1 Office of Clinical Research and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and Departments of 2 Medicine and 3 Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein. To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos. No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright's stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed. We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads. We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process. In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice. However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45-60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells. Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis.
phagocytosis; macropinocytosis; myristoylated, alanine-rich, C kinase substrate
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INTRODUCTION |
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THE MYRISTOYLATED, alanine-rich, C kinase substrate (MARCKS) is a member of a small family of protein kinase C (PKC) substrates widely expressed in different cell types and tissues (reviewed in Refs. 1 and 5). The myristate moiety targets MARCKS to cellular plasma membranes, an association that is further influenced by the phosphorylation state of the protein through changes in the electrostatic potential of a polybasic domain that contains the PKC phosphorylation sites (24, 25, 46, 47). In the presence of calcium, unphosphorylated MARCKS binds calmodulin with high affinity, whereas PKC-dependent phosphorylation of MARCKS inhibits this interaction (12, 19, 27, 50). MARCKS also cross-links actin filaments in a phosphorylation- and Ca2+/calmodulin-dependent manner (18). As a consequence of these membrane-, calmodulin-, and actin-binding capacities, a role for MARCKS in processes that depend on membrane turnover and cytoskeletal remodeling, including cell motility (28) and secretion, has been suggested (38).
Macrophages express high levels of MARCKS (2), apparently localized to
the plasma membrane and punctate podosomes, where it colocalizes with
talin and vinculin (36). The presence of MARCKS in areas in which actin
filaments associate with the cytoplasmic face of the plasma membrane
has also been described (18), and it is enriched at the leading edge of
motile fibroblasts (28). Mutation of MARCKS PKC phosphorylation sites
has been reported to induce defects in cell motility (28); because of
the similarity between the mechanisms for cell motility and
phagocytosis, these authors suggested that MARCKS might play a role in
phagocytosis. In addition, several recent papers have provided
additional evidence for a role for MARCKS and the MARCKS-like protein
(MLP; see Ref. 43 for nomenclature), also called F52, MacMARCKS, and
MARCKS-related protein (MRP), in phagocytosis. Allen and Aderem (3)
reported that MARCKS phosphorylation was induced by zymosan and that
MARCKS and PKC colocalized in the nascent phagosome.
Zhu et al. (57) reported that expression of mutated forms of MLP
inhibited phagocytosis of unopsonized zymosan by macrophage-like cell
lines. They also reported that the dominant-negative form of MLP
inhibited macrophage binding to sheep red blood cells (SRBC) coated
with complement iC3b (26).
These results prompted us to examine the role of MARCKS in phagocytosis and the related process, fluid phase endocytosis (FPE), in wild-type macrophages and fibroblasts and those derived from MARCKS-deficient mice (6, 7, 42). Our results indicate that MARCKS-deficient macrophages were almost identical in morphology and phagocytic and macropinocytic activity to the cells derived from their wild-type littermates. Similarly, MARCKS-deficient fibroblasts exhibited apparently normal morphology and rates of FPE. The only reproducible difference exhibited by the macrophages concerned the phagocytosis of zymosan at 45-60 min, which was modestly (8.5-17.6%) but significantly decreased in the MARCKS-deficient cells. These data suggest that MARCKS deficiency alone is not sufficient to result in dramatic changes in these cellular functions; instead, the previous data showing dramatic dominant-negative effects of mutant MLP on phagocytosis (26, 57) may be due to interference with the function of MLP alone or of both MARCKS and MLP, or with pathways independent of these proteins.
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MATERIALS AND METHODS |
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Mice
Mice heterozygous for Macs, the mouse gene encoding MARCKS (42), were kept in a barrier facility and were interbred to generate the offspring used in these experiments. The first gestational day (day 0.5) was defined by the presence of a vaginal plug the morning after mating. Fetuses at days 14-17 were used to obtain livers for macrophage generation and at day 14 were used for the preparation of mouse embryo fibroblasts (MEF). Genotyping of fetuses was performed as described previously (42). Briefly, tail DNA (10 µl, 5-15 µg) was digested with Hind III and probed with a 2.1-kb Hind III-Sst I fragment of the Macs genomic clone, which recognizes both the 2.2-kb Hind III fragment of the wild-type gene and the 3.3-kb Hind III fragment of the Neo-disrupted gene.Culture of Hematopoietic Progenitors From Fetal Liver
Fetal liver-derived macrophages were prepared as described previously (8, 29). In selected cases, cultures were grown in the presence of recombinant human macrophage colony stimulating factor (M-CSF; R&D Systems, Minneapolis, MN).Macrophages were harvested with the neutral protease Dispase II (Boehringer Mannheim, Indianapolis, IN) as described previously (8) and were recultured overnight in 8-well Lab-Tek tissue culture chambers (Nunc, Thousand Oaks, CA) or 96-well plates (Falcon) or were centrifuged onto microscope slides for morphological and cytochemical analyses, as described previously (8).
Culture of Primary MEF
MEF were cultured from day 14 mouse fetuses, as previously described (34). The embryos from which the fibroblasts were derived were genotyped as described in Mice. Fibroblasts were grown in DMEM (GIBCO BRL) supplemented with 10% (vol/vol) FCS, 4 µl/l 2-mercaptoethanol (Sigma Chemical, St. Louis, MO), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were grown at 37°C, and were used at passage 4.Morphological Analysis
To study the morphology of the macrophages in suspension, 1.5 × 105 cells were deposited onto a microscope slide with a cytocentrifuge (Shandon, Pittsburgh, PA). The morphology of attached cells was studied by culturing them in eight-well Lab-Tek chambers (75 × 103 cells/well). All cells were stained with the Diff-Quik stain set (Baxter Healthcare, McGaw Park, IL). Cells were analyzed and photographed with a Laborlux-12 microscope (Ernst Leitz, Wetzlar, Germany) equipped with a PM-C35B camera (Olympus America, Lake Success, NY).Cytochemistry
Cytopreps prepared as described above were used to test for nonspecific esterase activity, as described by Yam et al. (55), and for peroxidase activity, as described by Kaplow (21), to assess the purity of the macrophage population.Actin Staining
Cells (75 × 103/well) were plated on eight-well Lab-Tek tissue culture chambers and incubated overnight as described in Morphological Analysis. Cells were then incubated for 24 h in the presence of 1 µg/ml lipopolysaccharide (LPS; Sigma Chemical) or in the absence of LPS and then incubated for 30 min with either phorbol 12-myristate 13-acetate (PMA; Sigma Chemical; 100 nM) in 0.01% (vol/vol) DMSO (Fisher Scientific, Pittsburgh, PA) or DMSO alone (0.01%) at 37°C. Cells were washed briefly with PBS and fixed for 15 min with 3.7% formaldehyde in PBS. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 7 min, washed 3 times with PBS, and then incubated in a solution of rhodamine-phalloidin (Molecular Probes, Eugene, OR) in PBS at 37°C for 30 min. After being stained, the cells were washed three times in PBS, and the slides were mounted in a Slow-Fade antifade agent (Molecular Probes). Cells were then analyzed by fluorescence microscopy with a Nikon Eclipse E600 microscope, equipped with 20× and 40× Plan Fluor objectives (Southern Micro Instruments, Atlanta, GA) and a Nikon FDX-35/Nikon U-III multipoint sensor system camera set.Phagocytosis Assays
Cells (75 × 103/well) were plated on eight-well Lab-Tek tissue culture chambers and incubated as described above. Before the phagocytosis assays, the cells were washed with serum-free RPMI 1640 and incubated for 1 h in RPMI 1640 supplemented with 0.2% (wt/vol) BSA. To evaluate Fc receptor (FcR)-mediated phagocytosis, SRBC (Cappel Research Products, Durham, NC) were incubated for 1 h with a subagglutinating titer of rabbit anti-SRBC (Cappel Research Products; 1:2,500 dilution) at room temperature with gentle rotation, as described previously (4), and 400 µl of a 0.01% suspension of IgG-coated SRBC in RPMI 1640-0.2% BSA were added to the chambers. The phagocytosis assay was carried out for 1 h at 37°C. Uningested cells were eliminated by vigorous washing with PBS, and bound but uningested SRBC were eliminated by quickly dipping the slides in 0.15 M ammonium chloride. Slides were stained with the Diff-Quik stain set, and ingested SRBC were counted by light microscopy. Each condition was analyzed in duplicate, and 250 cells were scored per sample. Results were expressed as the phagocytic index (PI; no. of ingested SRBC per 100 cells multiplied by the percentage of phagocytic cells). Opsonized SRBC were labeled with sodium [51Cr]chromate (5 mCi/ml; DuPont NEN, Boston, MA), as described previously (40, 51). A 10% SRBC solution (100 µl) was added to each well of 96-well plates, into which 5 × 104 macrophages/well had been plated the day before. An additional 100 µl of medium containing PMA, to give final concentrations of 50, 100, 500, 1,000, and 1,600 nM, or 0.01% DMSO was added, and phagocytosis was assayed for 1 h at 37°C. Unbound SRBC were removed by aspirating the medium, and bound but uningested SRBC were removed by lysing with 200 µl of 0.15 M NH4Cl for 5 min at 37°C, followed by three washes with 200 µl of PBS at 4°C. Monolayers were then solubilized with 200 µl of 0.5% SDS, and 150 µl were then counted on a gamma counter (Beckman Instruments).Phagocytosis mediated by the receptor for fraction C3b of the complement system (C3bR) was studied by using C3b-coated Saccharomyces cerevisiae, prepared as described previously (20). Briefly, heat-killed yeast particles were opsonized in mouse serum at a ratio of 107 yeasts/0.4 ml serum for 1 h at 37°C. Opsonized particles were resuspended in RPMI 1640-0.2% BSA; 400 µl of medium containing 2 × 106 yeasts/ml were added to the macrophages, prepared as described above; and phagocytosis was carried out for 1 h at 37°C. Slides were profusely washed with PBS to remove all uningested yeast and fixed in methanol. Slides were stained with the Diff-Quik stain set, mounted, and scored under light microscopy, as described above for SRBC.
The phagocytosis of zymosan was performed as described previously (41, 57). Briefly, a stock suspension of zymosan (Sigma Chemical) was prepared at 5 mg/ml in PBS-0.2% sodium azide and kept at 4°C. A working solution of 500 µg/ml in DMEM-0.2% BSA was prepared fresh on the day of the experiment (400 µl/chamber; ~107 particles of zymosan/chamber). Chambers were incubated at 4°C for 45 min, and unbound zymosan was washed out with cold DMEM-0.2% BSA. Chambers were then incubated at 37°C for different times, and after being washed with PBS, cells were fixed with 10% formalin at 4°C for 15 min, washed with PBS, and scored under phase-contrast microscopy after being mounted in water. In each experiment, 250-500 cells were counted.
Nonspecific phagocytosis was assessed by latex bead ingestion. Latex beads (500 µl of 0.997-µm beads; Polysciences, Warrington, PA) were washed three times with 1 ml of RPMI 1640 and resuspended in 1 ml of RPMI 1640-0.2% BSA. A 50-µl aliquot of this suspension was added to each chamber, in a final volume of 500 µl. Cells were incubated with the beads for 1 h at 37°C, and uningested beads were removed by vigorous washing with PBS. Cells were fixed with 3.7% formaldehyde in PBS for 10 min, washed again with PBS, and then stained with the Diff-Quik stain set. Phagocytosis was assessed by light microscopy, as described above.
Measurement of FPE
Macrophages. Cells prepared as described above were plated at 0.4-15 × 105 cells/well in five 24-well plates, one plate to be used for assays performed at 2°C and the other four to be used for time points at 37°C.
FITC-conjugated dextran (FD; molecular wt = 70,000; Molecular Probes) was used as the marker of fluid phase uptake in these experiments; the relative fluorescence due to internalized FD in individual cells was measured with a flow cytometer. On the day of the experiment, an aliquot of PMA stored in DMSO as a 16 mM stock solution was thawed and diluted to a final concentration of 100 nM in 10% FCS-RPMI 1640 medium containing 1 mg/ml FD. The carrier DMSO was diluted into the FD-containing medium in the same fashion. Cells were incubated with medium containing 1 mg/ml FD and either DMSO or PMA for 5, 10, 20, or 30 min at 37°C. Typically, a plate of cells was placed in a 37°C incubator, then removed and placed on wet ice after the appropriate incubation at 37°C. In parallel, a plate of cells was incubated with FD for 30 min on wet ice. All subsequent washings and sample manipulations were performed on ice or in the cold room. The FD solution was aspirated from the wells, and the cells were washed once with PBS containing 1 mg/ml BSA and twice with PBS. Cells were then treated with 0.2 ml of Dispase II. After the cells were incubated for at least 10 min at 2°C, 0.165 ml of 10% (vol/vol) FCS in PBS was added to the wells. Wells were also gently scraped with a rubber policeman to maximize recovery of cells. The cell suspension was then transferred to a Falcon tube (type 2052) containing 0.135 ml of 3.7% formaldehyde in PBS. Racks of samples were wrapped in foil and stored at 4°C until fluorescence-activated cell sorting (FACS) analysis, which was completed within 24-48 h of each experiment by using either a FACStar Plus or a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), which were equipped with argon lasers operating at 488 nm and 50 mW. FITC fluorescence was determined with a 530-nm filter. Fluorescence intensity was recorded, and data were analyzed with either the Consort 32 or the LysysII analysis and software package. The mean and median fluorescence intensities per cell for at least 2,000 cells were determined for each sample.MEF. Two days before each experiment, MEF were plated in normal growth medium at a concentration of 3 × 105 cells per well in 24-well plates (Costar, Cambridge, MA) and incubated overnight at 37°C. Eighteen to 24 h later, the cells were washed three times in PBS and then serum starved by incubating them in serum-free DMEM containing 1% BSA, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin overnight (16-20 h) at 37°C. At appropriate times after the addition of each agent, FD was added to each well (final concn, 1 mg/ml). After various times of exposure to FD, the dye solution was aspirated and each well was immediately washed with ice-cold PBS containing 1 mg/ml BSA (United States Biochemical, Cleveland, OH); this was followed by two washes with ice-cold PBS. After the last PBS wash, 200 µl of 0.05% (wt/vol) trypsin (JRH Biosciences) in PBS were added. The plates were incubated on ice for ~10 min, until the cells were released from the plastic. Then 165 µl of PBS containing 10% (vol/vol) FCS were added to each well to inactivate the trypsin, and the contents of each well were transferred to a separate tube (Falcon 2052; Becton Dickinson). Formaldehyde (135 µl of a 3.7% solution in PBS containing 10% FCS) was added to each tube, and the tube contents were mixed gently; the final formaldehyde concentration was 1%. The cells were then stored at 4°C until analysis. FACS analysis was performed within 24-48 h of each experiment as described above. The mean and median fluorescence intensities per cell for ~5,000 cells were determined for each sample.
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RESULTS |
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Morphological and Cytochemical Characterization of Fetal Liver-Derived Macrophages
Several analyses to establish the macrophage nature of the cells generated by these cultures were performed, as described previously (8). When in suspension, the cells were generally mononuclear in nature, with abundant vacuolated cytoplasm (Fig. 1, A and C); however, a small number of multinucleated cells were also present, presumably the result of the cell-fusion phenomenon observed when cells from the monocyte-macrophage lineage are maintained in culture for long periods of time (53). Nuclei were round or slightly oval, exhibited mature chromatin, and frequently appeared in an eccentric position in the cell. Although cell size and the degree of cytoplasmic vacuolization differed from cell to cell in the same preparations, it was not possible to determine any consistent morphological differences between the cells derived from the MARCKS knockout mice and their wild-type littermates. When the morphology of adherent cells was studied (by staining the Lab-Tek chamber slides), the cells spread on the glass slide and exhibited long pseudopodia, as expected, but there were no discernible differences between the wild-type and knockout macrophages (Fig. 1, B and D).
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To further characterize these cells as macrophages, nonspecific esterase and peroxidase activities were tested by cytochemistry. Of the adherent cells obtained after treatment of the cultures with Dispase II, >90% were positive for nonspecific esterase activity, whereas 100% were negative for peroxidase staining, confirming that the cells present in the cultures were almost exclusively mature macrophages (37) (data not shown).
Actin Staining
Actin was visualized by staining the cells with rhodamine-phalloidin and observing them by fluorescence microscopy. The cells were cultured in the presence of 1 µg/ml LPS or in the absence of LPS for 24 h and then stimulated with either 100 nM PMA in 0.01% DMSO or 0.01% DMSO for 30 min. Under basal conditions (in the absence of LPS), wild-type macrophages exhibited spikelike structures (microspikes) extending from the periphery of the cells and rhodamine-phalloidin-stained actin was evenly distributed throughout the cytosol (Fig. 2A). PMA treatment (30 min in the absence of LPS) did not affect the microspike structures, but rhodamine-phalloidin staining showed that actin began to accumulate in the perinuclear area (Fig. 2B). After LPS stimulation, the microspike structures had disappeared and rhodamine-phalloidin-stained actin showed a slightly different distribution, with a higher accumulation in the periphery of the cell and a radial distribution, probably corresponding to ruffles on the cell surface (Fig. 2C). LPS plus PMA treatment induced an almost complete disappearance of both the microspike structures and the actin in the perinuclear area, producing the same radial distribution of actin in the cytosol and increased staining at the edges of the cells (Fig. 2D). When similar studies were performed in parallel with MARCKS-deficient macrophages, no morphological differences between the two sets of cells were observed. The cells derived from the knockout animals exhibited the same basal distribution of rhodamine-phalloidin and the same morphological responses after PMA and/or LPS stimulation (Fig. 2, E-H).
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Phagocytosis
The phagocytic activities of both wild-type and MARCKS knockout macrophages through the FcR were studied, with IgG-coated SRBC as a target. Ingested IgG-coated SRBC were counted, and the PI was calculated as the number of IgG-coated SRBC ingested per cell multiplied by the percentage of phagocytic cells. More than 90% of the cells were phagocytic in each sample studied. Wild-type macrophages showed a PI of 722 ± 40 (mean ± SD; n = 13), whereas the PI for knockout macrophages was 712 ± 67 (n = 16) (Fig. 3, A and B). These values agree with those previously reported by Falk and Vogel (11) for bone marrow-derived macrophages. A Student's t-test comparison of these means revealed no significant difference between them.
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FcR-mediated phagocytosis was also evaluated by using
51Cr-labeled SRBC as the
substrate. Macrophages derived from four wild-type and four knockout
animals were analyzed in the presence of increasing concentrations of
PMA. As shown in Fig. 4, there were
decreases in the phagocytic activities of both the wild-type and MARCKS knockout macrophages with increasing concentrations of PMA. In both
cases, the profiles of inhibition overlapped, indicating that there
were no significant differences in the phagocytic activities of the
MARCKS knockout and wild-type macrophages under these conditions.
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The high levels of phagocytosis observed in these cells and the fact
that TKL cell conditioned medium (TKL-CM) can contain a certain amount
of interferon-, which could induce an increased basal level of
phagocytosis (52) prompted us to examine whether cells cultured in a
more defined medium supplemented with recombinant M-CSF would have a
lower rate of basal phagocytosis. These experiments indicated that the
basal level of phagocytosis was not affected by culturing the cells in
the more defined medium (data not shown). However, as a precautionary
measure, cells were maintained in TKL-CM-free medium for 24 h before
all subsequent experiments.
Because IgG-coated SRBC are phagocytosed only through the FcR, we also studied, in selected samples, phagocytosis mediated through various additional receptors, including C3bR and the mannose receptor. We also compared the nonspecific phagocytic activities of the wild-type and MARCKS knockout macrophages by using latex beads.
When C3bR-mediated phagocytosis in wild-type macrophages was studied, >95% of the cells were found to be phagocytic, with an average of >10 yeast particles ingested per cell (Fig. 3C). The large number of particles ingested made it impossible to precisely quantify the uptake by microscopic examination of the cells. Therefore, PI was not calculated in these experiments. The MARCKS knockout macrophages also showed a high level of phagocytic activity, with an average of >10 particles per cell (Fig. 3D). Thus the lack of MARCKS had no discernible effect on the level of C3bR-mediated phagocytosis in these macrophages.
Unopsonized latex beads were used to assess nonspecific phagocytosis. As was the case with C3bR-mediated phagocytosis, it was impossible to quantify the exact number of particles inside each cell because the number of latex beads ingested by the wild-type macrophages was so high (Fig. 3E). The phagocytic activity of the knockout macrophages on latex beads was not discernibly different from that observed in the wild-type cells (Fig. 3F).
Phagocytosis of unopsonized zymosan is mediated either through the
mannose receptor or through other unknown receptors. Quantitation of
the phagocytosis of zymosan particles by wild-type macrophages over
time resulted in an almost linear curve for 60 min (Fig. 5A),
after which no further increase was noted. A similar pattern was
observed for the MARCKS knockout cells, which also had a linear response over time; however, these cells exhibited decreased
phagocytosis at 45 and 60 min (P = 0.0008 and 0.003, respectively, by Student's t-test; 17.6 and 12.7% decreases,
respectively). A repeat of this assay with larger numbers of cells at
45 and 120 min confirmed a modest decrease (8.5%;
P = 0.001 compared with wild-type
cells) in the average number of particles accumulated by the
MARCKS-deficient macrophages at 45 min, which nonetheless reached
nearly normal values of maximal accumulation after 120 min (Fig.
5B; decrease of 3.4%;
P = 0.046).
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FPE
Because FPE is strongly stimulated by phorbol ester activation of PKC in macrophages (32) and because this stimulation is dependent on actin polymerization (30, 32, 58), it seemed possible that MARCKS might have a role in regulating this response. To determine whether FPE was affected in the MARCKS knockout macrophages, both wild-type and knockout cells were incubated in medium containing fluorescent dextran, and then the relative increase in intracellular fluorescence was measured by FACS analysis of the cells. In our initial experiments, we observed that treatment of macrophages with 32-320 nM PMA stimulated FPE over basal levels. Interestingly, high doses of the carrier (0.01% DMSO) also modestly stimulated FPE (234 ± 3 vs. 188 ± 29 relative fluorescence units for treated and untreated cells, respectively). We chose to use 100 nM PMA in our subsequent experiments. Fetal macrophages were incubated with 1 mg/ml FD for the lengths of time indicated on Fig. 6 at 37 or 4°C, harvested, and analyzed by flow cytometry. There were marked increases in relative fluorescence over time under basal conditions, which were essentially identical in all macrophages regardless of genotype (Fig. 6). In contrast, PMA stimulated the uptake of FD in the macrophages, but there were no significant differences among the mean values for the Macs wild-type and knockout genotypes (Fig. 6). The large SEs shown in Fig. 6 resulted from variability in the fluorescence values obtained in experiments performed on different days. When individual experiments were plotted separately, there remained no difference between the two genotypes, but the SEs were much smaller.
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We also performed FPE assays on MEF derived from day
14 mouse embryos that were +/+, +/, or
/
for Macs. MEF from
several embryos of each genotype were used. Control experiments
established that FD uptake exhibited the expected dependence on time,
temperature, and concentration in these cells and that the return of
either internalized FD or horseradish peroxidase (HRP) to the medium was unaffected by PMA treatment (data not shown). The mean basal rates
of FD uptake in MEF cells under control conditions were essentially
identical for cells of all three genotypes (Fig.
7). In addition, PMA treatment inhibited FD
uptake in all three groups of cells to the same extent, i.e., to 58%
of control for the cells derived from the +/+ animals, to 58% of
control for the +/
cells, and to 55% of control for the
/
cells (Fig. 7). This extent of inhibition by PMA was
approximately the same as that seen in other types of fibroblasts (data
not shown). There were no apparent differences in actin staining of MEF
from normal and knockout animals under control and PMA-treated
conditions (not shown).
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DISCUSSION |
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Phagocytosis (15) and phorbol ester-stimulated FPE (32) represent two
macrophage functions that are dependent on actin polymerization. The
polymerization of actin can be observed in macrophage pseudopodia as
they surround a particle or an aliquot of medium before engulfment.
Actin polymerization is also notable in the ruffles that fuse to form
macropinocytic vesicles. Both of these processes are inhibitable with
cytochalasins, fungal metabolites that inhibit actin polymerization.
Because MARCKS is highly expressed in macrophages (2), is induced in
macrophages and related cell types by "priming" stimuli (2, 17,
48), binds actin in a
Ca2+/calmodulin- and
phosphorylation-dependent manner (18), and has been localized to sites
where actin filaments associate with the cytoskeletal face of the
plasma membrane (36), it has been proposed that MARCKS may play a role
in regulating the observed actin cytoskeletal remodeling in macrophages
(28). In addition, several recent papers have implicated MARCKS or its
homologue MLP (also called F52, MacMARCKS, or MRP) in the
receptor-mediated phagocytosis of unopsonized zymosan, uptake of which
is mediated through either mannose receptors, -glucan receptors, or
other uncharacterized receptors expressed by macrophages. For example, Zhu et al. (57) showed that a mutant form of MLP lacking the polybasic
phosphorylation site domain, when expressed in a macrophage-like cell
line, completely abolished the phagocytosis of unopsonized zymosan
particles, whereas the phagocytosis of low-density lipoprotein remained
normal. In a second study, they also demonstrated that dominant-negative MLP expression abolished macrophage binding to
complement iC3b-coated SRBC (26). In addition, Allen and Aderem (3)
showed that zymosan induced PKC-dependent phosphorylation of MARCKS
in LPS-primed macrophages, and that PKC
and MARCKS colocalized in
the phagosome membrane during phagocytosis. These results prompted us
to study the putative role of MARCKS in phagocytic processes, taking
advantage of macrophages from MARCKS-deficient mice previously
generated in this laboratory.
Because MARCKS knockout mice survive only a few hours after birth (42), it is impossible to obtain macrophages from adult mice by traditional techniques. A method previously used in our laboratory to generate fetal liver-derived macrophages (8) allowed us to obtain large numbers of cells. The morphology of these cells was indistinguishable from that of macrophages from adult mice. Furthermore, the morphology of the macrophages derived from the fetal livers of MARCKS-deficient animals was essentially identical to that exhibited by wild-type macrophages processed in parallel. The proliferative capacities of the wild-type and MARCKS-deficient precursors isolated from fetal liver were also equivalent, with similar cell recoveries after 2 wk in culture (data not shown). By cytochemical analysis, both the wild-type and knockout macrophages achieved similar degrees of differentiation, showing positivity for nonspecific esterase activity and lacking peroxidase activity.
The actin cytoskeleton of macrophages is significantly perturbed on treatment with either PMA or LPS (32, 33, 39). Because MARCKS exhibits actin cross-linking and bundling activities that are modulated by PKC-dependent phosphorylation and Ca2+/calmodulin (18), we also asked whether MARCKS deficiency would result in changes in the actin distribution within the macrophages, either under basal conditions or after stimulation with phorbol esters to activate PKC. Our results showed that, in the MARCKS-deficient macrophages, the actin distribution in the cell was the same as that in the wild-type macrophages and that the patterns of redistribution of actin observed after PMA and/or LPS stimulation were also the same. These results suggest that the MARCKS interaction with actin is not regulating its distribution in these cells under our experimental conditions. Nonetheless, it is possible that in the MARCKS-deficient cells, MLP is compensating for its absence; this point eventually may be clarified with macrophages from mice deficient in both MARCKS and MLP.
Macrophages are actively endocytic cells, capable of internalizing both particles and fluids from the extracellular environment with great avidity. Phagocytosis is stimulated through a number of different receptors on the surfaces of these cells, and engulfment of particulate matter is thought to proceed by a zippering mechanism (45). A thin sheet of plasma membrane from the phagocyte is pulled over the surface of the particle as successive binding sites on the particle surface are engaged by the receptors on the phagocyte membrane. This process is mediated through a variety of receptors, including those that recognize complement protein C3b, mannose residues in the particle, or immunoglobulins that have attached particle antigens. In addition, macrophages exhibit a high level of FPE or macropinocytosis, a process that is stimulated by phorbol esters and other activators of membrane ruffling (32, 33, 45). Both phagocytosis and FPE are inhibited by cytochalasins, indicating that actin turnover is involved in these processes (30, 32, 58). Thus FPE and phagocytosis are two actin-dependent processes in which MARCKS could play a role.
Receptor-mediated phagocytosis is induced from several types of receptors present on the surfaces of macrophages, including FcR, which interacts with immunoglobulin, C3bR, and the mannose receptor. The signaling pathways from these receptors to the phagocytic machinery are not clear at present. However, roles for tyrosine phosphorylation (13, 14) and PKC activation (16, 56) in FcR-mediated phagocytosis have been suggested. Study of the phagocytic activity of MARCKS-deficient macrophages via FcR showed that the capacity of these cells to ingest IgG-coated SRBC was equivalent to that of wild-type macrophages. Also, the effects of PMA on FcR-mediated phagocytosis were the same for cells from both genotypes. It has been reported that PMA inhibits FcR-mediated phagocytosis (10). We evaluated the effect of increasing concentrations of PMA on FcR-mediated phagocytosis to determine whether the absence of MARCKS resulted in a different response. However, the inhibition profiles for both wild-type and knockout macrophages were the same, suggesting that even if PKC activation is implicated in the pathways that lead to the inhibition of FcR-mediated phagocytosis, the absence of MARCKS does not affect this process, either in the basal state or after exposure to PMA. In addition, similar findings were obtained when phagocytosis mediated by complement fraction 3b was assayed with C3b-coated yeast particles and nonspecific phagocytosis was measured with unopsonized latex beads.
FPE is another actin-dependent process that is stimulated in
macrophages after treatment with specific cytokines or PMA (33, 44). In
contrast to phagocytosis, in which actin polymerization is localized to
the areas of the membrane forming the phagocytic cup, in FPE the actin
polymerization occurs at the leading edges of ruffles that are formed
across the surface of the macrophage, suggesting that a more diffuse
mediator of the polymerization signal may be at work. We found that the
basal levels of FPE in the wild-type and MARCKS-deficient macrophages
were essentially identical. PMA stimulation of the cells increased the
rate of internalization of the fluorescent dextran marker as expected from previous studies (22, 31, 32, 35). However, there were no
discernible differences in the rates of PMA-stimulated FPE among the
genotypes studied. In contrast to what was found for the macrophages,
PMA inhibited FPE in MEF derived from these fetal mice. However, as for
the macrophages, no differences among the
Macs +/+, +/, and
/
genotypes could be appreciated, either before or after
PMA treatment.
We ascribed particular importance to the phagocytosis of zymosan, given previous reports that a dominant-negative mutant form of MLP could completely abolish the phagocytosis of zymosan in macrophage cell lines (57). On the other hand, when macrophages were derived from mice deficient in MLP, there was no effect on their phagocytic activity against zymosan (9, 49, 54). The same group also reported no effect on zymosan phagocytosis of a dominant-negative mutant form of MLP expressed in macrophage cell lines (49). In our hands, despite exhibiting normal phagocytosis mediated by several different receptors, macrophages derived from mice deficient in MARCKS took up slightly less zymosan than control cells at early time points (45 and 60 min), differences that were nonetheless reproducible and statistically significant. These results support a role for MARCKS in phagocytosis, at least of zymosan. The differences observed at 45 and 60 min disappeared after longer incubation times (120 min), by which time the total number of particles ingested had reached a plateau. Thus the only significant differences observed were in the rate of particle uptake rather than in the total number of particles ingested. It may be possible to magnify these differences by using cells derived from transgenically rescued MARCKS-deficient mice in which a postulated dominant-negative transgene was used (23). However, it may be necessary to use cells derived from double knockouts of MARCKS and MLP to demonstrate more clearly a role for this family of proteins in phagocytosis.
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ACKNOWLEDGEMENTS |
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We are grateful to Mary Misoukonis from the Durham VA Medical Center for her help with the cytochemistry techniques and to J. Michael Cook and Alan Fisher from the Duke Comprehensive Cancer Center Flow Cytometry Facility for performing the FACS analyses. We also thank Drs. J. Bonner and D. Germolec for helpful comments on the manuscript.
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FOOTNOTES |
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Present address of R. T. Sperling: Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. J. Blackshear, A2-05 National Institute of Environmental Health Sciences, 111 Alexander Dr., Research Triangle Park, NC 27709 (E-mail: black009{at}niehs.nih.gov).
Received 7 May 1998; accepted in final form 2 April 1999.
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