dynamics of lung macrophage activation

Dynamics of lung macrophage activation in response tohelminth infection

Mark C. Siracusa,* Joshua J. Reece,* Joseph F. Urban Jr.,† and Alan L. Scott*,1

*The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of PublicHealth, Johns Hopkins University, Baltimore, Maryland, USA; and †Beltsville Human Nutrition Research Center,Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland, USA

Abstract: Most of our understanding of the devel-opment and phenotype of alternatively activatedmacrophages (AAMs) has been obtained from stud-ies investigating the response of bone marrow- andperitoneal-derived cells to IL-4 or IL-13 stimula-tion. Comparatively little is known about the devel-opment of AAMs in the lungs, and how the complexsignals associated with pulmonary inflammation in-fluence the AAM phenotype. Here, we use Nippo-strongylus brasiliensis to initiate AAM developmentand define the dynamics of surface molecules, geneexpression, and cell function of macrophages iso-lated from lung tissue at different times postinfec-tion (PI). Initially, lung macrophages take on afoamy phenotype, up-regulate MHC and costimu-latory molecules, express reduced levels of TNFand IL-12, and undergo proliferation. Cells iso-lated between days 8 and 15 PI adopt a dense,granular phenotype and exhibit reduced levels ofcostimulatory molecules and elevated levels of pro-grammed death ligand-1 (PDL-1) and PDL-2 andan increase in IL-10 expression. Functionally,AAMs isolated on days 13–15 PI demonstrate anenhanced capacity to take up and sequester anti-gen. However, these same cells did not mediateantigen-specific T cell proliferation and dampenedthe proliferation of CD3/CD28-activated CD4� Tcells. These data indicate that the alternative acti-vation of macrophages in the lungs, although initi-ated by IL-4/IL-13, is a dynamic process that islikely to be influenced by other immune and non-immune factors in the pulmonary environment. J.Leukoc. Biol. 84: 1422–1433; 2008.

Key Words: nematode � hygiene hypothesis � pulmonary � alterna-tively activated � arginase � YM1 � FIZZ1

INTRODUCTION

The mucosal immune environments have developed thecapacity to discriminate between potential pathogens andthe vast array of harmless antigens to which they are con-stantly exposed [1]. The ability to distinguish between dan-gerous and nondangerous challenges is carried out, in large

part, by specialized subsets of dendritic cells (DC) andmacrophages [2, 3]. Although many studies have focused ondefining the specialized activation states of DC within mu-cosal surfaces, much less attention has been paid to themore abundant macrophage populations [2, 4].

In contrast to IFN-�-mediated, classical activation ofmacrophages, alternative activation is induced by IL-4and/or IL-13 signaling through STAT6 [5, 6]. Alternativelyactivated macrophages (AAMs) are typically characterizedas anti-inflammatory in nature and are thought to provideprotective qualities by contributing to repair and remodelingof tissues [5, 7]. The majority of the studies characterizingthe molecular and functional phenotype of AAMs has usedperitoneal- or bone marrow-derived cells exposed to IL-4and/or IL-13 in vitro [8, 9]. Although informative and im-portant for establishing the basic tenets of alternative acti-vation, these studies have not accounted for the additionalsignals that macrophages receive in an inflammatory envi-ronment, which are likely to modulate the IL-4/IL-13-in-duced phenotype. In addition, for those studies that haveused cells alternatively activated in situ during stronglypolarized, Th2-immune responses, the majority has inferredactivation within tissues by measuring the increased tran-scription of genes encoding the AAM signature moleculesarginase I (arg1), resistin-like � (fizz1), and the chitinase-like molecules YM1 and AMCase [10, 11]. Thus, our un-derstanding of AAMs is likely to be somewhat limited by theexperimental approaches used to study them.

In this study, we define the dynamics of the alternateactivation of macrophages within the lungs of mice as aresult of an infection with the rodent hookworm Nippo-strongylus brasiliensis (Nb). We describe the morphological,surface-associated, transcriptional, and phenotypic changesthat take place as a result of a dynamic transition of mac-rophages into the alternatively activated phenotype. Thesestudies offer the first insight into the macrophage popula-tions during alternative activation in the context of a com-plex Th2, inflammatory environment.

1 Correspondence: Department of Molecular Microbiology and Immunology,Bloomberg School of Public Health, Johns Hopkins University, 615 NorthWolfe Street, Baltimore, MD 21205, USA. E-mail: [email protected]

Received March 24, 2008; revised July 9, 2008; accepted July 28, 2008.doi: 10.1189/jlb.0308199

1422 Journal of Leukocyte Biology Volume 84, December 2008 0741-5400/08/0084-1422 © Society for Leukocyte Biology

MATERIALS AND METHODS

Animals and infection

Six- to 8-week-old male wild-type (WT) and OVA-specific DO.11.10 T cell-transgenic mice, all on a BALB/c background, were obtained from JacksonLaboratories (Bar Harbor, ME, USA). Mice were housed in barrier filter-topcages, maintained on a 12-h light/dark cycle, and provided food and water adlibitum. All animal procedures described in this study were performed underthe approval of the Johns Hopkins Animal Care and Use Committee (Balti-more, MD, USA), in accord with the guidelines set by the National ResearchCouncil’s Guide for the Care and Use of Laboratory Animals. Infectious,third-stage larvae of Nb were harvested from fecal culture via a Baermannapparatus [12]. The larvae were washed in PBS and counted, and 500 parasiteswere introduced to a s.c. site in the ruff of the neck.

Antibodies

The following anti-mouse antibodies were purchased from eBioscience (SanDiego, CA, USA): CD80-PE (12-0801-81), MHCI-PE (12-5998-81), MH-CII-PE (12-5321-81), CD40-PE (12-0401-81), CD86-PE (12-0861-81), pro-grammed death ligand-1 (PDL-1)-PE (12-5982-81), PDL-2-PE (12-5986-81),Thy1.2-allophycocyanin (APC; 17-0902-83), Ter-119-APC (17-0902-83),CD3-APC (17-0031-82), CD28 (16-0281-85), and CD3ε (16-0031-81). PE-conjugated mouse IgG1� (555-749), Imag APC particles DM (E30-221), andFITC-conjugated BrdU antibody set (556028) were obtained from BD Phar-Mingen (San Jose, CA, USA). Anti-mouse CD11c-APC (120-002-001), B220-APC (130-091-843), and GR1-APC (130-091-931) were obtained from Mil-tenyi Biotec (Auburn, CA, USA). Anti-F4/80 (ab6440) was obtained fromAbcam (Cambridge, MA, USA), and Alexa Fluor 488 goat anti-rat IgG(A11006) was obtained from Invitrogen (Carlsbad, CA, USA).

Bronchial alveolar lavage (BAL)

Surgeries were performed as described previously [13]. In short, animals wereanesthetized with 300 mg/kg body weight with Avertin (2,2,2-tribromoethanol,Acros Organics, Morris Plains, NJ, USA), administered i.p. Pericardium andtrachea were exposed by dissection. A lateral incision was made through thetrachea, and a 19-gauge, flat-tipped needle was inserted and tied off. BAL wasperformed by flushing the lungs (3�) with 1 ml sterile PBS at 24°C. BAL fluidwas pooled and spun at 1500 rpm for 3 min at 4°C. Cells were incubated in 0.5ml RBC lysing buffer (Quality Biological, Gaithersburg, MD, USA) at 24°C for2 min and washed twice with PBS at 4°C. Cytology slides were processed usinga Shandon Cytospin 3 (Shandon, Pittsburgh, PA, USA), and slides were stainedwith Hemacolor (Harleco, Gibbstown, NJ, USA).

Lung monocyte preparations

Lungs were perfused with 10 ml sterile PBS (24°C) prior to removal byinserting a 25-guage needle into the right ventricle of the heart. Lungs wereremoved carefully to assure the exclusion of contaminating tissue. Lung tissuewas suspended in 5 ml RPMI 1640 containing 1 mg/ml collagenase type II(1701-015, Gibco, Carlsbad, CA, USA) and 30 �g/ml DNase I (10104159001,Roche, Indianapolis, IN, USA), minced thoroughly, and incubated at 37°C for30 min. After dispersing the remaining tissue fragments with repeated pipet-ting, 3 ml fresh collagense/DNase solution was added, and the samples wereincubated for 15 min at 37°C. Suspensions were then passed through a100-�m mesh nylon cell strainer (352360, BD Falcon, Bedford, MA, USA),and the resulting cells were pelleted at 1500 g (4°C). Pellets were resuspendedin 5 ml of ACK (150 mm NH4Cl, 10 mm KHCO3, 0.1 mm EDTA) lysing bufferfor 2 min at room temperature, passed through a fresh strainer, and washedtwice in FACS-staining buffer (PBS, 2% heat-inactivated FCS). Pellets wereresuspended in 200 �l staining buffer containing Fc Block (553142, BDPharMingen) and incubated on ice for 5 min. Anti-CD11c was then added, andcells were incubated on ice for 30 min. Cells were washed twice in stainingbuffer, and resulting pellets were resuspended in Imag APC particles DM andincubated on ice for 30 min. Isolation of CD11c� cells was performed accord-ing to the manufacturer’s protocol using the BD Imagnet System (BD PharM-ingen). Cell counts were performed on a standard hemacytometer with deadcells excluded by trypan blue stain (15250-061, Gibco).

Flow cytometry

Cells were incubated with the appropriate fluorochrome-conjugated antibodiesin FACS-staining buffer for 20 min on ice in the dark. Data were acquired byrunning samples on a FACSCalibur flow cytometer using CellQuest software(BD Biosciences, Mountain View, CA, USA). Data were analyzed using FloJosoftware (Tree Star, Inc., Ashland, OR, USA).

Histology

A 19-gauge, flat-tipped needle was inserted into the trachea, and the lungswere inflated slowly with zinc-buffered formalin fixative (Z-fix, Anatech Ltd.,Battle Creek, MI, USA). The lungs were removed and incubated in a 40-foldvolume of Z-fix. Inflated and fixed lungs were embedded in paraffin, andsagittal, 10 �m sections were obtained from four different levels of lung. Lungsections and cytology slides were examined using a Nikon Eclipse E800 lightmicroscope (Nikon Inc., Melville, NY, USA), and images were acquired usingSPOT RT charge-coupled device imager and software (Diagnostic Instruments,Inc., Sterling Heights, MI, USA).

Electron microscopy

Lung-derived cells were fixed with 2% glutaraldehyde for 2 h at 24°C, followedby postfixation with 2% osmonium tetroxide for 2 h at 24°C. The cells wereembedded in Epon 812, sectioned, poststained with 2% uranyl acetate andlead citrate, and viewed on a Philips CM 120 electron microscope at 100 kV.

Immunohistochemistry

For epitope retrieval, slides were incubated in 20 �g/ml proteinase K solutionfor 15 min at 37°C. Sections were allowed to cool to room temperature for 10min and washed (2�) for 2 min with PBS. Surfactant protein A (SP-A) stainingwas performed using (1:4000) polyclonal rabbit anti-SP-A (AB3420, ChemiconInternational, Temecula, CA, USA), which was detected using a biotinylatedgoat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA), peroxidasestrepavidin, and 3,3� diaminobenzidine (Vector Laboratories). For dual fluo-rescence staining of macrophages, SP-A was resolved with Fluorescein/Avidincell sorting grade Avidin D (DCS; Vector Laboratories), and after an avidin/biotin-blocking step (Vector Laboratories), biotin-conjugated CD11c (13-0114-85, eBioscience) binding was detected with Texas Red/Avidin DCS.Stained sections were mounted in Vectashield containing 4�-6-diamidino-2-phenylindole (DAPI; Vector Laboratories).

Gene expression analysis

CD11c-positive cells were harvested, flash-frozen in liquid nitrogen, and storedat –80°C. RNA was isolated as outlined by Reece et al. [13]. RNA quality wasdetermined by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100(Agilent Technologies, Palo Alto, CA, USA). An RNeasy total RNA cleanupprotocol (Qiagen, Valencia, CA, USA) was performed, followed by spectropho-tometric assessment of RNA concentration. CD11c� cells from five animalswere processed independently for each day of infection.

Processing of templates and the hybridization protocol for the 430 2.0 ArrayGeneChip (Affymetrix Inc., Santa Clara, CA, USA) were in accordance withmethods described in the Affymetrix GeneChip Expression Analysis TechnicalManual, Revision Three [14]. The hybridization results were assessed using theGCS3000 laser scanner (Affymetrix Inc.) at an emission wavelength of 570 nmat 2.5 �m resolution. Intensity of hybridization for each probe pair wascomputed by GCOS 1.2 software (Affymetrix Inc.). For more detailed methods,please refer to the website of the Malaria Research Institute, Gene Array CoreFacility, at the Johns Hopkins Bloomberg School of Public Health (http://jhmmi.jhsph.edu). Affymetrix CEL file data were preprocessed for use with theprobe level GeneChip Robust Multi-Array analysis (GC-RMA) [15] option inGeneSpring 7 software (Agilent Technologies). The data discussed in thispublication have been deposited in the National Center for BiotechnologyInformation Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo).

Real-time RT-PCR

Lung RNA (1 �g) was reverse-transcribed using the SuperScript first-strandsynthesis system for RT-PCR (Invitrogen) using an oligo(dT) primer. Quanti-tative real-time RT-PCR was performed using Applied Biosystems 7500 real-

Siracusa et al. Dynamics of AAM activation 1423

time PCR system, TaqMan� Gene Expression Assays-On-Demand, and Taq-Man Universal Master Mix (Applied Biosystems, Foster City, CA, USA). Thefollowing assays (Applied Biosystems) were used: Arg1 (Mm00475988), Ym1(Mm00657889), Fizz1 (Mm00445109), IL-12p40 (Mm00434165_m1), TNF-�(Mm00443558_m1), IL-6 (Mm00446190_m1), IL-1� (Mm00434228_m1), IL-10(Mm00438616_m1), TGF-ß (Mm00441724_m1), IFN-� (Mm00833861_s1), andIFN-� (Mm00439546__s1). Reactions were performed using 1 �l cDNA in a25-�l sample vol and the following thermal cycler profile: 10 min denaturationat 95°C, 50 cycles of 15 s denaturation at 95°C, followed by 1 min extensionat 60°C. Analysis was performed using the 7500 system SDS software package(Applied Biosystems).

BrdU staining

Animals were given BrdU (B5002, Sigma Chemical Co., St. Louis, MO, USA)via drinking water (0.8 mg/ml) from day 0 of infection until they weresacrificed. CD11c� mononuclear cells were isolated from WT and Nb-infectedmouse lungs as described above. Isolated cells were resuspended in 500 �l0.15 NaCl, fixed with 1.2 ml 95% ethanol, resuspended in 1 ml 1% parafor-maldehyde and 0.01% Tween-20 solution, and kept overnight at 4°C. Isolatedcells were then resuspended in a DNase digestion solution for 30 min at 35°Cand subsequently stained with 20 �l anti-BrdU-FITC or an isotype controlantibody. BrdU incorporation was determined by flow cytometry.

T cell proliferation

CD11c� mononuclear cells were isolated from WT and Nb-infected mouselungs as described above. CD4� T cells were isolated using a CD4 T cellisolation kit (Miltenyi Biotec), according to the manufacturer’s protocol.Briefly, a single-cell suspension of splenocytes was incubated in ACK lysingbuffer, washed, and stained with a biotin-labeled, anti-CD4 T cell antibodycocktail. Cells were then washed and incubated with the recommended con-centration of antibiotin microbeads. Stained and bead-labeled splenocyteswere then washed and passed through an LS column (130-042-401). IsolatedCD4 T cells were stained with Cell Trace CFSE (Molecular Probes, Eugene,OR, USA) by placing 2.0 � 107 isolated T cells in 2 ml PBS containing 3 �gCFSE for 6 min at room temperature, and unbound CFSE was quenched bywashing cells in supplemented DMEM. CFSE-labeled CD4 T cells (1�105)were incubated with CD11c-positive lung cells (1�105) in a 96-well flat-bottom plate. T cells were stimulated with 20 ng/ml recombinant (r)IL-2(34-8021-82, eBioscience), 5 �g/ml plate-bound anti-CD3ε, and 5 �g/mlsoluble anti-CD28. Cells were incubated for 4 days at 37°C. CFSE proliferationprofiles were analyzed by flow cytometry after staining with CD4-APC and7-amino-actinomycin (7-AAD).

Antigen uptake

Antigen uptake was evaluated using the Vybrant phagocytosis assay (Molec-ular Probes). CD11c� lung macrophages were isolated from the lungs of miceon days 0, 4, and 13–15 postinfection (PI). Briefly, 1 � 106 lung macrophages/well were plated and allowed to adhere to 96-well flat-bottom plates. Fluores-cent Bioparticles were added to experimental wells and allowed to incubate for2 h at 37°C. Trypan blue was added to quench Bioparticles that had not beentaken up by macrophages. Plates were read at 520 nm emission and 480 nmexcitation on a HTS 7000 Plus Bio Assay Reader with HT Soft 2.0 software(Perkin Elmer, Waltham, MA, USA).

Antigen-specific proliferation

On days 0 and 13–15 PI, lung macrophages were isolated as described above.DC were also isolated from the spleens of normal BALB/c animals as describedpreviously [16]. Briefly, spleens were minced into fine pieces and digested incollagenase. Liberated cells were spun through FCS supplemented with EDTA.Spleen cells were then separated over a Nicodenz gradient (density, 1.075g/ml), and the DC-rich, low-density fraction was kept. Contaminating cellswere removed by staining with APC-conjugated anti-CD3, anti-Thy1.2, anti-GR1, anti-B220, and anti-Ter119 and then incubated with Imag APC particlesDM. Depletion of APC-stained cells was performed, according to the manu-facturer’s protocol, using the BD Imagnet System. Isolated macrophages andDC were pulsed with 10 �g/ml OVA peptide 323–339 (27024, AnaSpec, SanJose, CA, USA) for 60 min at 37°C. Pulsed cells were washed (3�) insupplemented DMEM. OVA-specific CD4 T cells were isolated from the

spleens of DO11.10-transgenic mice and loaded with CFSE as describedabove. Pulsed macrophages and DC (1�105) were incubated with CD4 T cells(1�105) in the presence of 20 ng/ml rIL-2 in a 96-well v-bottom plate at 37°Cfor 4 days. CFSE proliferation profiles were analyzed by flow cytometry afterstaining with CD4-APC and 7-AAD.

RESULTS

BAL-derived lung macrophages

To gain an initial insight into the cellular changes induced bythe passage of Nb larvae through the lungs, the cellular com-ponent of the BAL was analyzed during the lung (days 2 and 3),intestinal (days 4 and 8), and postexpulsion (days 13 and 15)phases of infection. As described previously [17, 18], macro-phages were the dominant cell type found in the BAL fluidduring the first 9–12 days PI, after which, eosinophils becomemore numerous (data not shown). There was a demonstrableincrease in the number of BAL macrophages at day 2 PI, theday larvae arrive in the lungs, but the increase did not reachstatistical significance until day 4 PI (P0.05; Fig. 1A). BALmacrophage numbers returned to near-preinfection levels bydays 8 and 13 PI. Consistent with previous reports [19, 20],flow cytometric analysis of the BAL-derived mononuclear cellsindicated that a vast majority of the cells was CD11c�,MHCII�, F4/80� macrophages (data not shown).

Accompanying the changes in numbers, a significant pro-portion of the BAL-derived macrophages adopted distinct mor-phological changes. As early as day 2 PI, 50% of the mac-rophages in the BAL exhibited a highly vacuolated, foamyphenotype (Fig. 1, B and F) that was not observed in theuninfected controls (Fig. 1, B and E). The proportion of exten-sively vacuolated macrophages decreased to 20% at day 3(data not shown), and by days 4–8 PI, only 10% exhibited aless-pronounced, vacuolated appearance. On day 13 PI, 2–3days after the adult parasites had been expelled from theintestine, although the number of macrophages in the BAL hadreturned to near preinfection levels, 50% of the BAL mac-rophages were enlarged cells containing dense granular mate-rial in the cytoplasm (Fig. 1, B and G). In contrast to thetransient nature of the foamy phenotype, the granular pheno-type persisted with 50% of the macrophage population ex-hibiting the granular phenotype through day 100 PI, 89 daysafter the animals had cleared the infection and 97 days afterthe larvae were resident in the lung (Fig. 1B). To rule out thepossibility that an unapparent infection was contributing to thechanges in macrophage phenotype, uninfected littermateshoused in the same facilities were screened for a panel ofpathogens by serology and monitored by histology over time.The serology was negative, and there were no changes inmacrophage phenotype in any of the control animals (data notshown).

Electron microscopic analysis of the foamy macrophagesisolated from day 2 PI showed the cytoplasm to be dominatedby numerous large vacuoles that contained material of varyingdensities (Fig. 1H). This ultrastructural appearance was similarto the macrophages identified in the lungs as a result of severalinfectious agents including HIV and tuberculosis [21, 22].Ultrastructural analysis of days 13–15 PI granular macro-

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phages (Fig. 1I) revealed the presence of several forms of densegranules. Some of the granules were composed of large multi-lamellar structures (large arrowhead) that resemble surfactantprotein (SP), reported to accumulate in macrophages duringacute lung injury [23] and in disease states such as pulmonaryalveolar proteinosis [24], and others that contained membrane-like structures but were more dense and did not resembletypical SP accumulation (small arrowheads). Immunohisto-chemical analysis of day 13 PI macrophages showed that thecontents of some of the granules were comprised of SP-A (Fig.1L) and SP-D (data not shown).

Macrophages in the parenchyma of the lung

To determine the extent to which the profiles derived from theBAL reflected the number and phenotype of macrophages inthe parenchyma of the lung, histological sections were pre-pared from lungs isolated on days 2 and 13 PI. Foamy (Fig. 1J)and granular macrophages (Fig. 1K) were readily identified inhistological sections on days 2 and 13, respectively. Foamy and

granular cells were found to be distributed uniformly through-out the parenchyma of the lung (arrowheads). A systematicexamination of the histological sections strongly suggested thatthe extent and duration of the changes in the lung macrophagepopulation were underestimated significantly by BAL sam-pling. For example, on day 13 PI, when BAL results suggestedthat the response was waning and the macrophage numberswere near-preinfection levels (Fig. 1A), the histological anal-ysis indicated that macrophage levels were actually elevated(data not shown). Thus, to obtain a more accurate assessment ofthe cellular dynamics, CD11c� cells were isolated from wholelung at 0, 2, 4, 8, and 13 days PI.

Taking advantage of the elevated levels of CD11c present onlung macrophages [13, 20, 25, 26], a protocol using CD11cmagnetic beads was developed to isolate cells highly enrichedfor macrophages from lung digests. On day 2 PI, the number ofmacrophages in the lungs was not significantly different frompreinfection levels (Fig. 1C), but the composition of cells at day2 PI differed from that seen in the BAL in that only 20%

Fig. 1. Nb infection results in an increase in the number of lung macrophages and induces a change in their morphology. (A) BAL-derived macrophages werecounted on days 0, 2, 4, 8, and 13 post-Nb infection. (B) Cytospin preparations of BAL-derived cells were evaluated for the percentage of vacuolated/foamy (openbars) or granular (closed bars) macrophages on days 0, 2, 4, 8, 13, and 100 post-Nb infection. (C) Changes in the number of CD11c� macrophages isolated fromwhole lungs using CD11c-magnetic beads on days 0, 2, 4, 8, and 13 post-Nb infection. (D) Evaluation of the percentage of vacuolated/foamy (open bars) or granular(closed bars) macrophages in the CD11c� cells isolated from whole lungs on days 0, 2, 4, 8, and 13 after Nb infection. (E–G) Representative light microscopyimages of the morphologies of BAL-derived macrophages from days (E) 0, (F) 2 (vacuolated/foamy), and (G) 13 (granular) PI lungs. (H and I) Transmission electronmicroscopy of BAL-derived macrophages collected on (H) day 2 or (I) day 13 PI. Multilaminate large granules (large arrowheads) and smaller granules (smallarrowheads) are indicated. (J and K) Representative histological sections from days 2 and 13 PI, illustrating the distribution of (J) vacuolated/foamy and (K) granularmacrophages, respectively, in the parenchyma of the lung, and lung macrophages are indicate by arrowheads (original, 40�) and in the magnified insets. (L)Immunofluorescence staining of lung macrophages for CD11c (red) and SP-A (green). Histological sections of lungs isolated at days 0 and 13 PI wereimmunostained for CD11c and SP-A and counterstained with DAPI (blue; original, 40�). *, P 0.05; **, P 0.01.


exhibited the foamy phenotype (vs. 50% in BAL; Fig. 1D).On day 4 PI, the number of lung macrophages increasedsignificantly (P0.01) to 3 � 106, and 20% of the cellsexhibited a foamy phenotype. There was a decrease in thenumber of macrophages on day 8 PI that coincided with anunambiguous transition from the foamy to the granular pheno-type. The level of granular cells was maintained at between15% and 20% of the total macrophage population through day13 PI. In contrast to the BAL results, where peak macrophagenumbers were seen at day 4 PI (Fig. 1A), the highest numberof macrophages was isolated from day 13 PI lungs at 6 �106. It was concluded that the BAL-based analysis faithfullyrepresented neither the quantitative nor the qualitativechanges taking place within the parenchyma of the lungpost-Nb infection. Consequently, subsequent studies to definesurface phenotypes and expression profiles were carried outusing cells isolated from whole lungs.

AAM surface phenotype

A surface expression profile of the CD11c� macrophages iso-lated from uninfected lungs revealed constitutive production ofF4/80, MHCI, MHCII, PDL-1, CD80, ICAM-1 and CD1d butno significant expression of PDL-2, CD40 or CD86 (Fig. 2).Although the CDllc� cells from uninfected lungs could bedivided into populations expressing low and high levels ofMHCII, at days 2, 4, 8, and 13 PI, a vast majority of the cellswas in the MHCIIhi population. Surface expression of MHCI,ICAM-1, and CD1d was elevated during infection and re-mained elevated after the worms were expelled from the intes-tine. The costimulatory molecules CD80, CD86, and CD40 aswell the classic macrophage marker F4/80 showed a markedincrease in expression on day 4 PI, followed by a reduction tonear-preinfection levels on days 8 and 13 PI. In contrast, thelevels of PDL-1 and PDL-2 were low or remained near baselineduring the first few days PI but showed elevated expression ondays 8 and 13 PI.

Dynamics of AAM gene expression

Expression analysis was performed on CD11c� macrophagesisolated from the lungs on days 0, 2, 4, and 8 PI. Althoughmultiple attempts were undertaken to obtain RNA from mac-rophages isolated on day 13 PI lungs, we were unable to isolateRNA of sufficient quality to use in expression analysis. In total,the transcription of 2319 distinct genes was up-regulated sig-nificantly in the CD11c� lung macrophages over the threetime-points tested, with a peak of �1600 genes on day 4 PI(Fig. 3A). Between 31% and 48% of the up-regulated geneswere expressed uniquely at each time PI. Overall, 40% of theup-regulated genes were expressed at two or more time-points, and 277 were common to all three time-points. Atotal of 1383 genes was found to be down-regulated at leasttwofold during days 2, 4, and 8 PI (Fig. 3B) compared withmacrophages isolated from uninfected lungs, with 61 com-mon to all 3 days PI.

Consistent with the results from a previous study [13],CD11c� macrophages from the post-Nb lungs robustly up-regulated genes encoding proteins associated with alternativeactivation including ARG1, FIZZ1, YM1, YM2 (ym1, ym2),

and AMCase (chia), triggering receptor expressed on myeloidcells 2 (trem2b), and the scavenger receptors Marco (marco)and Msr1 (msr1; Fig. 3C) [13, 27–30]. Interestingly, argII,which has been shown to negatively regulate proinflammatoryqualities in activated macrophages by limiting NO production[31, 32], was also strongly up-regulated on days 4 and 8 PI.

There was also a rapid and sustained induction in genesencoding surface lectins, scavenger receptors, enzymes, andother proteins involved in lipid metabolism. Included among

Fig. 2. Surface phenotype of lung macrophages. CD11c� macrophages wereisolated from whole lung tissue on days 0, 2, 4, 8, and 13 post-Nb infection andexamined for changes in the levels of key surface-associated molecules by flowcytometry as outlined in Materials and Methods. A representative isotypecontrol is illustrated in white, day 0 surface levels are represented in gray, andthe PI surface levels are represented in black, according to the day noted. Theprofiles are representative of five biological replicates.


Fig. 3. Gene expression analysis of Nb-induced lungmacrophages. CD11c� macrophages were isolated fromwhole lung tissue on days 0, 2, 4, and 8 PI and processedfor Affymetrix GeneChip analysis as outlined in Materialsand Methods. The hybridization results were processedusing GC-RMA, and a minimum probe fluorescence in-tensity threshold was set at 150 for the significantlyregulated genes. (A and B) Ven diagrams of up-regulatedand down-regulated genes on days 2, 4, and 8 PI. (C)Fold-changes (FC) in the expression of selected genes byCD11c� lung macrophages isolated at days 2, 4, and 8 PIcompared with cells isolated from the lungs of uninfectedanimals; n � 3 biological replicates per time-point. FC of2, X; FC of 2–3, �; FC of 3–5, ��; FC of 5–10,��; FC of 10–20, ��; FC of �20, ��.


these were the class D scavenger receptor CD68 and a receptorfor oxidized low-density lipoproteins (oldr1), which play keyroles in the uptake of lipids and apoptotic cells from theenvironment [33–35], leukotriene B4 12-hydroxydehydroge-nase (ltb4dh) and acyl-CoA oxidase 1 (acox1), enzymes knownto be involved in the catabolism of lipids. This pattern of geneexpression is consistent with the development of the foamymacrophage phenotype typically associated the incorporationof lipids during conditions such as atherosclerosis and pulmo-nary alveolar proteinosis [36, 37]. On day 8 PI, macrophagesdramatically up-regulated genes encoding the complementcomponent C1q, Oldr1, and 15-Lipoxygenase (alox15), all ofwhich enhance the uptake of apoptotic bodies along with SP-Aand SP-D in the airways [33, 34, 38]. Also of interest was thesignificantly enhanced transcription of genes encoding CCL4,CCL8, CCL9, CCL11, CCL12, CCL17, and CCL24.

The increase in the CD11c� population in the post-Nb lung(Fig. 1, A and H) could be a result of proliferation of theresident lung macrophages, cell recruitment, or a combinationof proliferation and recruitment. Although the source of theincrease in lung macrophages remains to be defined, the cellsisolated from days 2 and 4 PI showed significant increases ingenes encoding proteins that regulate the cell cycle, suggestingthat early after infection, proliferation contributes, at least inpart, to the increase in macrophage numbers. The capacity ofthe lung macrophages to proliferate was confirmed by demon-strating that the CD11c� cells isolated from lungs on days 2and 4 PI incorporated more BrdU than the cells isolated fromuninfected lungs (Fig. 4). At day 2 PI, over 6% of the CD11c-positive cells had incorporated BrdU, and that number in-creased to over 9% by day 4 PI.

Among the significantly down-regulated genes were multiplegenes known to be involved in the classical activation ofmacrophages, including IFN-�R2 (ifngr2), the immunity-re-lated GTPase (Irgm1), inducible NO synthase (inos), Kruppel-like factor 4 (klf4), and heat shock protein 1b (hspa1b) [39–41].

Changes in cytokine expression

Real-time RT-PCR was performed on CD11c� lung macro-phages isolated from days 0, 2, 4, and 8 PI to validate thechip-based expression levels of the macrophage-associatedcytokine genes IL-12, TNF-�, IL-6, IL-1�, IL-10, TGF-�,IFN-�, and IFN-� (Fig. 5). There were modest but statisticallysignificant decreases in the expression of TNF-� at days 2, 4,and 8 PI and of IL-12, IL-6, and TGF-� at day 4 PI. IL-1� andIL-10 transcription levels increased as the Nb infection pro-

gressed. There was no detectable transcription of IFN-� on day2 PI.

Antigen uptake and T cell proliferation

The Nb-induced changes in the morphology, surface pheno-type, and gene expression profile of lung macrophages sug-gested that there could also be alterations in cell function. Totest the functional capabilities of these cells, we isolated

Fig. 4. BrdU incorporation into CD11c�

lung macrophages isolated from whole lungtissue on days 0, 2, and 4 post-Nb infection.BrdU was administered to mice via drinkingwater (0.8 mg/ml) on day 0 and continueduntil the day of sacrifice. Cells were immu-nostained with anti-BrdU or an isotype con-trol, and the level of BrdU incorporation wasevaluated by flow cytometry.

Fig. 5. Cytokine gene expression in CD11c� lung macrophages isolated fromwhole lung tissue on days 0, 2, 4, and 8 post-Nb infection. RNA isolation andprocessing for real-time RT-PCR analysis of IL-12, TNF-�, IL-6, IL-1�, IL-10,TGF-�, IFN-�, and IFN-� were carried out as outlined in Materials andMethods. Each bars represents the mean (SE) of five biological replicates ateach time-point (*, P0.05; **, P0.01).


CD11c� cells from the lungs at days 0, 4, and 13–15 PI todetermine their ability to take-up antigen and inhibit T cellproliferation. The CD11c� cells isolated from days 13–15post-Nb lungs have a significantly enhanced ability to take upantigen when compared with the CD11c� cells isolated fromuninfected and day 4 post-Nb lungs (Fig. 6A). Interestingly,the macrophages isolated from days 13–15 PI also showed anincreased capacity to inhibit CD4 T cell proliferation in re-sponse to plate-bound CD3 and soluble CD28 when comparedwith macrophages isolated from WT and day 4 post-Nb lungs(Fig. 6B). We tested the possibility that this enhanced abilityof days 13–15 macrophages to sequester antigen along withtheir elevated levels of MHCII expression (Fig. 2) correlatedwith an increased capacity to induce antigen-specific CD4� Tcell proliferation. Lung macrophages isolated from day 0-un-infected and days 13–15 post-Nb lungs were pulsed with OVApeptide (323–339) and incubated with CFSE-labeled CD4�

DO.11.10-transgenic T cells. In contrast to the robust prolif-eration observed when DC presented the peptide, the T cellsincubated with macrophages isolated from days 13–15 PI lungsshowed limited proliferation (Fig. 6C). However, this limitedability to promote T cell proliferation was not significantlydifferent from that measured from the lung macrophages iso-lated from uninfected lungs (Fig. 6C). These data illustrate thatdays 13–15 PI macrophages show a lack of ability to mediateantigen-specific T cell proliferation, despite increased antigenincorporation and elevated MHCII levels.

DISCUSSION

Infection with Nb results in a number of immediate and long-term changes to the immunological status of the lungs, includ-ing quantitative and phenotypic changes in the macrophagepopulation [10, 13, 17, 42]. In the current study, we describetwo distinct morphological forms that develop within the lungmacrophage population after a hookworm infection—a vacuo-lated, foamy phenotype that was supplanted within a few daysPI by a persistent population of enlarged macrophages thatcontained dense granules in the cytoplasm (Fig. 1). It is notclear if the origin of these cells is connected. Although it ispossible that foamy macrophages transform into the cells withthe granular morphology, it is equally likely that these distinctcell types represent at least two different populations whosephenotypes are influenced by the dynamics of the cellular andcytokine changes that persist in the PI lung environment [42].

Macrophages with the granular phenotype, which first ap-peared between days 4 and 8 PI in the parenchyma andbetween days 8 and 13 in the BAL, are readily detectiblethrough day 100 PI (Fig. 1) [43], suggesting they are anenduring element of the post-Nb lung. It is not clear if thegranular phenotype is maintained as a consequence of long-lived cells, or it represents scavenging of granules releasedafter cell death. Lung macrophages are distinguished by theirlongevity, with estimates of their half-life ranging between 30days and �2 years [44–46]; thus, it is conceivable that the

Fig. 6. Nb-induced CD11c� lung macro-phages have an increased phagocytic ca-pacity but are poor mediators of T cell ac-tivation. Macrophages were isolated fromwhole lung tissue using CD11c magneticbeads. (A) Changes in the phagocytic ca-pacity of CD11c lung macrophages isolatedat days 0, 4, and 15 PI. (B) PI CD11c� lungmacrophages modulate T cell proliferation.CD4� T cells (1�105) were loaded withCFSE and stimulated for 4 days with plate-bound anti-CD3 plus soluble anti-CD28 inthe presence of 1 � 105 CD11c� lungmacrophages isolated from lungs at day 0,day 4, or day 13 PI. The level of prolifera-tion was evaluated by flow cytometry. Adotted gray line represents the results of unstimulated T cells. Results are representative of three to five independent experiments. (C) CFSE-stainedDO11.10-transgenic CD4� T cells (1�105) were incubated with spleen-derived DC of CD11c� lung macrophages isolated from lungs at day 0 or day 13 PI. TheDC and lung macrophages pulsed with 10 �g/ml OVA peptide (323–339) for 60 min prior to adding the transgenic T cells (**, P0.01).


granular cells observed at 100 days PI are the same cellsformed within a few days after Nb traversed the lungs.

The composition of the granules appears to be complex.Although it is apparent that SPs make up a portion of macro-phage granules, some macrophages also stained positive forPrussian blue (data not shown), suggesting the sequestration ofiron, possibly through the uptake of RBCs. During the first 2–3days of infection, hemorrhage is evident as a consequence ofthe mechanical damage caused by the Nb larvae as they enterthe lungs [13] and could be the source of the iron. In addition,coincident with the development of the granular phenotype, alarge number of cells infiltrating the lungs undergo apoptosis,a significant percentage of which appears to be eosinophils(data not shown). Therefore, it is likely that macrophage gran-ularity is a result of the incorporation of SPs and cellular debrisreleased as a result of the structural damage and cellularinfiltration caused by the larvae’s migration through the air-ways. Histological staining for Fizz1 and YM1 revealed thatFizz1 is distributed uniformly throughout the cytoplasm of thegranular macrophages and does not appear to be concentratedin granules (data not shown). In contrast, YM1 staining ispunctate in nature and is concentrated in specific areas of thecytoplasm, suggesting that YM1 could be a component of thegranules (data not shown). Additional work will be required toconfirm the distribution of YM1 within the cells.

Measuring the cellular composition of the BAL fluid hasbeen used traditionally as a rapid method to monitor cellularlung infiltrates, typically during acute inflammation. The re-sults presented here suggest that BAL-based analysis of pul-monary macrophage populations does not accurately reflect thequantitative and qualitative changes in the macrophage popu-lations during acute inflammation or after inflammation wasresolved. Although BAL-based analysis may be useful as arapid, low-resolution measure of the cellular content of thelungs during inflammation, a more extensive protocol, such asthe bead-based isolation protocol used here, may be requiredto gain an accurate evaluation of cellular changes in inflamedand postinflammatory lungs.

One confounding issue associated with the CD11c bead-separation protocol is the coisolation of lung DCs (estimated at5% of total cells) that could have influenced the outcome ofthe expression and functional analyses. In C57/BL6 mice, acombination of autofluorescence and MHCII levels has beenproposed to differentiate lung DC and macrophage populations[47], but these criteria do not appear to be suitable for theseparation of these cell types from the lungs of BALB/c mice(data not shown). Although F4/80 has been used traditionallyas a macrophage-specific surface marker, its use in the lung iscompromised by low levels of F4/80 on lung macrophages andexpression levels that vary in the context of inflammation (Fig.2) [48, 49]. To increase the purity of lung macrophage prepa-rations for procedures such as adoptive transfers, it may benecessary to add a step to specifically deplete the lung DCs[50].

Gene expression analysis of lung-derived macrophages con-firmed earlier observations that there is a rapid and persistentinduction of AAMs in the pulmonary environment as a result ofa helminth infection [10, 13]. Expression profiles of day 2 PIcells isolated from whole lung showed induction of the genes

encoding ARG1, FIZZ1, YM1, and AMCase and the lectinMSR1, all hallmark indicators of alternative activation [5].AAMs are induced by a combination of the rapid induction ofTh2 cytokines [13] and tissue injury [51] caused by the Nblarvae as they enter the lungs and then sustained by thelong-term changes to the immunological environment of thelung [42].

Mature macrophages have long been considered end-stagecells with no or limited capacity to proliferate. However, recentevidence indicates that under certain conditions, lung macro-phages do have the capability to divide [52], and the expres-sion and cellular data from this study support this idea. Day 2PI macrophages expressed elevated levels of a number cellcycle-associated genes (Fig. 3) and incorporated BrdU, sug-gesting that at least a portion of the increases in macrophagesobserved on day 4 PI is a result of resident cell proliferation.However, this capacity to proliferate appears to be short-lived,as the cell cycle genes are no longer elevated by day 8 PI. Lungmacrophages are derived from blood monocytes and take ontheir unique characteristics only after a period of developmentin the pulmonary environment [52]. Therefore, it is possiblethat the capacity to proliferate provides an immediate source ofdifferentiated lung macrophages that can play a role in limitingthe extent of inflammation and promoting repair of cellular andstructural damage.

The AAM phenotype is induced by engaging the IL-4R orIL-13R and is accompanied by the dramatic up-regulation ofgenes encoding ARGI, FIZZI, YMI, and AMCase [5]. In addi-tion to the traditional AAM expression profile, the macro-phages isolated on days 4 and 8 PI expressed the gene encod-ing ArgII (Fig. 3), a molecule not typically associated withalternative activation. Although it is possible that ARGII pro-duction represents a novel marker of alternative activation inthe lung, it is also likely that argII expression is a reflection ofan environment, where the macrophages are receiving signalsthat modify the IL-4/IL-13 AAM phenotype. Clearance ofapoptotic cells may be one of those signals. Although argIIexpression is largely independent of Th2 cytokine signaling[53], it is strongly induced in macrophages during the incor-poration of apoptotic bodies, where it plays a role in inhibitingclassical macrophage activation [31]. In addition, post-NbAAMs strongly up-regulated complement components such asC1q and C3a, which are known to increase the ability of lungmacrophages to phagocytize and incorporate SPs and apoptoticgranulocytes [38].

Furthermore, the up-regulation of genes that encode proteinsinvolved in the metabolism of lipids such as CD68, Oldr1,PU.1, peroxisome proliferator-activated receptor (PPAR)�, andPex11� (a PPAR� target gene) also supports the notion thatTh2-independent stimuli are important cosignals in thepost-Nb lung. The importance of lipid metabolism and theactivation of PPAR� and other PPAR-associated factors haveoften been cited as a major factor responsible for initiatinganti-inflammatory qualities in macrophages [54, 55]. Recentstudies have also shown that PPAR� activation greatly en-hances alternative macrophage development and along withIL-4, is a dominant signal responsible for this phenotype [56,57]. Furthermore, it has been demonstrated that activatingPPAR� in the absence of IL-4 and IL-13 is sufficient to drive


M2 qualities in macrophages [58], suggesting it may play anindependent role in alternative activation. In particular, theactivation of these factors appears to be essential for theregulation of pulmonary inflammation by lung macrophagesand may represent a crucial regulatory component capable ofshifting lung macrophages to a protective phenotype [59, 60].

Over time, there were changes in the surface and cytokineprofiles of lung AAMs that suggested a transition to a moreanti-inflammatory state. The peak in surface expression ofCD40, CD80, and CD86 on day 4 PI was followed at day 8 PIby a marked reduction in these conventional costimulatorymolecules and an increase in PDL-1 and PDL-2 expression.These changes at the surface were accompanied by a signifi-cant increase in IL-10 transcription. Interestingly, PDL-1 hasbeen shown to be up-regulated by AAMs during helminthinfections and was demonstrated as an AAM mechanism of Tcell suppression [61, 62]

Lung AAMs also appear to exert control on the pulmonaryenvironment by increasing the transcription of genes encodingchemokines such as CCL11, CCL17, and CCL24, which areknown to be involved in cellular recruitment during Th2 im-mune responses [63–65]. CCL17, which was up-regulatedmore than ten-fold on each day of infection, has been shown tobe induced by AAMs as a result of IL-4 signaling [66]. CCL17is also known to be involved in the recruitment of Th2 cellsduring allergic responses and asthma, further illustrating itsimportance in driving Th2 responses in the lung [67–70].

Interestingly, day 13 post-Nb AAMs also possess a signifi-cantly enhanced ability to incorporate antigen when comparedwith the lung macrophages isolated from day 4 post-Nb and day0 uninfected lungs. The biological significance of this en-hanced ability to phagocytize on a per-cell basis becomes evenmore striking when one considers that there are approximatelysix times as many macrophages present in the lungs on day 13post-Nb than are found in uninfected controls. As expected,similar to control lung macrophages, post-Nb macrophagesshowed a limited ability to mediate antigen-specific CD4 T cellproliferation when compared with DC. These data suggest thatpost-Nb AAMs may act as an antigen sink capable of seques-tering large amounts of antigen and preventing its uptake byDC, thus preventing the initiation of a strong, adaptive immuneresponse. Furthermore, days 13–15 AAMs also showed a mod-estly enhanced capability to check the proliferation of CD4 Tcells activated with plate-bound anti-CD3 and soluble anti-CD28, illustrating another mechanism by which AAMs arecapable of dampening an adaptive immune response (Fig. 6).

In summary, alternative activation has often been viewed asan “off-on” phenotype downstream of IL-4 and IL-13 signaling.The results presented here illustrate that the development oflung AAMs is a dynamic process that appears to progress in astepwise manner. Lung macrophages are exposed to manydominant signals as a result of the structural damage andcellular infiltration initiated during and after a Nb infection.This is evident by the distinct sets of genes that are up- anddown-regulated during the various days of infection and wellafter initial Th2 cytokine signals are received. Included amongthese signals are the metabolism of oxidated lipids, the incor-poration of SPs, and the exposure to apoptotic bodies found inthe post-Nb lung. The notion of multiple signals contributing to

the progression of lung macrophages into the alternative phe-notype is illustrated further by acute transitions in gross phe-notype, surface marker characteristics, cytokine production,chemokine production, and general function between days 0and 15 PI. These data expand significantly upon past obser-vations and more fully define the dynamics of AAMs thatdevelop in the lung as a result of an infectious insult. Th2-independent signals are an important component of AAM invivo that are often overlooked and should be studied further innatural systems of infection. It is likely that the lack of Th2-independent signals in other systems of study is the reason whythe in vitro AAMs by IL-4 and IL-13 is considerably lessintense in nature than the AAMs during a natural infection [8].

ACKNOWLEDGMENTS

This work was supported by grants from National Institutes ofHealth (NIH), National Heart, Lung and Blood Institute (U01HL66623), NIH Training Grant T32AI007417, and the Heg-ner-Cort-Root Fellowship to M. C. S. We thank Jason Baileyand Anne Jedlicka (Johns Hopkins Malaria Research Institute,Gene Array Core Facility) for assistance with microarray ex-periments. We also thank Egbert Hoiczyk and Janet Folmer forelectron microscopy assistance.

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dynamics of lung macrophage activation