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The Journal of Immunology, 2002, 169: 6332-6342.
Copyright © 2002 by The American Association of Immunologists

Endocytic Internalization of Adenovirus, Nonspecific Phagocytosis, and Cytoskeletal Organization Are Coordinately Regulated in Alveolar Macrophages by GM-CSF and PU.11

Pierre-Yves Berclaz, Zsuzsanna Zsengellér, Yoko Shibata2, Kazuhisa Otake3, Steven Strasbaugh, Jeffrey A. Whitsett and Bruce C. Trapnell4

Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, OH 45229.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GM-CSF gene-targeted (GM-/-) mice have impaired pulmonary clearance of bacterial and fungal pathogens by alveolar macrophages (AMs). Because AMs also clear adenovirus from the lung, the role of GM-CSF in endocytic internalization of adenovirus by AMs was evaluated. Pulmonary clearance of adenovirus was severely impaired in GM-/- mice compared to wild-type (GM+/+) mice as determined by Southern analysis of viral DNA. Internalization of adenovirus by AMs was deficient in GM-/- mice in vivo and in vitro as determined by uptake of fluorescently labeled adenovirus or by PCR quantification of adenoviral DNA internalized within AMs. An AM cell line previously established from GM-/- mice (mAM) had impaired internalization of adenovirus and transferrin-coated 100-nm latex beads compared to MH-S, a GM+/+ AM cell line. Phagocytosis of 4-µm latex beads was also impaired in mAM cells as determined by confocal and fluorescence microscopy. Retroviral vector-mediated reconstitution of PU.1 expression in cultured GM-/- AMs restored phagocytosis of 4-µm beads, endocytosis of adenovirus, and transferrin-coated 100-nm beads (independent of integrin {alpha}V and transferrin receptors, respectively), and restored normal cytoskeletal organization, filamentous actin distribution, and stimulated formation of filopodia. Interestingly, mRNA for the phosphoinositide 3 kinase p110{gamma} isoform, important in macrophage phagocytic function, was absent in GM-/- AMs and was restored by PU.1 expression. These data show that GM-CSF, via PU.1, regulates endocytosis of small (~100 nm) pathogens/inert particles and phagocytosis of very large inert particles and suggests regulation of cytoskeletal organization by GM-CSF/PU.1 as the molecular basis of this control.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alveolar macrophages (AMs)5 play a central role in innate immune lung host defense, partly through their ability to internalize and eliminate microbial pathogens and inhaled particulates from the lung surface (1). A broad range of pathogens and particles are internalized by AMs through distinct mechanisms including receptor-mediated endocytosis and both receptor-mediated and nonspecific phagocytosis (2). Recently, an important role has been suggested for AM-mediated clearance of virions during pulmonary infection by influenza in humans (3) or adenovirus in mice (4). Adenovirus is a small (60–100 nm) nonenveloped DNA virus that is internalized by receptor-mediated endocytosis (5). Although best-studied in epithelial cells (5), internalization of adenovirus by mononuclear phagocytes is believed to occur by integrin receptor-mediated endocytosis (5, 6, 7). Integrin {alpha}M mediates attachment but not internalization of adenovirus virions in monocytes (6), while integrin {alpha}V also mediates internalization via clathrin-dependent endocytosis in both monocytes and epithelial cells (5, 6, 7, 8). Internalization of adenovirus requires phosphoinositol 3 kinase (PI3K) (9), a family of molecules involved in endocytosis, phagocytosis, cell adhesion, cell migration, and cytoskeletal organization (10, 11). Terminally differentiated AMs internalize adenovirus rapidly in vivo and in vitro (12) and are responsible for clearance of the majority of adenovirus administered to the murine lung (4). Adenovirus internalized by human, rat or murine AMs is similarly degraded with a half-life of ~6 h while in epithelial cells <5% is degraded by 48 h (4). Macrophages internalize larger microbial pathogens whose size is on the order of several microns, e.g., bacteria, mycobacteria, and fungi, by phagocytosis mediated by cell-surface receptors (2, 13). Internalization of large inert particles, e.g., latex beads, by macrophages occurs by nonspecific phagocytosis involving the extension of filopodia from the cell surface which surround and engulf the particle without known involvement of specific receptors (2). Although endocytosis and phagocytosis represent distinct mechanisms of internalization, both are critically dependent on the complex orchestration of interactions between overlapping sets of cytoskeletal elements that include actin and numerous other structural and regulatory proteins (2, 5, 6, 7, 9, 14, 15, 16, 17, 18, 19).

GM-CSF is a hemopoietic growth factor that primarily affects myeloid lineage cells (20) but also stimulates the growth of some nonhemopoietic cells, e.g., pulmonary type II alveolar epithelial cells (21). In addition to stimulating proliferation of myeloid progenitors, GM-CSF regulates a number functions in mature myeloid cells including cell adhesion (22), complement- and Ab-mediated phagocytosis (23), and pathogen destruction (24). Targeted ablation of the murine GM-CSF gene (GM-/- mice) revealed a critical role for GM-CSF in lung surfactant homeostasis (25, 26). GM-/- mice develop pulmonary alveolar proteinosis (PAP) characterized by age-dependent, progressive accumulation of surfactant lipids and proteins within AMs and alveolar spaces (25, 26). Interestingly, PAP in GM-/- mice is histologically similar to a form of PAP in humans (27) recently demonstrated to be strongly associated with neutralizing anti-GM-CSF autoantibodies (28, 29). GM-/- mice have increased susceptibility to pulmonary infection by bacteria and fungi (30, 31) and AMs of GM-/- mice exhibit multiple abnormalities including impaired phagocytosis and killing of bacteria and fungi (30, 31, 32).

PU.1 is a transcription factor present in myeloid and B cells that is essential for production of macrophages and other hemologic lineage cells (33, 34) and stimulates differentiation of macrophage progenitors (35). PU.1 is present in AMs of mice expressing GM-CSF normally but is not found in AMs of GM-/- mice (32). High levels of PU.1 in AMs are restored by overexpression of GM-CSF only in the lungs of GM-/- mice, thus demonstrating that GM-CSF levels in the lungs regulate PU.1 levels in AMs (32). Based on these and other data, GM-CSF-stimulation of high PU.1 levels in AMs has been proposed as the principal mechanism regulating terminal differentiation of AMs in the lung (32).

The present study is based on the hypothesis that GM-CSF regulates particle internalization by AMs via PU.1-dependent pathways. To address this hypothesis, we assessed the role of GM-CSF and PU.1 in receptor-mediated endocytosis of adenovirus or transferrin-coated beads as well as phagocytosis of large, inert latex beads by AMs of GM-/- and GM+/+ mice in vivo and in vitro. The role of GM-CSF and PU.1 in the regulation of cytoskeletal organization was also assessed during the process of viral and particle internalization by AMs in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice targeted at the GM-CSF locus were created previously (26), bred into the C57BL/6 background and maintained for several years (36) (referred to as GM-/- mice hereafter). C57BL/6 mice (Charles River Breeding Laboratories, Wilmington, MA) served as controls (referred to as GM+/+ mice hereafter). Mice were housed in a "barrier" facility as previously described (37).

AMs and AM cell lines

Primary AMs, obtained by bronchoalveolar lavage (BAL) (32), consisted of 98% macrophages as determined by differential cytometry and were >=95% viable as determined by trypan blue staining. mAM is an AM cell line derived previously from GM-/- mice without viral transformation (32). mAM cells constitutively expressing murine PU.1 (referred to as mAMPU.1+ hereafter) were created previously (32) by transduction with a retroviral vector expressing PU.1, and also green fluorescent protein (GFP) as a selectable marker (38). mAM cells expressing only the retroviral GFP marker (referred to as mAMGFP+ hereafter) were used as a transduction control. MH-S is a murine AM cell line (CRL-2019; American Type Culture Collection, Manassas, VA) with morphologic features and functions of normal, mature AMs derived previously from wild-type (GM+/+) mice (39). Cultured AMs were maintained as previously described (32).

Adenovirus

The adenovirus used in this study is a recombinant human serotype 5 derivative whose structure has been described (40). Virus preparation was done using endotoxin-free medium, supplements, and solutions (supplied routinely or by special arrangement from BioWhittaker, Walkersville, MD) (12, 41). Viral concentration was determined by the OD method and expressed as optical particle units (opu) (42). In some experiments, adenovirus was fluorescently labeled by covalent linking of the carbocyanin dye, Cy3 (Amersham, Arlington Heights, IL) to the capsid (12, 43). Fluorescently labeled adenovirus (Ad-Cy3) contains 2.5–3.2 dye molecules per virion (12) and retains >90% infectivity (43).

Pulmonary clearance of adenovirus

Adenovirus (4 x 1010 opu in 50–75 µl of indicator-free HBSS) was administered to mice by intratracheal instillation into the lung using a 30-gauge needle (44). Mice were sacrificed at 0.25 or 24 h by lethal pentobarbital injection and exsanguination, the lungs were removed en bloc, minced, and incubated (50°C, 16 h) in 10 mM Tris, pH 7.8, 10 mM EDTA, 0.5% SDS, 1 mg/ml proteinase K (lysis buffer). Lung lysates were extracted twice with buffer-saturated phenol/chloroform/isoamyl alcohol (25:24:1) and total DNA (murine lung and adenoviral) was collected by ethanol precipitation and resuspended in 10 mM Tris Cl, 1 mM EDTA, pH 8.0. Adenovirus DNA was quantified by Southern analysis (45). Briefly, total DNA (10 µg) was cleaved with BamHI, electrophoresed on 1% agarose, transferred to Hybond (Schleicher & Schuell, Keene, NH) and hybridized with a 32P-labeled adenovirus DNA fragment (adenovirus sequences 3328–6241; GenBank accession number M73260). Adenovirus DNA hybridization was quantified by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA) (44). As a loading control, blots were striped and rehybridized with a 32P-labeled surfactant protein C (SP-C) probe (46).

Quantification of adenovirus internalization by AMs in vivo and in vitro

Fluorescent adenovirus method: in vivo. Ad-Cy3 (4 x 1010 opu/mouse) was administered to mice as above (12). After 6 h, mice were sacrificed and AMs were recovered by BAL (HBSS, 0.05% EDTA; three aliquots of 1 ml each) and collected by low speed centrifugation (200 x g, 10 min, 4°C) (12). Cells were resuspended (PBS, 0.2% BSA, 0.01% sodium azide) and sediments were prepared by cytocentrifugation (400 x g, 5 min; Thermo Shandon, Pittsburgh, PA). Cells were air-dried, stained with Hoescht dye (Aventis, Lyon, France) as per the manufacturer, rinsed in PBS, dried, and viewed by fluorescence microscopy on a Nikon FXTA microscope (Melville, NY). A rhodamine filter was used to visualize Ad-Cy3 (appears yellow) internalized within AMs and a 4',6'-diamino-2-phenylindole (DAPI)/PE filter was used to visualize internalized Ad-Cy3 (appears red) and cell nuclei (appear blue). In some experiments, internalization of adenovirus by AMs in vivo was evaluated in lung sections as previously described (12). Briefly, mice were sacrificed as above 6 h after infection and the lungs were inflation-fixed (4% paraformaldehyde, PBS at 25-cm water pressure) for 1 min in situ, removed en bloc, and fixed for 24 h at 4°C. Lungs were then dissected along major bronchi, dehydrated in graded ethanols, embedded in paraffin, and sections (5 µm) were assessed by fluorescence (rhodamine filter) and phase contrast microscopy. Internalization of fluorescent virions was also evaluated by confocal microscopy (12).

Fluorescent adenovirus method: in vitro. To assess endocytic internalization of adenovirus by AMs in vitro, AM cell lines (mAMGFP+, mAMPU.1+, and MH-S) were cultured overnight in eight-well micro-chamber slides (Nalge Nunc International, Rochester, NY). Cells were exposed to Ad-Cy3 (5 x 109 opu/well) for 6 h and then washed extensively with PBS. Cells were visualized by fluorescence and phase microscopy as above.

Semi-quantitative PCR method. Internalization of adenovirus by AMs in vivo and in vitro was also quantified by using PCR amplification as previously described (47). For in vivo analysis, adenovirus was administered and AMs were recovered by BAL as above. For in vitro analysis, cells were incubated with adenovirus (1010 opu/106 cells) in six-well plates for 4 h. Following adenovirus exposure, noninternalized virus was removed by extensive washing and total cellular DNA (i.e., from cells and internalized adenovirus) was extracted. Adenovirus DNA was then quantified in total DNA by PCR amplification using adenovirus-specific primers with an automatic thermal cycler (PerkinElmer/Cetus, Norwalk, CT) (48). Each PCR contained 100 ng of total DNA and amplification, consisting of 94°C, 30 s; 55°C, 30 s; 72°C, 30 s, was continued for either 25 cycles (adenovirus DNA-specific primers) or 27 cycles (murine thyrotropin {beta} subunit (TSH-{beta}) gene-specific primers) followed by extension at 72°C for 10 min. The TSH-{beta} gene served as a control to ensure that similar amounts of total DNA were present in each reaction.

Histological evaluation of lipid accumulation in lung AMs

AM morphology and lipid accumulation were assessed in mice 2 or 6 wk of age (49). Briefly, mice were sacrificed as above and the lungs were inflated in optimum cryosectioning medium (OCT), removed en bloc, embedded in OCT and frozen at -80°C until use. Cryosections (15 µm) were cut, fixed in 30% buffered formalin and stained with oil-red-O (Sigma-Aldrich, St. Louis, MO), lightly counterstained with hematoxylin and viewed by light microscopy (Nikon FXTA microscope).

Quantification of mRNA transcript levels in AMs

mRNA transcript levels of murine PU.1, transferrin receptor (TR), PI3K subunits or, as a control, GAPDH, in cultured AMs were assessed using RT-PCT analysis (44) with gene-specific primers. Primer sets used included: PU.1 (5'-TCT GAT GGA GAA GCT GAT GG-3'; 5'-CTT GAC TTT CTT CAC CTC GC-3') and GAPDH (5'-CTG ACG TGC CGC CTG GAG AAA-3'; 5'-TTG GGG GCC GAG TTG GGA TAG-3'); TR (5'-GGC GAG ATG AAC ACT ATG TGA AGA-3'; 5'-AAG AGT GCA AGG TCT GCC TCA ACA-3'); p85{alpha} PI3K (5'-CAA GCC CAC TAC TGT AGC CAA CAA-3'; 5'-CTG GGT AGA GCA ACT TCA CAT CCA-3'); p85{beta} PI3K (5'-GTA CCA ACA AGA CCA GGT GGT GAA-3'; 5'-GTA TCT CCG CGA TGC GAG ACT TGA-3'); p101 PI3K (5'-CAG CAT GCT CTG GAG CGA TGC TT-3'; 5'-CAA CAG CAC AGT GAC AGT GGA ACT T-3'); p110{alpha} PI3K (5'-TGA GGC CAC ACT CGT CAC CAT CAA-3'; 5'-CTT CAC GGT TGC CTA CTG GTT CAA-3'); p110{beta} PI3K (5'-GTT CCA CGG TCA AGT TGG ATG GAA-3'; 5'-GGT ATT TAC CCA TGC GAC AGG ATA-3'); p110{delta} PI3K (5'-CAT GGA GTT CTG GAC CAA AGA GGA-3'; 5'-GGA GGC TGA TCT GGG AGT TAA TGA-3'); and p110{gamma} PI3K (5'-ACC GGT GGT TCT AAG AGA GGA CAA-3'; 5'-CTT TCT TCT GGT AGA GCA GGA GGA-3').

Quantification of AM cell surface markers by flow cytometry

Expression of macrophage cell-surface differentiation markers and integrins was assessed by flow cytometry. Primary AMs collected by BAL or cultured AMs collected by scraping in versene were resuspended and washed in FACS buffer (PBS, 0.2% BSA, 0.01% sodium azide). Aliquots of cells (105 cells/100 µl of FACS buffer) were incubated with Fc-block (rat anti-mouse CD16/32; BD PharMingen, San Diego, CA; 30 min, 4°C) and then with primary Ab (2 µg, 30 min, 4°C). Primary Abs included the rat anti-murine macrophage differentiation markers BM8, ER-MP20, and ER-MP12 (all from Bachem, Torrence, CA), and PE-conjugated, anti-murine integrin {alpha}V, {alpha}M, and {alpha}L Abs (all from BD PharMingen). Cells were washed twice in FACS buffer and kept on ice (anti-integrin primary Abs) or incubated (ER-MP12, ER-MP20, BM8; 30 min, 4°C) with a FITC-conjugated anti-rat IgG secondary Ab (BD PharMingen) and washed twice in FACS buffer. As controls, cells were assessed using primary isotype- and species-matched anti-mouse Igs. Cells were analyzed by single color flow cytometry using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and results were analyzed using CellQuest software on a Macintosh microcomputer. Fluorescence data were collected using logarithmic amplification on 10,000 viable cells as determined by forward light scattering.

Quantification of TR-mediated endocytosis

Murine TR-coated fluorescent latex beads (referred to as TF-beads) were prepared using 100-nm diameter, FITC-labeled latex microspheres (Molecular Probes, Eugene, OR) by incubation with murine transferrin (Sigma-Aldrich; 10 mg/ml, 37°C, 60 min) followed by washing (three times) and resuspension in PBS at a concentration of 1.5 x 109 beads/ml. TR-mediated endocytosis was quantified in cultured AM cell lines plated the day prior to analysis. Cells (105/well, plated in 35-mm dishes), were exposed to TF-beads at a concentration of 0.5 x 107/ml for 1 h. Cells were then washed extensively in FACS buffer to remove noninternalized particles, detached by brief trypsinization and evaluated by flow cytometry on a FACScan flow cytometer (BD Biosciences). Results were analyzed using CellQuest software (BD Biosciences) on a Macintosh microcomputer.

Quantification of phagocytosis by AMs using confocal and fluorescence microscopy.

Confocal microscopy method. The ability of mAMGFP+ and mAMPU.1+ to phagocytose very large latex beads was assessed using both confocal and fluorescence microscopy (50). Cells grown on cover slips were incubated with 4.0-µm latex beads (Molecular Probes) at a concentration of 0.5 x 106/ml beads/ml for 4 h and then noninternalized beads were removed by extensive washing. Cells were viewed on a Zeiss Axioplan 2 double laser fluorescence confocal microscope (Oberkochen, Germany) and images were handled using Zeiss Image Examiner software version 2.50.0929. Serial x-y sections of cells were obtained along the z axis at 0.3 µm- (mAMGFP+) or 0.5-µm (mAMPU.1+) intervals. At least 200 cells of each type were assessed for internalization of beads.

Fluorescence microscopy method. To quantify phagocytic internalization, bead-exposed cells were also viewed under phase contrast on a Nikon FXTA microscope at x40. The percentage of cells containing internalized beads and the mean number of beads per positive cell was determined by direct counting in each of 10 fields per coverslip on three coverslips per cell line. The phagocytic index (PI) was calculated using the formula PI = percentage of AMs with internalized beads x mean number of beads/positive AM (32).

Assessment of AM cell morphology and cytoskeletal organization by confocal microscopy

mAMGFP+ or mAMPU.1+ were cultured for 12 h on cover slips, fixed (4% paraformaldehyde in PBS, 10 min, 25°C) and immunostained with anti-murine phalloidin Ab (200 U/ml, 20 min, 25°C; BD PharMingen) to localize filamentous actin (F-actin) and washed in PBS (50). Cells were visualized using double laser fluorescence confocal microscopy. Image analysis was as above. Cells were also visualized using phase contrast microscopy as above.

Microarray analysis

Total RNA was purified from mAMGFP+ or mAMPU.1+ using TRIzol (Life Technologies, Gaithersburg, MD) and biotinylated cRNA probes were made (51) and hybridized to the microarray chip (GeneChip Murine Genome U74; Affymetrix, Santa Clara, CA) and washed using protocols supplied by the manufacturer. Chips were then scanned at an excitation frequency of 488 nm and the hybridization results were quantified using an Affymetrix 428 array scanner. Data were analyzed using Affymetrix Microarray Suite V4.0 software on PC microcomputer.

Statistical methods

Numerical data are presented as mean ± SE of the mean. Statistical comparisons were made by using Student’s t test. Calculations were performed with Sigma Plot (version 7.0) software on a PC microcomputer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pulmonary clearance of adenovirus is impaired in GM-/- mice

The role of GM-CSF in viral lung host defense was assessed by comparing clearance of adenovirus DNA after pulmonary administration of adenovirus in GM-/- and GM+/+ mice. Fifteen minutes after administration, amounts of adenoviral DNA were similar in the lungs of GM-/- and GM+/+ mice (Fig. 1Go). However, 24 h after administration, viral DNA levels were reduced by >90% in the lungs of GM+/+ mice but were unchanged in the lungs of GM-/- mice (11.1 ± 3.5 vs 100 ± 34.5%, respectively; p < 0.05). These data show that GM-CSF is necessary for adenovirus to be cleared from the lung during acute respiratory tract infection.



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  		FIGURE 1. Pulmonary clearance of adenovirus is impaired in GM-/- mice. Adenovirus was administered to the lungs of GM+/+ and GM-/- mice and its subsequent elimination was assessed using Southern blotting to quantify adenovirus DNA in total lung DNA. A, Southern hybridization analysis. Equal amounts of virus were administered to mice as indicated by similar hybridization of the adenoviral DNA probe (Ad DNA) in total lung DNA isolated shortly (0.25 h) after virus infection (left panels). One day later (24 h), the amount of adenovirus DNA was markedly reduced in the lungs of GM+/+ mice but not in the lungs of GM-/- mice. Each lane shows data from one mouse. Equal loading of total lung DNA was demonstrated by stripping and rehybridizing the blots to a murine surfactant protein C gene probe (Murine control). B, PhosphoImager analysis of Southern blots. Data obtained by Southern blotting with the adenovirus probe (represented in A) were quantified using a Molecular Dynamics PhosphorImager. Twenty-four hours after infection, adenovirus DNA levels were unchanged in the lungs of GM-/- mice compared to initial values, while levels were significantly reduced in the lungs of GM+/+ mice. Each bar represents the mean of four mice. This experiment was performed twice and representative data are shown.

 
Internalization of adenovirus by AMs is impaired in GM-/- mice

To determine whether impaired pulmonary clearance of adenovirus in GM-/- mice was due to a defect in AM function, internalization of adenovirus by AMs in GM-/- and GM+/+ mice was compared using several experimental methods. In one approach, infectious fluorescently labeled adenovirus (Ad-Cy3) was administered to the lungs and 6 h later AMs were recovered by BAL and evaluated by fluorescence and confocal microscopy (12). Adenovirus was readily detected in most AMs of GM+/+ mice, but not in AMs of GM-/- mice as determined by fluorescence (Fig. 2GoA) and confocal microscopy (not shown).



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  		FIGURE 2. Internalization of adenovirus by AMs is impaired in GM-/- mice. A, Assessment of internalization of adenovirus by AMs in vivo. Ad-Cy3 was administered to the lungs of 6-wk-old GM+/+ or GM-/- mice. Six hours after infection, AMs were recovered by BAL stained with Hoechst dye and viewed by fluorescence microscopy using a rhodamine filter (a and c) with which Ad-Cy3 appears yellow or a DAPI/PE filter (b and d) with which Ad-Cy3 appears red and cell nuclei appear blue. Ad-Cy3 was easily detected (arrows) within most AMs of GM+/+ mice but not in AMs of GM-/- mice (open arrows; c and d). Neutrophils (*) were observed in BAL cells of GM-/- mice more frequently than in BAL cells of GM+/+ mice (x920). This experiment was repeated four times with similar results and representative examples are shown. B, In vivo lipid accumulation in AMs was assessed by oil-red-O staining of lung sections of GM+/+ or GM-/- mice. In mice 6 wk of age, AMs (arrowheads) in the lungs of GM+/+ mice had minimal lipid accumulation (red color) (e) in contrast to AMs in GM-/- which were increased in size and had extensive lipid inclusions (f). In contrast, AMs in GM+/+ and GM-/- mice at 2 wk of age were morphologically normal and had little intracytoplasmic lipid. This experiment was done twice with similar results. C, Internalization of adenovirus by morphologically normal AMs (white arrowheads) was assessed in 2-wk-old GM+/+ and GM-/- mice. Six hours after pulmonary administration of Ad-Cy3, the lungs were removed and prepared as described in Materials and Methods and visualized by fluorescence (i and j) or phase (k and l) microscopy. Ad-Cy3 internalization was easily detected in AMs of GM+/+ mice (i) but not in AM of GM-/- mice (j) despite their normal morphology (g and h, k and l). This experiment was done twice with similar results; representative fields are shown. (x400, a–c; x250, e–h). D, Quantification of adenovirus internalization by AMs in vivo by using PCR amplification. Six hours after pulmonary administration of adenovirus, AMs recovered by BAL were evaluated for the presence of internalized adenovirus DNA using PCR amplification with virus-specific primers. Adenovirus DNA was readily detected in AMs of GM+/+ mice but not GM-/- mice or uninfected controls. Amplification of endogenous murine TSH-{beta} gene sequences (Murine control) demonstrated assessment of equal numbers of cells. Each lane represents results for a separate animal.

 
To exclude the possibility that reduced internalization of adenovirus by AMGM-/- was due to abnormalities attributable to intracellular or intraalveolar accumulation of lipoproteinaceous surfactant material, internalization studies were conducted in young mice prior to development of histologically significant PAP. As expected, oil-red-O staining demonstrated marked lipid accumulation in AMs in GM-/- but not GM+/+ mice at 6 wk of age (Fig. 2GoB, e and f). However, in mice at 2 wk of age, similar staining demonstrated no detectable abnormal lipid accumulation in AMs in either GM-/- or GM+/+ mice (Fig. 2GoB, g and h). Internalization of Ad-Cy3 by AMs administered as above was assessed in frozen lung sections by fluorescence microscopy (12). Importantly, despite their normal morphologic appearance and the absence of lipid accumulation, AMs in younger GM-/- mice still failed to internalize Ad-Cy3 in contrast to GM+/+ mice in which AMs readily internalized adenovirus (Fig. 2GoC).

Impaired internalization of adenovirus by GM-/- AMs was further assessed by quantifying the amounts of adenovirus DNA within AMs recovered by BAL 6 h after pulmonary administration of infectious unlabeled adenovirus (48). Adenovirus DNA was readily detected within AMs from GM+/+ but not GM-/- mice or uninfected controls (Fig. 2GoD). Together, these data demonstrate that GM-CSF is a critical regulator of endocytic internalization of adenovirus by AMs.

Expression of AM cell surface integrins and macrophage differentiation markers are altered in GM-/- mice

Because GM-CSF modulates integrin expression in mononuclear phagocytes (52, 53), expression of integrins known to mediate adenovirus binding (integrin {alpha}M, CD11b) or internalization (integrin {alpha}V, CD51) by mononuclear cells (5, 6, 8) were evaluated in primary AMs by flow cytometry. Integrin {alpha}V was detected at low levels on GM+/+ but not GM-/- AMs (Fig. 3Go). In contrast, integrin {alpha}M was detected on both GM+/+ and GM-/- AMs. Integrin {alpha}L (CD11a; a marker of GM-CSF-induced macrophage differentiation; Refs. 54 and 55) was present on GM+/+ but not GM-/- AMs suggesting that GM-/- AMs might not be fully differentiated. To further explore this possibility, several cell surface markers of macrophage differentiation were evaluated in primary AMs by flow cytometry. BM8, a marker of mature tissue macrophages (56), was readily detected on GM+/+ but not GM-/- AMs (Fig. 3Go). In contrast, ER-MP20, a marker of intermediate myeloid differentiation that is down-regulated in mature monocytes and macrophages (57, 58), was detected at low levels on GM-/- but not on GM+/+ AMs. ER-MP12, a marker present on most tissue macrophages, was present on GM+/+ and GM-/- AMs. Together, these data suggest that AMs in GM-/- mice may not have completed terminal differentiation.



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  		FIGURE 3. Cell surface integrins and macrophage differentiation markers are altered on AMs from GM-/- mice. Various cell surface integrin receptors involved in binding or internalization of adenovirus as well as macrophage differentiation-associated Ags on AMs of GM+/+ or GM-/- mice were assessed by flow cytometry. The specificity of the primary Ab (grey shading) is indicated in each panel. Isotype controls (black line) are also shown. This experiment was done three times with similar results. Representative data from one experiment is shown.

 
Constitutive expression of PU.1 in AMs of GM-/- mice corrects defective endocytic internalization of adenovirus

To determine whether impaired internalization of adenovirus by GM-/- AMs might be due to a defect in AM terminal differentiation, expression of the macrophage differentiation-inducing transcription factor PU.1 was assessed in cultured AMs using RT-PCR analysis. PU.1 mRNA was not present in cultured GM-/- AMs (mAMGFP+) but were restored by retroviral transduction (mAMPU.1+) and were also present in GM+/+ AMs (MH-S), serving as a positive control (Fig. 4GoA). Endocytic internalization of adenovirus by these AM cell lines was then assessed by two methods, detection of adenoviral DNA within the total DNA of virus-exposed cells by PCR (48) and direct visualization of internalization of fluorescently labeled virus (Ad-Cy3) (12). Internalization of adenovirus by MH-S cells, as a positive control, was readily demonstrated by the presence of adenovirus DNA (Fig. 4GoB) in virus-exposed cells. In contrast, mAMGFP+ cells poorly internalized adenovirus as demonstrated by reduced amounts of cell-associated adenovirus DNA in virus-exposed cells. PU.1 expression restored a normal capacity for adenovirus internalization as demonstrated by the presence of increased amounts of adenovirus DNA in mAMPU.1+ cells compared to mAMGFP+ similar to levels observed in MH-S control cells. Adenovirus DNA was not detected in the absence of viral exposure (data not shown). Internalization of adenovirus by MH-S cells was also demonstrated by direct visualization of Ad-Cy3 in virus-exposed cells (Fig. 4GoC). In contrast, mAMGFP+ Ad-Cy3 fluorescence was not detectable within mAMGFP+ cells but could be restored by PU.1 expression as seen by abundant Ad-Cy3 fluorescence within mAMPU.1+ cells.



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  		FIGURE 4. Retrovirus-mediated restoration of PU.1 expression in cultured AMs of GM-/- mice rescues their ability to internalize adenovirus independent of integrin {alpha}V. A, Assessment of PU.1 mRNA transcripts in cultured AM cell lines. PU.1 mRNA transcripts were quantified in cultured AMs from GM+/+ mice (MH-S) or GM-/- mice transduced with a retroviral vector expressing PU.1 (mAMPU.1+) or a control vector (mAMGFP+) using RT-PCR with PU.1-specific primers (PU.1). Similar assessment using murine GAPDH-specific primers demonstrated evaluation of equal amounts of total cellular RNA. B, Assessment of adenovirus internalization by cultured AMs in vitro using PCR. Cultured AMs were exposed to adenovirus for 6 h and internalization of adenovirus was assessed by quantifying adenoviral DNA in total cellular DNA using PCR with adenovirus-specific primers as described in Materials and Methods. Amplification of endogenous murine TSH{beta} gene sequences demonstrated assessment of equal numbers of infected cells (Murine control). Each lane represents results for a separate animal. C, Assessment of adenovirus internalization by cultured AM cell lines in vitro. Cultured AM cell lines (indicated) were exposed to Ad-Cy3, washed extensively, and evaluated by fluorescence (upper panels) and phase (lower panels) microscopy as described in Materials and Methods. Fluorescence photomicrographs shown were obtained using DAPI/PE filter as for Fig. 2Go. Ad-Cy3 (appears red) was easily detected within most wild-type macrophages (MH-S cells) (nuclei are blue). Ad-Cy3 uptake was markedly diminished in the absence of PU.1 (mAMGFP+ cells) and restored by PU.1 expression (mAMPU.1+ cells). D, Assessment of cell surface integrin expression on cultured AMs. Immunostaining of cultured AM cell lines was as described for primary AMs (Fig. 3Go). The specificity of the primary Ab (grey shading) is indicated in each panel. Isotype controls (black line) are also shown. These experiments were performed twice (A and B) or three times C) and representative data are shown.

 
Because PU.1 regulates expression of certain integrins in macrophages during differentiation (59, 60), expression of integrin {alpha}V and integrin {alpha}M was assessed in MH-S, mAMGFP+, and mAMPU.1+. These integrins were readily detected on MH-S cells (Fig. 4GoD) but both were reduced in amount on mAMGFP+ cells. PU.1 had a differential effect on expression of these integrins in mAM cells, further reducing levels of integrin {alpha}V and markedly increasing levels of integrin {alpha}M. To further explore the effect of PU.1 on integrin expression in these macrophages, microarray analysis was performed comparing RNA isolated from mAMGFP+ and mAMPU.1+ using the Affymetrix Murine Gene Chip. Levels of mRNA transcripts for a number of integrins were changed little by constitutive expression of PU.1 (Table IGo). However, integrin {alpha}V transcript levels were reduced 3.8-fold in mAMPU.1+ compared to mAMGFP+, consistent with the FACS data (Fig. 4GoD). Also consistent with FACS data, integrin {alpha}M transcripts were increased 6.8-fold in mAMPU.1+ compared to mAMGFP+. Interestingly, mRNA levels for integrin {beta}5 were reduced 22.9-fold while integrin {beta}2 and integrin {alpha}4 mRNA levels were increased 21.9- and 27.9-fold, respectively, in mAMPU.1+ compared to mAMGFP+ (Table IGo). These data show that PU.1 reduces integrin {alpha}V and stimulates integrin {alpha}M expression in AMs at the mRNA and protein levels. Thus, PU.1-mediated correction of impaired endocytic internalization of adenovirus by GM-/- AMs is not due to restoration of integrin {alpha}V on the AM cell surface.


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  		Table 1. Regulation of integrin mRNA levels in AMs of GM-/- mice by retrovirus-mediated PU.1 expressiona

 
TR-mediated mediated endocytosis of latex beads by AMs is impaired in GM-/- AMs and is restored by retroviral PU.1 expression

To quantify the regulation of endocytic function in AMs by GM-CSF/PU.1, endocytosis was further assessed using the well-characterized TR uptake mechanism (61) in mAMGFP+, mAMPU.1+, and MH-S cells. The TR gene was expressed in all three cell lines as shown by similar levels of transferrin receptor mRNA (Fig. 5GoA). As a control, endocytosis of transferrin-coated fluorescent latex beads by MH-S cells was readily demonstrated and quantified by flow cytometry (Fig. 5GoB). Endocytosis of transferrin-coated beads by GM-/- PU.1negative mAMGFP+ cells was markedly reduced. Importantly, PU.1 stimulated significantly increased endocytosis of transferrin-coated beads as seen in GM-/- PU.1positive mAMPU.1+ cells. These data show that PU.1 can bypass the GM-CSF requirement and restore endocytosis by a receptor-independent mechanism.



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  		FIGURE 5. TR-mediated endocytosis is impaired in cultured GM-/- AMs independent of TR expression and is restored by retroviral-mediated PU.1 expression. A, Assessment of TR mRNA transcript levels. Total RNA was isolated from cultured AM cell lines (indicated) and evaluated by RT-PCR using gene-specific primers for the murine TR or GAPDH as described in Materials and Methods. B, Cultured AM cell lines (indicated) were exposed to transferrin-coated, fluorescently labeled 100-nm beads, washed extensively, and evaluated by flow cytometry as described in Materials and Methods. The mean fluorescence intensity (n = 4 separate determinations for each group) is shown. PU.1 significantly stimulated endocytosis of TF-beads in mAMPU.1+ compared to mAMGFP+ cells (p < 0.001). Endocytosis of TF-beads was also positive in MH-S cells. This experiment was repeated twice and representative data are shown from one experiment.

 
Phagocytic internalization of very large latex beads is impaired in GM-/- AMs and can be restored by retroviral PU.1 expression

To further define the internalization defect in AMs of GM-/- mice, phagocytosis of very large (4 µm) latex beads by mAMGFP+ and mAMPU.1+ cells was assessed in vitro using confocal and phase microscopy (50). Despite examination of over 200 cells, phagocytosis of 4-µM beads by mAMGFP+ cells was not observed (not shown). In contrast, mAMPU.1+ readily phagocytosed beads as seen in confocal images of antiphalloidin-immunostained cells (Fig. 6GoA). Interestingly, numerous filopodia were seen extending from the cell surface toward and partly surrounding beads in mAMPU.1+ engaged in internalizing beads and both filopodia and the phagocytic cup strongly stained for F-actin (Fig. 6GoA). Filopodia were not seen in mAMGFP+ in cells juxtaposed to noninternalized beads (not shown). Nonspecific phagocytic internalization of large latex beads was also quantified by phase microscopy (50) and expressed as PI. As a positive control, MH-S cells readily internalized beads (Fig. 6GoB). In marked contrast, phagocytosis of large latex beads was severely impaired mAMGFP+ cells as indicated by a PI of 0. Importantly, phagocytosis was rescued by PU.1 as demonstrated by a significantly increased PI in mAMPU.1+. These data show that the defect in internalization by GM-/- AMs extends from receptor-mediated endocytosis of very small (~0.1 µm) viral pathogens to nonspecific phagocytosis of very large (4 µm) inert particles.



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  		FIGURE 6. PU.1 rescues phagocytosis of large inert particles by AMs from GM-/- mice. A, Confocal microscopy. mAMGFP+ or mAMPU.1+, grown on coverslips, were exposed to 4.0-µm latex beads, stained with anti-murine phalloidin Ab to visualize F-actin and viewed using confocal microscopy. In mAMPU.1+, internalized beads were seen in all fields while in mAMGFP+, no cells with internalized beads were seen despite examination of over 200 cells. Shown is a three-dimensional reconstruction of a typical mAMPU.1+ cell engulfing a latex bead (dark area marked by *). F-actin (red color) is readily seen beneath the cell membrane and adjacent to the latex bead (i.e., the actin cup) and within cytoplasmic processes (filopodia) extending towards and engulfing the latex bead. The cytoplasm shows green fluorescence due to expression of the retroviral GFP marker gene. The image has been rotated to view the cell along the z axis from above or below as indicated. B, Quantification of phagocytic internalization of large latex beads by cultured AMs. mAMGFP+, mAMPU.1+, or MH-S (as a positive control) were exposed to latex beads as above and visualized by phase contrast microscopy. The phagocytic index was calculated by multiplying the percentage of cells positive for internalized beads by the mean number of beads in positive cells. PU.1 significantly stimulated internalization of latex beads in mAMPU.1+ compared to mAMGFP+ (p < 0.001). Phagocytosis of beads was also positive in MH-S cells. This experiment was repeated twice and representative data are shown from one experiment.

 
AM cytoskeletal organization is regulated by PU.1 and is abnormal in GM-/- mice

Because numerous filopodia were observed in mAMPU.1+-engulfing latex beads, but not in mAMGFP+ cells, which failed to do so, AM morphology and cytoskeletal organization were further assessed using phase and confocal microscopy. mAMGFP+ grown on coverslips had a broad, flattened appearance with multiple large tubular processes attached to the coverslip and extending long distances from the cell body (Fig. 7Go). F-actin was present in the tubular structures and was also distributed relatively homogenously along the cell surface in contact with the coverslip. mAMPU.1+ had a markedly different appearance (Fig. 7Go). In comparison, mAMPU.1+ were round, slightly smaller but taller and lacked the large tubular processes seen in mAMGFP+. Resting cells also had numerous, short filopodia extending in all directions from the cell surface. F-actin was localized within filopodia and also along the contact surface. To further define the effects of PU.1 on cytoskeletal organization in AMs, microarray analysis in mAMGFP+ and mAMPU.1+ (described above) was used to assess potential changes in mRNA transcripts for major cytoskeletal elements and components of the endocytic internalization of adenovirus. No differences were observed in levels of mRNA transcripts for any of the actin isoforms and only minor changes were observed in several tubulin isoforms while others were similar (Table IIGo). Of the regulatory molecules known to be required for adenovirus internalization, only one subunit of PI3K was moderately increased by PU.1.



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  		FIGURE 7. PU.1 regulates cytoskeletal organization, cell shape and stimulates formation of filopodia in AMs from GM-/- mice. The effect of PU.1 on cytoskeletal organization and cell morphology was assessed using phase contrast microscopy (a and f) and laser confocal microscopy (b–e, g–j). mAMGFP+ appear large and flat under phase contrast microscopy (a) while mAMPU.1+ have a smaller and rounded (f). Cells stained with PE-anti-murine phalloidin antibody (b–e, g–j) were viewed en face (b–d, g–i) to visualize green fluorescence from GFP (b and g), red fluorescence from anti-phalloidin-stained F-actin (c and h) or both (d and i). Cells viewed in cross-section after three-dimensional image reconstruction showed the taller appearance of mAMPU.1+ compared to mAMGFP+ (e and j). (Magnification was similar in, a and f, b–e, and in g–j, indicated by bar). This experiment was repeated twice with similar results.

 

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  		Table 2. Regulation of mRNA levels for cytoskeletal elements known to be involved in adenovirus internalization or endocytosis by retrovirus-mediated expression of PU.1 in AMs of GM-/- micea

 
To further explore the possibility that GM-CSF, via PU.1, may regulate endocytic and phagocytic functions through regulation of PI3K expression, the mRNA transcript levels for regulatory and kinase domains of several PI3K family members was assessed. Transcripts for the PI3K regulatory subunits p85{alpha}, p85{beta} (Fig. 8GoA), and the associated kinase subunit p110{alpha} (Fig. 8GoB) were present in MH-S, mAMGFP+ and mAMPU.1+ cells. Interestingly, the PI3K kinase subunit p110{gamma} and its related adaptor subunit p101 were present in MH-S cells, absent in mAMGFP+ and restored by PU.1 expression in mAMPU.1+ cells. The PI3I kinase subunit p110{delta} exhibited a similar pattern of expression to the p110{gamma} subunit.



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  		FIGURE 8. Assessment of PI3K mRNA transcript levels in cultured AM cell lines. Total RNA was isolated from cultured AM cell lines (indicated) and evaluated by RT-PCR using gene-specific primers for various murine PI3K regulatory (p85{alpha}, p85{beta}, p101) and kinase (p110{alpha}, p110{delta}, p110{gamma}) subunits or GAPDH as described in Materials and Methods. This experiment was repeated twice with similar results.

 
Together, these data show that PU.1 coordinately regulates AM cell shape and cytoskeletal organization in parallel with both receptor-mediated endocytosis of very small pathogens and particles and nonspecific phagocytosis of large inert particles. Results suggest a mechanism in which GM-CSF/PU.1 modulates expression of cytoskeleton-organizing regulatory molecules, rather than expression of cytoskeletal structural proteins levels themselves, possibly through members of the PI3K family.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was designed to determine the role of GM-CSF in regulation of viral clearance by AMs. GM-CSF was required for efficient clearance of adenovirus from the lung during acute infection in mice. AMs of GM-/- mice were severely impaired in endocytosis of adenovirus in vivo and in vitro and were unable to phagocytose very large latex beads. Cultured GM-/- AMs did not express PU.1 in contrast to GM+/+ AMs which did. Further, GM-/- AMs expressed cell surface integrin and macrophage differentiation markers in a pattern suggesting incomplete terminal differentiation. Retrovirus-mediated PU.1 expression in cultured GM-/- AMs coordinately restored 1) endocytosis of adenovirus and transferrin-coated beads and phagocytosis of large latex beads; 2) a mature macrophage pattern of integrin and differentiation marker expression; 3) macrophage-like cell shape, distribution F-actin filaments, and filopodia formation; and 4) expression of a PI3K isoform known to be important in myeloid cell phagocytic functions and innate immune host defense in vivo (10, 11, 62).

The observations that adenovirus was efficiently internalized by GM+/+ AMs in vivo and in vitro and efficiently cleared from the lungs of GM+/+ mice are consistent with prior studies from our lab and others demonstrating that AMs are responsible for clearing the majority of adenovirus administered to the murine respiratory tract (4, 12). Impaired pulmonary clearance of adenovirus in GM-/- mice is consistent with previously reported defects in pulmonary clearance of bacterial and fungal pathogens in these mice (30, 31). Reduced clearance of adenovirus could be explained, at least in part, by reduced internalization by AMs which was demonstrated in vivo and in vitro. Defective internalization of adenovirus by GM-/- AMs was corrected by PU.1 expression but did not require integrin {alpha}V on the cell surface. Thus, while integrin {alpha}V was important in endocytic internalization of adenovirus by epithelial cells (7) and increased integrin {alpha}V expression levels augmented adenovirus uptake by monocytes and lymphocytes (8), integrin {alpha}V is not absolutely required for internalization by mature macrophages. Integrin {alpha}M mediated attachment, but not internalization, of adenovirus in monocytes and conferred attachment, but not internalization, to Chinese hamster ovary cells (6). However, integrin {alpha}M was present on primary AMs in the absence or presence of PU.1 expression. Thus, while PU.1 stimulated integrin {alpha}M expression in cultured AMs, it is unlikely to be the molecular mediator of correction of impaired adenovirus internalization by PU.1 in GM-/- AMs. Microarray analysis showed that PU.1 markedly stimulated mRNA transcripts encoding integrin {alpha}4, a molecule recently demonstrated to have an important role in the respiratory burst following exposure of murine neutrophils cells to fungal spores (63). Further studies will be required to determine whether integrin {alpha}4 has a role in internalization of adenovirus by AMs. Finally, while integrin {beta}5 was recently shown to be involved in the endosomal lysis step of adenovirus entry (17), integrin {beta}5 transcription is repressed by GM-CSF (53). mRNA transcripts for integrin {beta}5 were markedly reduced by PU.1 suggesting its lack of involvement in the PU.1-mediated correction. This observation is also consistent with recent data suggesting that GM-CSF regulates PU.1 expression in AMs (32) and further suggests that PU.1 may mediate the transcriptional repression of integrin {beta}5 stimulated by GM-CSF. Together, these observations do not support a critical role for integrin {alpha}V in adenovirus uptake by mature AMs beyond serving as a point of virion attachment.

Phagocytosis of very large beads was also defective in GM-/- AMs suggesting a generalized defect of internalization in these cells. This concept is consistent with recent data demonstrating that GM-/- AMs also have defects in phagocytosis of Gram-positive and Gram-negative bacteria, Pneumocystis carinii and zymosan as well as nonspecific endocytosis of very small (0.1 um) latex beads (31, 32, 64). That PU.1 stimulated changes in cell shape, formation of filopodia, and altered the pattern of F-actin distribution shows that PU.1 is important in the regulation of AM cytoskeletal organization. Notwithstanding, PU.1 did not markedly alter expression of mRNA transcripts encoding the major cytoskeletal structural elements. The finding that PU.1 stimulated mRNA levels of class IB PI3K p110{gamma} isoform is interesting because this catalytic subunit has been implicated in the innate immune function of macrophages and neutrophils. In PI3K p110{gamma}-/- mice, macrophages and neutrophils are poorly recruited in a Listeria septic peritonitis infection model resulting in severely impaired clearance of viable bacteria from the peritoneal cavity (10). Further, granulocytes from these mice exhibit reduced migration in response to G protein-coupled receptor agonist exposure in vitro (62). Although a mechanistic basis had not been well-defined, it has been suggested that PI3K p110{gamma} may mediate actin rearrangement via small GTPases at the plasma membrane (11). This concept is based on 1) phenotypic similarities in p110{gamma}-/- and GTPase Rac2-/- mice including abnormalities in granulocyte chemotaxis; and 2) the knowledge that phosphoinositol (3, 4, 5)P3 can activate such GTPases (65) and the actin cytoskeleton is dynamically reorganized at the leading edge of migrating cells (66). It is noteworthy that cellular internalization of adenovirus also requires GTPase-mediated actin reorganization (15) and can be inhibited by the PI3K inhibitor wortmannin in epithelial cells (9) and macrophages (12). Further, PI3K is also required for macrophage pseudopod extension and phagocytosis of large particles as demonstrated in studies using the PI3K inhibitors wortmannin and LY294002 (67). Together, our results are consistent with a mechanism whereby GM-CSF/PU.1 regulates AM internalization through regulation of cytoskeletal organization possibly through modulating expression of one or more PI3K isoforms.

GM-CSF stimulation of levels of PU.1 in AMs has been proposed as an important mechanism regulating terminal differentiation of AMs in the lung (32). Data in the present study support this model by showing that 1) both GM-CSF and PU.1 regulate diverse aspects of AM structure and function and 2) PU.1 regulates the presence of cell surface markers of macrophage differentiation. Prior studies demonstrating that GM-CSF (68) and PU.1 (69) stimulate expression of large sets of partly overlapping genes in mononuclear phagocytic lineage cells also supports this model. This is also consistent with data showing that GM-CSF stimulates differentiation of normal AMs in vitro (55, 70, 71).


   Acknowledgments
 
We thank Hristos Glavinas for help with the adenovirus DNA quantitation, Susan Wert, and Matt Kofron for help with morphological evaluations, Sara Rankin for performing the microarray hybridization and Abu Hossain for preparation of adenovirus. Zissis Chroneos provided the mAM cell line.


   Footnotes
 
1 This work was supported by the Children’s Hospital Research Foundation (to B.C.T. and P.-Y.B.), Grants HL61646 (to J.A.W.) and HL69549 (to B.C.T.), Fondation Suisse deBourse en Medicine et Biologie (to P.-Y.B.), and M. Carvaja-Steuer, Kartagener Foundation (to P.-Y.B.). Back

2 Current address: Department of Pediatrics, Harvard Medical School, Boston, MA 02115. Back

3 Current address: Yamagata University School of Medicine, 2–2–2 Iida-Nishi, Yamagata 990-23, Japan. Back

4 Address correspondence and reprint requests to Dr. Bruce C. Trapnell, Division of Pulmonary Biology, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: Bruce.Trapnell{at}chmcc.org Back

5 Abbreviations used in this paper: AM, alveolar macrophage; PI3K, phosphoinositol 3 kinase; PAP, pulmonary alveolar proteinosis; BAL, bronchoalveolar lavage; GFP, green fluorescent protein; opu, optical particle unit; TR, transferrin receptor; PI, phagocytic index; F-actin, filamentous actin. Back

Received for publication December 13, 2001. Accepted for publication October 2, 2002.


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 Introduction
 Materials and Methods
 Results
 Discussion
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