The three clones designated as ZK1, ZK2 and ZK6 were obtained by limiting dilution and examined for their growth characteristics, surface phenotype, and functional properties. PCR genotyping of these cell lines confirmed that they are SR-AI/II^-/- and MARCO^-/- (Fig. 1). SR-AI/II wild-type allele exhibited a 325-bp PCR product, whereas SR-AI/II^-/- mutant allele showed a 434-bp PCR product. MARCO wild type allele exhibited ca. 500-bp PCR product, and MARCO^-/- mutant exhibited ca. 850-bp PCR product. All of the three cell lines are stable and Mycoplasma-free by Mycoplasma PCR ELISA test (Roche, Indianapolis, IN) during culture in the past 24 months.

All three ZK cell lines replicate rapidly in regular RPMI medium or DMEM with 10% FBS in the absence of a specific growth factor supplement. Doubling time (mean generation time = mgt) was calculated according the formula: N = N₀2^T/mgt. On the average, doubling time of ZK cell lines is between 12 h to 16 h in RPMI complete medium (Fig. 2). Like their parental primary AMs isolated from the MS^-/- mice, all of the cell lines are adherent but trypsin-sensitive for passage.

Light microscopic examination of Diff Quik, a modified Wright-stained cytospin slides of ZK cells showed cells of considerable homogeneity in size and shape. All ZK cells had large, dark, round or oval nuclei with abundant pale cytoplasm containing small granules and numerous cytoplasmic vacuoles (Fig. 3). Only few cells showed double nuclei that may indicate cell division. Primary AMs isolated from wild-type mice and MS^-/- mice by BAL contained at least 95% macrophages, and ZK cells demonstrated the same morphology as these primary AMs.

Phenotypic analysis of ZK cells revealed the presence of macrophage markers F4/80 and Mac-1 (CD11b), which are expressed only by mature macrophages, but not on polymorphonuclear leukocytes, lymphocytes, or fibroblasts 3233. Immunofluorescent labeling demonstrated that more than 99.5% of ZK1 cell population expressed F4/80 and CD11b (Fig. 4). Moreover, the surface expression of F4/80 and CD11b on ZK1 cells was greater than that seen on the AMJ2-C11 cell line (relative fluorescence of F4/80 and CD11b was 155.9 vs 57.3 and 114.5 vs 92.1 compared to AMJ2-C11, respectively). Similar results were observed in ZK2 and ZK6 cells (data not shown). In addition, all three clones ZK1, ZK2 and ZK6 adhered to plastic tissue culture surfaces, a feature characteristic of macrophages. However, similar to primary AMs isolated from MS^-/- mice, these ZK cells did not attach to the plastic as tightly as wild-type macrophages. These ZK cells and primary AMs deficient in MARCO and SR-AI/II receptors were readily detached by vigorously pipetting after cold PBS treatment for 5–10 min.

ZK1, ZK2 and ZK6 cell lines exhibited sheep erythrocyte receptor- and FcγR-mediated phagocytosis of opsonized sheep red blood cells (SRBCs) that are typical of macrophages 3435. In contrast to opsonized SRBCs, only rare unopsonized SRBCs appeared bound to ZK1 cells; most cells did not have any unopsonized SRBCs attached. After binding, these unopsonized SRBCs were easily lysed away (Fig. 5A). Approximately 80% of ZK1 cells were positive for FcγR-mediated phagocytosis of opsonized SRBCs (Fig. 5B). Similar results were seen in ZK2 and ZK6 clones (data not shown).

To determine whether MS^-/- macrophages were able to bind and phagocytose unopsonized particles, uptake by ZK clones of unopsonized fluorescent latex beads was compared with that of primary AMs from MS^-/- mice and wild-type mice. Flow cytometry analysis revealed that all three ZK clones exhibited binding and phagocytic capacity of latex beads similar to primary AMs deficient in MARCO and SR-AI/II. All three clones showed significantly decreased uptake of fluorescent latex beads compared to the wild type primary AMs (Fig. 6A). Observed differences in phagocytic capacity between these clones and parental primary AMs from MS^-/- mice may reflect the heterogeneity seen in populations of primary alveolar macrophages.

In contrast to findings in AMs from wild-type mice, uptake of fluorescent latex beads by MS^-/- AMs and ZK cells is not inhibited by scavenger receptor ligands such as polyinosinic acid (PolyI) and dextran sulfate (DS, 500 kDa) at 10 μg/ml (Fig. 6B), nor by fucoidan (data not shown). Notably, dextran sulfate actually enhanced slightly the uptake of fluorescent latex beads by ZK1 cells (Fig. 6B). Furthermore, to determine whether the size of dextran sulfate molecules alters the effect on ZK1 cells' uptake of latex beads, we tested different sizes of DS in the binding/phagocytosis assay. The results indicated that only dextran sulfate with smaller molecular weight (5-8-kDa) inhibited binding, whereas larger 100-500-kDa dextran sulfate did not show inhibition of binding, but even enhanced binding (Fig. 6C).

ZK cell lines also exhibited phagocytosis of titanium dioxide (TiO₂) and fluorescently labeled heat-killed Staphylococcus aureus. However, similar to primary AMs from MS^-/- mice, all three ZK cell lines demonstrated marked reduced phagocytosis activity compared to primary AMs from their wild type mice (Fig. 7A and Fig. 7B). These results indicated that ZK cells persist in macrophage phagocytic capacity functionally, but like primary AMs deficient in MARCO and SR-AI/II, ZK cells were significantly impaired in phagocytosis compared to the WT AMs due to deficiency in scavenger receptors.

SR-AI/II-deficient mice 6, and MARCO-deficient mice, constructed by Soininen and colleagues 39, both on the C57BL/6 background were maintained in Harvard School of Public Health animal facility under pathogen-free conditions. Both MARCO and SR-AI/II-deficient (MS^-/-) mice were obtained by cross-breeding of the MARCO^-/- mice with SR-AI/II^-/- mice in our facility in accordance with NIH guide lines. Wild type C57BL/6 (WT) mice were purchased from Charles River Laboratories International, Inc (Wilmington, MA). Male mice between 9 to 14 weeks of age were used in the experiments. Animal experiments were conducted under a protocol #03827 approved by Harvard School of Public Health institutional animal use review committee.

Titanium dioxide (TiO₂) was obtained from Baker Chemicals (Phillipsburg, NJ). Latex beads (1.0 μm in diameter) that show green fluorescence after excitation at 488 nm were purchased from Interfacial Dynamics Co (Portland, OR). Fluorescently-labeled, heat-killed bacteria (Staphylococcus aureus) were obtained from Molecular Probes (Eugene, OR). All particles were suspended in balanced salts solution (BSS^- [124 mM NaCl, 5.8 mM KCl, 10 mM dextrose and 20 mM HEPES]) at stock solution and were probe sonicated for ~40 s (Model W-p200, setting 4; Ultrasonics, Plainview, NY) immediately prior to use. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from American Type Culture Collection (ATCC, Manassas, VA); RPMI 1640 medium and PBS were purchased from BioWhittaker (Walkersville, MD); Gentamycin, penicillin/streptomycin, macrophage-colony stimulating factor (M-CSF), and Polybrene were obtained from Sigma (St. Louis, MO); MethoCult™ GF M3434, semi-solid medium was obtained from StemCell Technologies (Vancouver, Canada); Diff-Quik, a modified Wright-Giemsa stain was obtained from VWR (Boston, MA); Bovine washed red blood cells (RBCs) was purchased from Quad Five (Ryegate, MT); Rabbit IgG fraction of anti-sheep red blood cell was purchased from Rockland Inc (Gilbertsville, PA); Fluorescein isothiocyanate (FITC)-labeled anti-mouse F4/80 and FITC-labeled anti-mouse CD11b (Mac-1) were purchased from eBioscience Inc (San Diego, CA); FITC-labeled FcγIII/II receptor, and FITC-labeled rat isotype controls, IgG2a and IgG2b, and mAb 2.4G2 were obtained from BD BioScience Pharmingen (San Diego, CA). All chemical reagents not otherwise specified were obtained from Sigma-Aldrich.

AMJ2-C11 cell line was purchased from ATCC, and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and gentamycin (50 μg/ml). ZK cell lines in this study were cultured in RPMI 1640 complete medium, in which RPMI medium was supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml).

Alveolar macrophages (AMs) were obtained by repeated brochoalveolar lavage with phosphate-buffered saline (PBS) from C57BL/6 mice or MARCO^-/- and SR-AI/II^-/- mice after being euthanized by intraperitoneal pentobarbital injection. Brochoalveolar lavage fluid (BAL) was immediately placed on ice, centrifuged at 200 × g for 10 min and the pellets were washed twice with cold PBS. Cell counts and viability were determined by using a hemocytometer and trypan blue dye exclusion. Cytocentrifuge preparations were stained with Diff-Quik, a modified Wright-Giemsa stain to allow differential analysis. All BAL samples contained greater than 95% AMs. AMs were adjusted to 1 × 10⁶ cells/ml in DMEM conditioned medium (CM), which contained DMEM medium supplemented with 15% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), 10 mM HEPES, and 10 ng/ml of mouse macrophage colony stimulating factor (M-CSF).

The AMJ2-C11 cell line was the source of the J2 retrovirus used 30. The virus was prepared as described previously 30 with some modifications. Supernatants from exponentially growing cultures of AMJ2-C11 were collected and cell debris was removed by centrifugation (3000 g, rotor GH-3.8, Beckman) for 15 min. After clarification, the supernatants were filtered through a 0.45-μm membrane (Millipore, Bedford, MA), diluted 1/1 with DMEM conditioned Medium containing 6 μg/ml of polybrene (final concentration), and added to the primary cultures for overnight at 37°C.

Primary AMs were isolated by BAL and stimulated to proliferate in DMEM conditioned medium at a density of 1 × 10⁶ cells/ml in a 6-well plate (Costar, Cambridge, MA), and incubated for 24 h at 37°C with 5% CO₂. Nonadherent cells were washed off with warm medium 6–8 h later. After an additional incubation for 24 h in DMEM conditioned medium, the nonadherent cells were washed off again, resulting in a population of cells with predominantly macrophage (>99%) morphology as determined with the modified Wright's stain. Adherent AM monolayers were infected with J2 virus supernatants mixed with DMEM conditioned media and polybrene. After 24 h, excess virus was removed and cells were exposed to a second round of J2 infection under the same conditions to improve transfection efficiency. Clusters of rapidly proliferating cells were evident ca. 10 days after initial infection. After 1 month, cells were grown in regular DMEM medium without M-CSF. The virus transformed cells progressively increased in number. Several AM cell lines rapidly growing in semi-solid medium (0.96% methylcellulose) were isolated and subsequently recloned by limiting dilution method. Three clones designated as ZK1, ZK2, ZK6 were characterized using the assays described below.

ZK cells (5 × 10⁴ cells/slide) were cytocentrifuged onto glass slides, air-dried and stained with Diff-Quik, a modified Wright-Giemsa stain. ZK cells were identified as alveolar macrophages by their large, dark nuclei and abundant pale, granular cytoplasm often containing numerous vacuoles. Primary AMs were isolated from the wild type mice and MS^-/- mice as controls.

Total genomic DNA was isolated from cultured ZK cells as well as from wild type (WT) mouse tails according to the protocols described in the DNeasy Tissue Kit from QIAGEN (Valencia, CA). Primers used for SR-AI/II were: forward primer (for both WT and SR-AI/II^-/- mice), 5'-CAAGTGATA CATCTCAAGGTC-3'; reverse primer for WT, 5'-CTGTAGATTCACGGACTCTG-3', which amplify a 325-bp product, and reverse primer for SR-AI/II^-/-, 5'GAGGAGTAGAAG GTGGCGCGAA-3', which amplify a 434-bp product. Primers used for MARCO WT allele are 5'-CAGCTGGGTCCATACCAGC-3' (forward primer) and 5'-CTGGAGAGCCTCGTTGACC-3' (reverse primer), which amplify a ca. 500-bp product; Primers used for MARCO mutant allele were 5'-CCACGCTCATCG ATAATTTCAC-3' (forward primer) and 5'-GCCTGCAGTGGCCGTCGTTTTA-3'(reverse primer), which amplify a ca. 850-bp product. The PCR was conducted on a MJ Research PTC-200 (Watertown, MA) under the following conditions (4 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C, followed by 5 min at 72°C). PCR products (10–15 μl/lane) were resolved in a 2.0% agarose gel.

ZK-1, ZK-2 and ZK-6 cells were seeded in six-well plates at 2 × 10⁴ cells/ml with RPMI complete medium and incubated at 37°C in 5% CO₂-humidified air. At various time intervals (14 h and 18 h incubation), three wells of each cell line were detached by repeatedly pipetting after treatment with cold PBS for 10 min, and the cells from each well were counted with a hemacytometer. Cell viability was determined by trypan blue dye exclusion. Doubling time (mean generation time = mgt) was calculated according the formula: N = N₀2^T/mgt. N is the number of cells at any time T; N₀, is the number of cells at an initial point.

Expression of CD11b (Mac-1) and F4/80 antigens was demonstrated by immunofluorescence staining of cell suspensions. To make a single cell suspension from cultured cell lines, cells were harvested by repeatedly pipetting with cold PBS. Cells were washed twice in a staining buffer (PBS containing 0.1% fetal bovine serum and sodium azide), and resuspended at 2 × 10⁷/ml. Fifty microlitters of each cell suspension were incubated with the following mAbs: pre-incubate the cells with 0.5 μg of mAb 2.4G2 (anti-mouse CD16/CD32, to block binding of FcγR) per million cells for 5–10 minutes on ice prior to staining, then add 0.25 μg/ml of FITC-labeled anti-mouse F4/80 or FITC-labeled anti-mouse CD11b to the cells in each tube, mix gently and incubate for 30 min in the dark. All staining procedures were carried out at 4°C. Cells were then washed 3 times and analyzed using a Coulter ELITE flow cytometer (Coulter Corporation, Miami, FL). At least 10,000 cells were collected for each histogram. Positive staining was determined by comparison with relevant FITC labeled rat IgG isotype.

Sheep red blood cells (SRBC) were opsonized by mixing fresh SRBC with appropriately titered rabbit anti-SRBC antiserum (Rockland, Gilbertsville, PA) for 30 min at room temperature, then the erythrocytes were washed, plated on monolayer of macrophages at a ratio of 20:1. Fc-receptor-dependent phagocytosis of IgG-coated bovine erythrocytes by ZK cell lines was performed as described by Mbawuike IN et al. 40 with some modification. Briefly, ZK cells were plated at 1 × 10⁶ cells/well in a 6-well plate containing a sterile 22 × 22 mm micro cover glass (VWR Scientific) per well in RPMI complete medium. After adherence overnight at 37°C, the unattached cells were washed away by medium exchange, then replaced with 2 ml RPMI medium to each well containing ca. 2 × 10⁷/ml of SRBC and incubated at 37°C for 1 h. After removal of free SRBC by medium exchange and lysis by osmotic shock, the cells on the cover glass were fixed with 2% paraformaldehyde and stained with Diff-Quik stain. The percentage of ZK cells which phagocytosed SRBC was scored by light microscopy. Cells were deemed positive for phagocytosis if they ingested more than five SRBC. Cells uptake of unopsonized SRBC and cells without lysis were used as controls.

Alveolar macrophage and ZK cell lines were collected and resuspended at 2 × 10⁶ cells/ml in a balanced salt solution (BSS⁺) containing NaCl (124 mM), KCl (5.8 mM), dextrose (10 mM) and HEPES (20 mM), adding CaCl₂ (0.3 mM) and MgCl₂ (1.0 mM). In a 96-well ultra low adherent plate (Corning Inc., Corning, NY): fifty microliters of macrophages (100K cells) were mixed with 50 μl of TiO₂ (final conc. 50 μg/ml) or fluorescent latex beads/bacteria (ratio of particles to cells: 50:1) and incubated at 37°C for 40 min. Cells and particles were mixed once more during the assay at 20 min. In some assays, MØs were preincubated with scavenger receptor inhibitors, polyinosinic acid (Poly-I, 10 μg/ml) and dextran sulfate (DS, 10 μg/ml) for 10 min at room temperature. After incubation, the plate was put on the ice and was added 200 μl of BSS⁺ buffer to each well, then transferred to flow tubes for flow cytometry. Propidium iodide (PI) was added to each tube to separate live cells from dead cells. MØ uptake of particles was measured using the increase in the mean right angle scatter (RAS) caused by these granular materials. All cell types tested showed almost identical baseline RAS values (no differences between the macrophage lines), which increased after particle uptake. Fluorescent latex beads or bacteria binding is expressed as relative fluorescence.

Experiments were performed, at minimum, in triplicate and all results represent the mean ± SD. We determined statistical significance between groups using the Student t test. For three or more groups, differences among groups were evaluated using One-way ANOVA on each group and followed by Dunnett's Multiple Comparison Test. A value of P < 0.05 was considered significant difference. All statistics and graphs were performed using Prism software, version 3.0 (GraphPad, San Diego, CA).