C chemokines were also elevated with C10 inthe BAL from A. fumigatus-challenged mice (see Fig. 1), and bothhave been shown to be major participants in other eosinophil-me-diated airway allergic models (36, 40). Immunoneutralization ofC10 in ABPA mice significantly inhibited the levels of eotaxin andMCP-1 present in BAL and whole lung homogenates before and at24 h after intratrachealA. fumigatuschallenge (Fig. 7).

C10 modulates pulmonary levels of IL-13 and IL-10 duringallergic inflammation

Because IL-13 (41) and IL-10 (42) have been ascribed modulatoryroles in the Th2-type cytokine response, changes in both cytokineswere examined in the ABPA model. Interestingly, recent data haverevealed that IL-10 appears to suppress the production of Th1 andTh2 cytokines, and the bronchial inflammation associated with ex-

perimental ABPA (13). The experimental model of ABPA exam-ined in the present study was characterized by a robust increase inIL-13 levels within whole lung homogenates to 75 ng/ml at 24 hand approximately 100 ng/ml at 48 h afterA. fumigatuschallenge.(Fig. 8). Immunoneutralization of C10 significantly reduced IL-13levels at the 48-h timepoint. In contrast, at 24 and 48 h afterAs-pergillus challenge, IL-10 levels were reduced significantly bygreater than 50% in both treatment groups (Fig. 8).

C10 does not affect systemic IgE levels

A hallmark of clinical ABPA is increased systemic IgE levels (43).Similar to the clinical condition, the murine model of ABPA usedin the present study showed similar dramatic changes in serumlevels of IgE (Fig. 9). Although lower levels of IgE were observedbefore and at 48 h afterAspergillusintratracheal challenge, thesechanges did not reach statistical significance. These findings areinteresting when considering previous studies showing that aller-gic responses toA. fumigatusproceed even in the absence ofIgE (12).

DiscussionABPA complicates asthma (4), and results in lung destruction dueto a complex interaction between pulmonary resident cells such asepithelium, alveolar macrophages, mast cells, fibroblasts, andsmooth muscle, and infiltrating leukocytes such as eosinophils andT cells (19). The evolution of this cellular mileau within the al-lergic lung is dependent upon the expression of adhesion mole-cules and numerous soluble mediators, including the recently de-scribed chemotactic cytokines or chemokines (18, 40). The present

FIGURE 6. Histologic appearance of airways inA.fumigatus-sensitized mice before and at 24 and 48 hafter intratrachealA. fumigatusAg challenge. Sensitizedmice received 0.5 ml of anti-C10 antiserum or normalrabbit serum (i.e., control) via an i.p. injection 2 h beforethe Ag challenge. Lung sections were prepared fromparaformaldehyde-fixed lungs removed from eachmouse immediately after i.v. methacholine provocation(seeMaterials and Methods). Representative sectionsfrom anti-C10- and normal serum-pretreated mice areshown as follows:A, normal serum-pretreatedA. fu-migatus-sensitized mouse before intratracheal Ag chal-lenge; B, anti-C10-pretreatedA. fumigatus-sensitizedmouse before intratracheal Ag challenge;C, normal se-rum-pretreatedA. fumigatus-sensitized mouse 24 h afterintratracheal Ag challenge;D, anti-C10-pretreatedA. fu-migatus-sensitized mouse 24 h after intratracheal Agchallenge;E, normal serum-pretreatedA. fumigatus-sen-sitized mouse 48 h after intratracheal Ag challenge;F,anti-C10-pretreatedA. fumigatus-sensitized mouse 48 hafter intratracheal Ag challenge. Original magnificationwas3200.

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study demonstrates that the endogenously generated C10 chemo-kine modulates many of the pulmonary features of experimentalABPA. Among the chemokines analyzed in BAL and whole lunghomogenates, C10 was present at the highest levels during theinflammatory response to an intratrachealA. fumigatuschallenge.Based on the present in vitro observations, this increase may havebeen the consequence of C10 production by alveolar macrophages,vascular smooth muscle, and fibroblasts within the inflamed lung.Compared withA. fumigatus-sensitized and challenged mice thatreceived normal rabbit serum, the neutralization of C10 using ananti-C10 antiserum abrogated the bronchial hyperresponsiveness

and eosinophilia that followed intratrachealA. fumigatusdeliveryin sensitized mice. Diminished C10 levels also significantly atten-uated the presence of lymphocytes in the BAL, and the levels ofMCP-1, eotaxin in BAL, and whole lung homogenates. DecreasedC10 was also associated with significantly lower IL-13 in wholelung homogenates, but lung levels of IL-10 and serum IgE werenot changed. Thus, C10 exerts a prominent role during the allergicairway response toA. fumigatuschallenge.

Sources of C10 include a diverse array of cells from macro-phage (26) to trigeminal ganglia (44). Based on our previous stud-ies of a Th2-type pulmonary granulomatous response in the lung

FIGURE 7. Eotaxin and MCP-1 levels in BAL and whole lung homogenates fromA. fumigatus-sensitized mice before and at 24 and 48 h afterintratrachealA. fumigatusAg challenge. Sensitized mice received 0.5 ml of anti-C10 antiserum or normal rabbit serum (i.e., control) via an i.p. injection2 h before the Ag challenge. Eotaxin and MCP-1 were analyzed by specific ELISA. Data are mean6 SE from a minimum of five mice per timepoint inboth treatment groups.p, p # 0.05 compared with the corresponding control mice that received normal rabbit serum beforeA. fumigatusAg challenge.

FIGURE 8. IL-10 and IL-13 levels in whole lung homogenates fromA. fumigatus-sensitized mice before and at 24 and 48 h after intratrachealA.fumigatusAg challenge. Sensitized mice received 0.5 ml of anti-C10 antiserum or normal rabbit serum (i.e., control) via an i.p. injection 2 h before theAg challenge. IL-10 and IL-13 were determined using specific ELISA. Data are mean6 SE from a minimum of five mice per timepoint in both treatmentgroups.p, p # 0.05 compared with the corresponding control mice that received normal rabbit serum beforeA. fumigatusAg challenge.

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(38), we postulated that a number of cell types in the lung mightalso be capable of generating C10. Accordingly, alveolar macro-phages, VSMCs, and fibroblasts from lung were constitutivegenerators of C10, and all of these cells augmented C10 productionfollowing exposure to cytokines. Coinciding with a previous report(26), IL-4 was a potent stimulator of C10 production by alveolarmacrophages. However, in cultures of pulmonary fibroblasts andVSMCs, IL-1b was the most potent stimulant of C10 production.This finding is notable because of prior observations showing thatIL-1 is rapidly induced following intrapulmonaryA. fumigatuschallenge in mice (45). Thus, pulmonary resident cells followingexposure to inflammatory or immune cytokines are capable of aug-menting C10 synthesis, and this response possibly explains themajor changes in C10 observed duringA. fumigatus-induced air-way inflammation.

The enhanced generation of C10 followingA. fumigatuschal-lenge appears to provide an important signal for the recruitment ofinflammatory leukocytes into the lungs. The infiltration of eosin-ophils and T cells into the lungs is a major feature of allergicairway inflammation, but the precise role of these leukocytes inallergic disease is still being explored. Although the profound sup-pression of eosinophilia using anti-IL-5 (10), anti-MIP-1a, or anti-RANTES Abs (36) did not reduce the airway hyperresponsivenessin allergic mice, eosinophils are a significant source of cationic andmajor basic proteins that are toxic to the airway epithelium (19),and these cells are involved in tissue remodeling during chronicpulmonary inflammation (46). As mentioned above, CD41 T cells,particularly those that express Th2 cytokines such as IL-4 andIL-5, are abundant and appear to contribute to the airway inflam-mation and hyperresponsiveness associated with many experimen-tal allergic responses in the lungs, including ABPA (7, 47). Indeed,clear evidence shows that the attenuation of T cell recruitment tothe allergic lung through anti-MCP-1 (36, 40) or anti-MCP-3 (48)Ab treatments results in a pronounced decrease in airway inflam-mation and hyperresponsiveness. Based on previous studies dem-onstrating that C10 is a chemoattractant for CD41 T cells (26) andour present data showing that C10 is chemotactic for eosinophils,the immunoneutralization of C10 during experimental ABPA pre-sumably attenuates the movement of both cell types into the al-lergen-challenged lungs. In addition, the absence of recruited leu-kocytes in the BAL and airways of anti-C10 Ab-treated mice mayalso explain, in part, the decreased C10 levels measured in thesemice. Thus, the immunoneutralization of C10 during the allergicresponse toA. fumigatusabolishes the recruitment of key immuneeffector cells, and stems the augmentation of C10 synthesis withinthe lung.

Other observations in the ABPA model during the suppressionof endogenous C10 activity indicated that C10 possibly modulatesthe levels of other mediators previously shown to be present duringor to contribute to the development of allergic responses. MCP-1and eotaxin are two examples of a proallergic mediator (36, 40,49). Although the diminution of MCP-1 and eotaxin levels ob-served in the present study may reflect the reduction in recruitedleukocytes in the lungs, pulmonary resident cells are also excellentsources of both chemokines (32, 50). Furthermore, the lack ofendogenous C10 may have directly affected the ability of thesecells to generate MCP-1 and eotaxin. IL-13 is a product of T cellsand alveolar macrophages (51) that induces Ig production, B cellproliferation, and monocyte differentiation. In addition, IL-13 andIL-4 share the same cell activation pathway (52), which may ex-plain why allergic responses toA. fumigatusdevelop normally inmice genetically deficient in IL-4 (8). Consistent with the decreasein lymphocyte recruitment in anti-C10 antiserum-treated mice, lessIL-13 was also detected in whole lung homogenates from thesemice. IL-10 appears to exert a prominent role during pulmonaryresponses toA. fumigatusthat relates to the ability of this cytokineto balance the Th1 and Th2 cytokine responses in this model (13).The presence of IL-10 also suppresses the oxidative burst re-sponse, but enhances the phagocytic activity of mononuclear cellsexposed toAspergillus(53). In the present study, IL-10 levels inwhole lung homogenates were significantly attenuated as the al-lergic response progressed, but additional changes in IL-10 werenot observed in the ABPA model when C10 was inhibited. Therelative impact of reduced pulmonary IL-10 during the progressionof the allergic response toA. fumigatusis not presently apparent,and warrants further study. In addition, the mechanism throughwhich C10 modulates the synthesis of MCP-1, eotaxin, and IL-13within the allergic lungs deserves further attention.

In patients with clinical ABPA, elevations of IgE correlate bothwith allergic inflammation of the airways and with bronchial hy-perresponsiveness (2). Whereas a central role for IgE in the patho-genesis of the eosinophilic inflammation as well as in the obstruc-tive airway physiology associated with ABPA has been suggested,experimental ABPA proceeds normally in IgE-deficient mice (12).The present data demonstrate that although C10 effectively abol-ishes many of the features of experimental ABPA, this chemokinedoes not appear to affect serum levels of IgE. Therefore, thesefindings suggest that C10 may not participate in the sensitizationprocess toA. fumigatusAgs.

Chemokines have been shown to exert defined effects on leu-kocyte recruitment to the allergic airways and the development ofairway hyperresponsiveness in experimental models (36, 40, 54).The role of chemokines in the pathogenesis of ABPA is less welldefined, but the results from the present study show that the C10chemokine exerts a prominent role in the development of lunginflammation and bronchial hyperresponsiveness. Further exami-nation of the role of these types of chemokines in clinical andexperimental ABPA may be warranted.

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FIGURE 9. Serum IgE levels inA. fumigatus-sensitized mice beforeand at 24 and 48 h after intratrachealA. fumigatusAg challenge. Sensitizedmice received 0.5 ml of anti-C10 antiserum or normal rabbit serum (i.e.,control) via an i.p. injection 2 h before the Ag challenge. Sera from bothtreatment groups were analyzed by specific ELISA for total IgE. Data aremean6 SE from a minimum of five mice per timepoint in both treatmentgroups.

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