©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
MacMARCKS Mutation Blocks Macrophage Phagocytosis of Zymosan (*)

(Received for publication, May 11, 1995; and in revised form, June 7, 1995)

Zixin Zhu Zhihua Bao Jianxun Li (§)

From the Department of Microbiology and Immunology, College of Medicine, The University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A major protein kinase C substrate, MacMARCKS (F52, MPR), was examined for its role in phagocytosis. In macrophage-phagocytosing zymosan particles, MacMARCKS was concentrated around nascent phagosomes as detected by immunofluorescent microscopy. The effector domain of MacMARCKS contains the phosphorylation sites, a calmodulin binding site, as well as a putative actin binding site. Stable J774 macrophage cell lines constitutively expressing effector domain deletion mutants of MacMARCKS were generated. When given zymosan particles, these transfectants showed approximately a 90% reduction in their phagocytic capacity. The receptor-mediated endocytosis of acetylated low density lipoproteins, however, was not affected by the mutant. These results strongly suggest the involvement of MacMARCKS in macrophage phagocytosis.


INTRODUCTION

Macrophage phagocytosis requires efficient signal transduction from the phagocytic receptors to the actin-based cytoskeleton. Such signals induce a massive membrane-cytoskeleton rearrangement(1) , providing the motile force to drive the internalization of particles. This process is accompanied by the formation of pseudopodia (3, 4) or membrane ruffles(5) , and F-actin is essential for the internalization of ligand-coated particles(4, 6, 7) . However, the signals that induce actin-cytoskeleton rearrangements and the mechanism of such a rearrangement are not yet identified. A number of cytoskeleton-associated proteins, such as talin (8) and paxillin(9) , have been implicated in this process. Tyrosine phosphorylation is part of the signal transduction pathway(s)(10) .

Another major signal transduction pathway involved in macrophage phagocytosis requires protein kinase C (PKC).()Activation of PKC with phorbol esters profoundly enhances phagocytosis mediated both by C3 receptors (11) and Fc receptors(12, 13) . However, little is known about the events after PKC activation, nor is it known how the PKC signal is transduced to the cytoskeleton to initiate the internalization of particles. One approach to these questions is to investigate the role of PKC substrates in these processes.

Among the PKC substrates, the MARCKS (myristoylated alanine-rich C kinase substrate) family has received much attention. MARCKS is a membrane-associated, actin-binding protein with ubiquitous distribution (reviewed in (14) and (15) ). Its membrane association and actin binding activities are regulated both by Ca/calmodulin and by PKC-mediated phosphorylation(16, 17) . As an actin-binding protein, it is involved in a number of actin-cytoskeleton-related cellular activities, such as motility (18) and secretion(19) .

Besides MARCKS, macrophages also express another member of the MARCKS family of PKC substrate, MacMARCKS/F52/MPR(20, 21, 22) . The synthesis and phosphorylation of MacMARCKS in macrophages are dramatically increased when macrophages encounter bacteria(20) , a finding that suggests the potential role of this protein in macrophage defense against infection. A comparison of primary structure shows similar domain organization between MacMARCKS and MARCKS. They both contain a myristoylated membrane targeting domain that is important for the membrane association of these proteins(20, 23, 24) . These two proteins also share an almost identical basic domain that contains PKC phosphorylation sites and calmodulin and actin binding sites(20, 21, 25) . This basic domain is therefore called the effector domain. Invitro experiments have shown that MacMARCKS binds calmodulin in a phosphorylation-dependent manner(20, 21) . Such regulated calmodulin binding activity has been demonstrated with a similar protein, neuromodulin(26) . However, whether MacMARCKS interacts with actin is yet to be determined.

The in vivo function of MacMARCKS is the focus of the current study. We report that MacMARCKS protein is highly enriched on phagosomes containing zymosan particles. We further generated an effector domain deletion mutant of MacMARCKS (ED). Expression of this mutant showed a dominant negative effect and dramatically reduced the phagocytic activity of J774 macrophage cells toward zymosan particles.


EXPERIMENTAL PROCEDURES

Materials

The J774.a1 macrophage cell line was obtained from ATCC and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Zymosan A (Catalogue No. Z4250) and fluorescein-conjugated phalloidin (Catalogue No. P5282) were purchased from Sigma. Fluorescein-conjugated zymosan particles (Catalogue No. Z2841), Dil-conjugated low density lipoprotein (LDL) (Catalogue No. L3484), and Texas Red-conjugated goat anti-rabbit IgG (Catalogue No. T2767) were purchased from Molecular Probes. Rabbit anti-MacMARCKS antibody was originally generated in Dr. A. Aderem's laboratory (Rockefeller University). Anti-Fc (2.4G2) and anti-CD11b (M1/70) were kindly provided by Dr. R. Steinman (Rockefeller University).

Expression of Mutant MacMARCKS in J774 Macrophage Cell Line

To obtain a cDNA construct encoding the effector domain-deleted MacMARCKS, two fragments flanking this domain were generated using polymerase chain reaction. One fragment encoded amino acids 1-85, and another encoded amino acids 109-200 of MacMARCKS. The two fragments were then ligated through the MluI sites. The mutant DNA was then inserted into a eukaryotic expression vector (p463) (27) that was kindly provided by Dr. M. Nussenzweig (Rockefeller University). The final DNA construct was then electroporated into J774 cells as described(28) . The transfected cells were subjected to G418 (800 µg/ml) selection 48 h later. After selection, positive clones were randomly picked, and the expression was examined by two-dimensional isoelectrofocusing and SDS-polyacrylamide gel electrophoresis (IEF-SDS-PAGE)(29, 30) . Then the proteins were transferred to PVDF membrane (Millipore Catalogue No. IPVH304F0) followed by immunoblotting with anti-MacMARCKS antibodies. The anti-MacMARCKS antiserum recognizes both mutant protein and the wild type protein with the same efficiency (data not shown). For simplification, ED is used to refer the effector domain deletion mutant of MacMARCKS.

As a control, cDNA encoding full-length MacMARCKS was also inserted into vector p463 and transfected into J774 cells using the above method. Again, for simplification, FM is used to refer to the full length MacMARCKS.

Uptake of Zymosan Particles and Immunofluorescence

Parental J774 cells, FM control cells, and ED mutant cells were cultured on coverslips overnight. Zymosan particles were added to the culture at a final concentration of 25 µg/ml. The cells were allowed to bind zymosan particles for 45 min at 12 °C, and ingestion was allowed to proceed for 15 min at 37 °C. After fixation with 10% formalin in phosphate-buffered saline (PBS) for 15 min at 4 °C, the cells were permeabilized with acetone at -20 °C for 5 min. MacMARCKS was visualized with affinity-purified rabbit anti-MacMARCKS antibody followed by Texas Red-conjugated goat anti-rabbit IgG as described(31) . To visualize F-actin at the same time, fluorescein-conjugated phalloidin was added to cells at a concentration of 10 ng/µl together with secondary antibody.

To assay the attachment index of macrophages, the cells were incubated with 25-µg zymosan particles at 12 °C for 45 min. After three washes in PBS, the cells were fixed, and attached zymosan particles were counted. To assay the phagocytic activity of macrophages, the cells were given zymosan particles for 1 h at 37 °C to achieve maximum uptake (32) and to verify that ED mutant cells did not phagocytose more slowly than the FM control cells and parental J774 cells. After fixation, the internalized zymosan particles were counted in samples from three parallel experiments. At least 300 cells were counted in each sample. The internalized zymosan particles can be distinguished easily by their refractile appearance in phagocytic vacuoles(33) .

Flow Cytometry Analysis of Phagocytosis

This assay was adopted from Liao et al.(34) . J774 cells cultured in 60-mm dishes were allowed to ingest fluorescein-conjugated zymosan particles for 1 h at 37 °C. Most of the excess zymosan particles was removed by three washes in PBS. The cells were then treated with trypsin/EDTA for 1.5 h at 37 °C to free any bound zymosan from the cell surface and cells from the plate. After fixation with 10% formalin, the cells were analyzed using flow cytometry at 488 nm. Free zymosan particles were distinguished from cells containing zymosan by their different light scattering behavior.

Assay of the Endocytosis of AcLDL

This assay was adopted from Suzuki et al.(35) . J774 cells in 60-mm dishes were starved in serum-free Dulbecco's modified Eagle's medium for 30 min before adding acetylated LDL (AcLDL). Dil-conjugated AcLDL was then added to the culture to a final concentration of 10 µg/ml. The cells were cultured for an additional 30 min before being washed in PBS twice and fixed in 10% formalin. The uptakes of Dil-AcLDL were determined by flow cytometry.


RESULTS

MacMARCKS Associates with Phagosomes in J774 Macrophage Cells

We first examined the subcellular localization of MacMARCKS during uptake of zymosan particles. The J774 cell line was used in this study for two reasons. First, J774 cells express endogenous MacMARCKS. Second, they have been used in previous studies of the phagocytosis of zymosan(33) . Unopsonized zymosan particles are phagocytosed by macrophages through multiple receptors such as complement and mannose receptors, as well as through potential unidentified receptors(33, 36) . Thus, these particles are particularly suitable for simultaneously monitoring phagocytosis via multiple receptors.

To visualize MacMARCKS protein, polyclonal rabbit anti-MacMARCKS antibodies were affinity-purified as described(31) , and their specificity was demonstrated by three criteria. First, the antibodies immunoprecipitated a single smear band from P-labeled total J774 cell lysate (Fig. 1A). Second, they recognized a single protein (doublet) on a Western blot of total J774 cell lysate resolved on SDS-PAGE (Fig. 1A). MacMARCKS has been shown to appear as a smear band or doublet on SDS-PAGE(20, 37) . Third, the affinity-purified antibodies did not stain free zymosan particles outside the cells (Fig. 1B).


Figure 1: A, J774 cells were labeled with P, and MacMARCKS was specifically immunoprecipitated (IP) with affinity-purified rabbit polyclonal anti-MacMARCKS antibodies as described. The affinity-purified antibodies also specifically recognized MacMARCKS (doublet) on immunoblot (Blot) of total cell lysate resolved by SDS-PAGE. B, unopsonized zymosan was added to J774 cells on a coverslip at 12 °C for 45 min. The cells were moved to 37 °C to start phagocytosis. After 15 min, the cells were fixed and stained for both MacMARCKS and actin as described under ``Experimental Procedures.'' The arrowheads indicate the phagosomes that were decorated with MacMARCKS and actin. The arrows indicate mature phagosomes that lacked staining.



J774 cells on coverslips were allowed to phagocytose unopsonized zymosan for 15 min. Using affinity-purified antibodies, MacMARCKS was seen concentrated beneath the phagosomes at the periphery of the cells (Fig. 1B, MacMARCKS, arrowheads). Using fluorescein-conjugated phalloidin, F-actin was also seen concentrated beneath these same phagosomes (Fig. 1B, Actin, arrowhead). The phagosomal association of MacMARCKS was selective. A number of perinuclear-distributed mature phagosomes were seen in these cells (Fig. 1B, arrows). However, MacMARCKS and actin were not concentrated around those phagosomes. F-actin has been shown to associate only with nascent phagosomes(8) , and our results thus suggest that MacMARCKS-decorated phagosomes are also nascent phagosomes. This phagosomal association of MacMARCKS prompted us to further examine whether it is involved in macrophage phagocytosis.

Expression of MacMARCKS Mutant Proteins in J774 Macrophage Cells

The effector domain of MacMARCKS is a basic domain that contains the PKC phosphorylation sites(20) , calmodulin binding site (25) , and a putative actin binding site(17) . Thus, deleting this domain is likely to generate a nonfunctional MacMARCKS that might serve as a dominant negative mutant in vivo to compete with endogenous wild type MacMARCKS.

A cDNA-encoding effector domain deletion mutant of MacMARCKS (ED) (Fig. 2A) was generated and introduced into J774 cells as described under ``Experimental Procedures.'' J774 cells expressing the ED mutant were selected by subjecting the cells to selection with 800 µg/ml G418. Three stable ED cell lines were randomly picked and expended as ED1, ED2, and ED3. Two-dimensional IEF-SDS-PAGE was used to separate ED mutant (pI 3.84) from endogenous MacMARCKS (pI 4.37), and the expression level of these proteins was determined using immunoblot with anti-MacMARCKS antibodies. The ratios of endogenous MacMARCKS to ED mutant were 1:1.06 for ED1, 1:0.81 for ED2, and 1:0.96 for ED3 (Fig. 2C).


Figure 2: A, a diagram illustrating the full-length MacMARCKS (FM) and effector domain deletion mutant of MacMARCKS (ED). B, lysates from FM control cells and parental J774 cells were resolved by SDS-PAGE and transferred to PVDF membrane. Expression level of MacMARCKS protein was determined by immunoblotting with rabbit anti-MacMARCKS antiserum. C, lysate from ED mutant cells was subjected to two-dimensional IEF-SDS-PAGE and transferred to PVDF membrane. Endogenous MacMARCKS (Endo) and ED mutant proteins were detected by immunoblotting with anti-MacMARCKS antiserum.



As a control, cDNA-encoding full-length MacMARCKS (FM) was also transfected into J774 cells. Because FM is identical with the endogenous MacMARCKS in J774 cells, we could not determine their expression separately. However, the total amount of MacMARCKS in FM control cells was about twice as much as in parental J774 cells (Fig. 2B), suggesting that FM was expressed to the same level as endogenous MacMARCKS.

J774 Cells Expressing the ED Mutant of MacMARCKS Are Impaired in the Phagocytosis of Zymosan Particles

In the resting state, ED mutant cells, FM control cells, and their parental J774 cells appeared round and refractile, with no obvious difference in their appearance (Fig. 3, upper panel). Once the zymosan particles were added, FM control cells and parental J774 cells quickly spread out and started to phagocytose. However, little phagocytosis occurred in ED mutant cells, despite the apparent attachment of zymosan particles to the surface of these cells (Fig. 3, lower panel). During the 1-h incubation with zymosan, the ED mutant cells remained round and refractile and therefore appeared smaller than those control cells that spread out and phagocytosed zymosan particles (Fig. 3).


Figure 3: Cells on coverslips were incubated with unopsonized zymosan at 12 °C for 45 min followed by 1 h at 37 °C. The samples were then fixed and photographed as shown (lower panel). The upper panel shows the cells before they were given zymosan. The binding activities were measured by incubating cells with zymosan at 12 °C for 45 min without proceeding to 37 °C (images not shown). The attached and ingested particles were counted in samples from three parallel experiments (n = 3). At least 300 cells were counted in each sample. The phagocytic/attachment indexes (number of ingested/attached zymosan particles per 100 cells) are shown under corresponding images.



The phagocytic index (number of ingested particles per 100 cells) was obtained by counting the ingested particles(33) . The ingested zymosan particles appear refractile, while those outside the cells are dark and can be counted easily. We observed that parental J774 cells phagocytosed unopsonized zymosan with a phagocytic index of 325 ± 58 (n = 3) (Fig. 3). A similar phagocytic index of unopsonized zymosan by J774 cells had been reported earlier(33) . However, the ED mutant cells showed a 10 reduction in phagocytic capacity toward unopsonized zymosan. The phagocytic indexes were 16 ± 8 (n = 3) for ED1, 23 ± 5 (n = 3) for ED2, and 19 ± 9 (n = 3) for ED3 (Fig. 3), whereas FM control cells showed approximately same phagocytic index of 349 ±48 (n = 3) as parental J774 cells (Fig. 3). This observation suggests that the reduction of phagocytosis indeed resulted from the expression of ED mutant, not from the integration of transfected foreign DNA.

To minimize the possible miscounting due to human error, a modified flow cytometry method described by Liao et al.(34) was used to measure the phagocytosis of zymosan particles. In brief, fluorescein-conjugated zymosan particles were given to ED mutant cells, FM control cells, and parental J774 cells. After 1 h, cell surface-bound zymosan particles were removed by extensive trypsin digestion, and cell-associated fluorescence was measured using flow cytometry. Microscopy showed that the cells appeared as a dark shadow around bright fluorescent zymosan particles (Fig. 4, upper panel). Most FM control cells and parental J774 cells contained more than one fluorescent zymosan particle, whereas most of the ED mutant cells remained dark (no zymosan inside) or contained only one particle. Analyzed with flow cytometer, parental J774 cells and FM cells showed fluorescence with relative average intensities of 29.9 and 37.1, respectively (Fig. 4, lower panel). With ED mutant cells, only a small percentage of cells showed fluorescence with relative average intensities of only 1.58 for ED1, 1.15 for ED2, and 1.32 for ED3.


Figure 4: Cells were allowed to ingest fluorescein-conjugated unopsonized zymosan for 1 h. The bound zymosan particles were released from the cell surface by extensive trypsin digestion. A fraction of the cells was examined under a microscope (upper panel). With both visible and fluorescent light sources on, the cells appeared as a dark shadow and zymosan as a bright spot (as the circle and arrow indicate). The lower panel shows the results of the flow cytometry analysis of these samples.



Both ED mutant cells and FM control cells showed similar binding capacity toward zymosan particles (Fig. 3) as their parental J774 cells. In addition, the surface expression of Fc and complement receptor (CD11b) were similar to parental J774 cells as well (data not shown). Therefore, we conclude that the ED mutant blocked phagocytosis at the internalization stage.

Receptor-mediated Endocytosis of AcLDL Is Not Affected by Mutant MacMARCKS

We examined whether ED mutant affected general membrane trafficking as measured by receptor-mediated endocytosis. The uptake of AcLDL is mediated both by the LDL receptor and macrophage scavenger receptor (38) through receptor-mediated phagocytosis. It was therefore used to measure the effect of MacMARCKS mutant on endocytosis. After endocytosing Texas Red-conjugated AcLDL for 30 min, the cells were washed, fixed, and subjected to flow cytometry analysis as described(35) . All ED mutant cells showed similar amounts of AcLDL uptake compared to FM control cells and parental J774 cells (Fig. 5). This similarity in the uptake by all the cell lines was confirmed by fluorescence microscopy (data not shown). Thus, the defect in phagocytosis caused by MacMARCKS mutant did not seem to affect receptor-mediated AcLDL uptake.


Figure 5: Cells were allowed to endocytose AcLDL for 30 min as described under ``Experimental Procedures.'' The cells were then fixed in 10% formalin and subjected to flow cytometry analysis.




DISCUSSION

The results of our study suggest that MacMARCKS plays an important role in macrophage phagocytosis. It has been demonstrated previously that the activation of PKC strongly enhances phagocytosis(11, 13) . As a major PKC substrate, MacMARCKS is a logical candidate to be examined for its potential involvement in this process.

The phagosomal association of MacMARCKS initially suggested that MacMARCKS might be involved in phagocytosis. This enrichment of MacMARCKS appears only on nascent phagosomes, whereas perinuclear-distributed mature phagolysosomes are devoid of MacMARCKS (Fig. 1B). Such a distribution of MacMARCKS very much resembles the selective enrichment of actin on nascent phagosomes(8) . Our data indeed showed that MacMARCKS and actin are colocalized on the same phagosomes (Fig. 1B). F-actin was demonstrated to be essential for macrophage phagocytosis (reviewed in (1) ). A number of other cytoskeleton-associated proteins, such as talin (8) and paxillin (9) , showed similar phagosomal association as well. Since MacMARCKS protein contains a putative actin binding domain, its phagosomal association and colocalization with actin encouraged a more detailed investigation into its potential role in phagocytosis.

A more direct suggestion of the role of MacMARCKS in phagocytosis comes from the expression of an effector domain deleted mutant of MacMARCKS (ED). The effector domain is a basic domain that contains PKC phosphorylation sites(20) , a calmodulin binding site(25) , and a putative actin binding site(17) . Deletion of this domain abolished PKC-mediated phosphorylation and calmodulin binding.()Our data showed that all three randomly selected ED mutant cell lines expressed approximately the same amount of mutant protein with a ratio to endogenous MacMARCKS of about 1:1. All three ED mutant cell lines showed approximately a 90% reduction in phagocytosis of zymosan particles compared with FM control cells. This result strongly suggests that MacMARCKS plays a role in macrophage phagocytosis and that the ED mutant acts as a ``dominant negative'' suppressor of MacMARCKS functions. In general, phagocytosis consists of two steps: binding and internalization of particles. Our data showed that there were as many bound zymosan particles on the mutant cells as on the control cells, implying a normal ligand binding of ED mutant cells (Fig. 3). Therefore, the ED mutant specifically blocks phagocytosis at the internalization step.

MARCKS protein has been shown to bind and cross-link actin filaments (17) and possibly regulates membrane-cytoskeleton interaction(14) . MacMARCKS contains an almost identical actin binding domain (within the effector domain) as MARCKS, and deletion of this domain might therefore affect actin-based cytoskeleton and thereby causes a defect in phagocytosis. That the expression of ED mutant did not affect receptor-mediated endocytosis of AcLDL seems to favor such a hypothesis, because the main difference between phagocytosis and endocytosis is their dependence on actin-based cytoskeleton. Phagocytosis depends on the actin-based cytoskeleton, whereas endocytosis does not. In addition, upon adding zymosan, ED mutant cells failed to spread out as normal J774 cells do, thus appearing smaller. Such spreading is important for macrophage phagocytosis, and the actin-based cytoskeleton is primarily responsible for such a spreading process(5) . The difference in spreading was more dramatic when Salmonella was added to these cells.Salmonella induced the formation of massive membrane ruffles in macrophages as part of its entry mechanism, and this process is actin-dependent(39) . We observed that Salmonella induced extension of lamellipodia in control cells as soon as 30 s, whereas no change was observed in ED mutant cells. Therefore, we speculate that the MacMARCKS may be involved in regulating membrane-cytoskeleton rearrangement during macrophage phagocytosis.


FOOTNOTES

*
This study was supported by American Cancer Society Grant IN-176-C and a grant from the UT Medical Group, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Microbiology & Immunology, The University of Tennessee, 858 Madison Ave., Memphis, TN 38163. Tel. 901-448-6763; Fax: 901-448-8462.

The abbreviations used are: PKC, protein kinase C; MARCKS, myristoylated alanine-rich C kinase substrate; PBS, phosphate-buffered saline; IEF-SDS-PAGE, isoelectrofocusing and SDS-polyacrylamide gel electrophoresis; LDL, low density lipoprotein; AcLDL, acetylated LDL; ED, effector domain deletion mutant of MacMARCKS; FM, full-length wild type MacMARCKS; PVDF, polyvinylidene difluoride.

J. Li, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. A. Aderem for providing us with MacMARCKS cDNA and antibody against MacMARCKS protein. We also thank Dr. M. Nussenzweig for providing expressing vector and Dr. R. Steinman for providing antibodies against Fc and CD11b. We appreciate Dr. M. Doctor and Dr. G. Majumdar for the flow cytometry measurement. We thank Dr. D. Armbruster, an author's editor, for help in editing this manuscript. Most of all, our thanks go to Dr. R. Steinman, Dr. S. D. Wright, and Dr. Y. Liu for their fruitful discussion and advice, as well as their comments on the manuscript.


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