ToxSci Advance Access originally published online on March 14, 2007
Toxicological Sciences 2007 97(2):398-406; doi:10.1093/toxsci/kfm050
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A Murine Scavenger Receptor MARCO Recognizes Polystyrene Nanoparticles
Environmental Nanotoxicology Section, Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan
1 To whom correspondence should be addressed. Fax: +81-29-850-2512. E-mail: sanae{at}nies.go.jp.
Received December 28, 2006; accepted February 27, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent toxicological studies indicate that nanoparticles or ultrafine particles (< 100 nm) are more toxic than fine particles (< 2 µm) because of their greater surface area. It is well known that alveolar macrophages play an important role in the first defense against various environmental particles and microorganisms. This is accomplished by binding to a macrophage receptor with collagenous structure (MARCO), one of several scavenger-type receptors expressed on the cell surface of macrophages. MARCO has been shown to mediate the ingestion of unopsonized environmental particles such as TiO2 and Fe2O3 (1.3 µm in diameter). However, very little is known about the cellular uptake of nanoparticles. In the present study, we investigated whether MARCO mediates the uptake of nanoparticles by using fluorescent-tagged polystyrene particles (20 nm, 200 nm, and 1 µm in diameter). COS-7 cells were transfected with either MARCO cDNA or an empty vector, and the association of the particles with the cells were observed by fluorescence microscopy and atomic force microscopy. MARCO-transfected cells associated with all three sizes of particles in a time-dependent manner, while no obvious binding of particles occurred after 5 h to the empty vector–transfected cells. The uptake of particles by MARCO-transfected cells was partially inhibited by polyG. These results suggest that macrophages associate with nanoparticles (20 nm) at least in part through MARCO and that MARCO plays a role in clearing nanoparticles which can deposit in the alveolar region.
Key Words: MARCO; nanoparticles; COS-7; atomic force microscopy; fluorescence microscopy.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Epidemiological studies show that exposure to ambient particulate matter (PM) is associated with increased pulmonary and cardiovascular morbidity and mortality (Churg and Brauer, 2000
; Pope et al., 2002; Sioutas et al., 2005). It is known that ultrafine particles (UFP), with a diameter less than 100 nm, constitute 1–8% of the mass of PM in the ambient air and considerably contribute to the health effects of PM (Chalupa et al., 2004). Recent toxicological studies indicate that UFP are more toxic than fine particles at an equal mass concentration because their greater surface area per mass render UFP more reactive to cells and biomolecules (Oberdorster et al., 2005). UFP have an ability to induce reactive oxygen species and lung injury (Nel et al., 2006). It has been shown that inhaled UFP influence lung physiology in humans (Schulz et al., 2005) and cause impairment of phagocytic activity in macrophages in vitro (Moller et al., 2002; Renwick et al., 2001, 2004). The large surface area of UFP may enhance their toxic potential by direct access to intracellular proteins and DNA within cells (Geiser et al., 2005; Vinzents et al., 2005).
UFP have a higher deposition rate in the peripheral lung compared with larger particles, and UFP are retained more efficiently in exhaustively lavaged lung (Oberdorster et al., 2005). Mills et al. (2006) have demonstrated that high levels of technetium 99m–labeled ultrafine carbon particles are retained in human lungs after inhalation. Moreover, technetium 99m–labeled ultrafine carbon particles translocated rapidly into the systemic circulation system in human subjects (Nemmar et al., 2002), and UFP appeared to penetrate the boundary membranes of lungs rapidly (Geiser et al., 2005). It is plausible that UFP or nanoparticles are small enough to migrate through lung tissue before they are recognized by alveolar macrophages (AMs). Thus, UFP may enter the circulation system and exert direct effects on the heart and blood vessels.
It is well known that macrophages play an important role in the first defense against various environmental particles and microorganisms. AMs bind and ingest unopsonized environmental particles and bacteria through scavenger receptors (Palecanda et al., 1999). Scavenger receptor class A is known to play a critical role in innate immunity and apoptotic clearance (Greaves and Gordon, 2005). Recently, a macrophage receptor with collagenous structure (MARCO) was identified as one of the scavenger receptor class A proteins expressed on the cell surface of macrophages (Elomaa et al., 1998). MARCO mediates binding and ingestion of unopsonized environmental particles such as TiO2, Fe2O3 with a median diameter of 1.3 µm, and latex beads with a median diameter of 1 µm (Palecanda et al., 1999). More recent studies revealed that MARCO is involved in silica-induced toxicity in primary AMs (Hamilton et al., 2006). However, very little is known about whether MARCO mediates cellular uptake of UFP.
The entry of particles into cells is largely dependent on particle size and surface chemistry. Large particles, usually those over 0.5 µm in diameter, are phagocytized (Ferin et al., 1991), while smaller particles are endocytosed (Conner and Schmid, 2003). There are several mechanisms for endocytosis: macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin- and caveolae-independent endocytosis. It has been reported that clathrin-mediated endocytosis has a particle upper size limit of 200 nm. Internalization of microspheres with a diameter < 200 nm involves clathrin-coated pits, whereas increasing particle size causes a shift to a mechanism that relies on caveolae-mediated internalization (Rejman et al., 2004). Geiser et al. (2005) reported that, unlike larger particles, UFP can cross any cellular membrane in the lung and in culture cells without involving either endocytosis- or actin-based mechanisms. Thus, it is of interest to know whether the uptake of UFP is mediated by MARCO.
In this study, we measured the uptake of fluorescent carboxylated–polystyrene particles with three different sizes (20 nm, 200 nm, and 1 µm) using MARCO-transfected cells and compared differences in the cellular association of these particles in these cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
TRIZOL was purchased from Gibco (Grand Island, NY). Lipopolysaccharide (LPS; phenol extraction, Escherichia coli) was purchased from Sigma (St Louis, MO). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). Cytochalasin D and nocodazole were purchased from Sigma and were dissolved in dimethyl sulfoxide (Sigma). DNase I and 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel (Tris-acetate buffer) were purchased from Invitrogen. Block Ace was purchased from Dainippon Pharm (Osaka, Japan). ED31 anti-MARCO–antibody was purchased from Serotec (Raleigh, NC). Horseradish peroxidase (HRP)–conjugated anti-rat IgG antibody was purchased from Santa Cruz Biotech. (Santa Cruz, CA). Enhanced chemiluminescence (ECL) was purchased from Amersham (Buckinghamshire, UK). A GeneAmp RNA PCR Kit was purchased from ABI (Foster City, CA). FluoSpheres carboxylate–modified microspheres red (
ex, 580 nm; em, 620 nm) and yellow-green (ex, 505 nm; em, 515 nm) in three sizes (20 nm, 200 nm, and 1 µm) were purchased from Molecular Probes (Eugene, OR). These negatively charged particles were suspended in medium and sonicated 30 s before use. Polyguanylic acid (polyG) and polycytidylic acid (polyC) were purchased from Sigma and dissolved in sterile H2O. Y-27632 and exoenzyme C3 (Clostridium botulinum) were purchased from Calbiochem (San Diego, CA) and dissolved in phosphate-buffered saline (PBS) or sterile H2O, respectively. pSG5 vector was purchased from Strategene (La Jolla, CA). A bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, IL). Wizard PureFection Plasmid DNA Purification System was purchased from Promega (Madison, WI). Bovine serum albumin (BSA) was purchased from Sigma. WST-8 assay kit was purchased from Dojindo (Kumamoto, Japan).
Cell culture.
COS-7 cells (CRL-1651, a monkey kidney cell line) were obtained from the American Type Culture Collection. J774.1 cells (RCB0434, a mouse macrophage cell line) were obtained from Riken (Tsukuba, Japan). MARCO- or empty vector–transfected COS-7 cells were cultured at 37°C in a 5% CO2 atmosphere in phenol-red free Dulbecco's modified Eagle's medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. J774.1 cells were cultured in RPMI 1640 (Invitrogen) containing 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Hanks' balanced salt solution was purchased from Invitrogen.
Plasmid constructs.
J774.1 cells were treated with 10 µg/ml LPS for 6 h to enhance expression of MARCO. Total RNA was extracted from the cells using TRIZOL. Amplification of cDNA encoding the full-length murine MARCO was performed in a DNA thermal cycler (PTC200, MJ Research, Watertown, MA) with the following primers: sense primer 5'-GTTTAGATCTATGGGAAGTAAAGAACTCCTCAAAG-3'; antisense primer 5'-GTTTAGATCTTCAGGAGCATTCCACACCCG-3' (Bgl II restriction sites underlined). The following conditions were used: initial denaturation at 94°C for 2 min and 10 cycles of denaturation at 94°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 3 min. The cDNA encoding the full-length murine MARCO was engineered into a pSG5 vector using Bgl II restriction sites. The plasmid was extracted using the Promega Wizard PureFection Plasmid DNA Purification System.
Cell transfections.
COS-7 cells were transfected transiently with the SG5-MARCO construct using Lipofectamine 2000 following the manufacturer's protocol. Western blot analysis and RT-PCR were used to confirm the expression of MARCO in MARCO vector–transfected cells; the empty vector–transfected cells were tested for MARCO expression as well.
For Western blotting, the MARCO- or empty vector–transfected cell monolayer was washed twice with PBS and lysed in the following buffer: 150mM sodium chloride, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 50mM N-(2-hydroxyethyl)piperazine-N'-(4-butanesulphonic acid), 1mM phenylmethylsulphonyl fluoride, 1mM sodium orthovanadate, 50mM sodium fluoride, 1mM p-nitrophenyl phosphate, 10 µg/ml aprotinin, 5mM benzamide, and 20nM calyculin A. The lysate was centrifuged at 10,000 x g, for 5 min at 4°C. Proteins in the supernatant were resolved by SDS-polyacrylamide gel electrophoresis (10% Tris-acetate gel) under reducing conditions and electroblotted onto a nitrocellulose membrane. The membrane was blocked using Block Ace and probed with ED31 anti-MARCO-antibody, followed by HRP-conjugated anti-rat IgG antibody. MARCO on the membrane was visualized using ECL.
For RT-PCR to examine the transfection, the MARCO- or empty vector–transfected cell monolayer was washed twice with PBS and total RNA was extracted from the cells using TRIZOL. Total RNA was treated with DNase I to digest single-and double-stranded DNA, thus protecting the RNA from contaminating DNA. mRNA levels of MARCO in the cells were analyzed by RT-PCR using a GeneAmp RNA PCR Kit. Primers for the detection of murine MARCO were 5'-AGGACCTCGAGGAGAGAAGG-3' (forward) and 5'-AATTCCTGTGTCACCCTTGC-3' (reverse). The amplification was performed in a DNA thermal cycler (PTC200, MJ Research) under the following conditions: initial denaturation at 95°C for 5 min and 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min. The PCR products were separated on 1.8% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light.
Fluorescence microscopy.
Five hours after transfection, the MARCO- or empty vector–transfected cells were plated at 4.5 x 105 cells per 35-mm dish and incubated overnight at 37°C. The cells were treated with 20-nm (6.8 x 1011 particles per dish), 200-nm (5.9 x 108 particles per dish), or 1-µm (5.4 x 106 particles per dish) fluorescent particles for periods ranging from 10 min to 18 h. After incubation, the cell monolayers were washed with fresh medium three times and then incubated in fresh medium. Association of the particles with MARCO- or empty vector–transfected cells was observed with phase contrast and fluorescence microscopy.
Viability assay.
Five hours after transfection, the MARCO- or empty vector–transfected cells were plated at 2 x 103 cells per 96-well culture dish and incubated overnight at 37°C. The cells were incubated with or without 20-nm, 200-nm, or 1-µm fluorescent particles for 18 h. The viability of MARCO- or empty vector–transfected cells was measured by WST-8 according to the manufacturer's instruction.
Atomic force microscopy.
MARCO-transfected cells were grown on glass coverslips and incubated overnight. The cells were further incubated (within 2 h) with 20-nm, 200-nm, or 1-µm fluorescent particles. The cells were washed three times with PBS and fixed in a 2.5% solution of glutalaldehyde in 0.1M phosphate buffer for 1 h at 4°C. The coverslips were rinsed with PBS and distilled water and then air dried. The images of association of the particles with the cells were analyzed by atomic force microscopy (AFM) (SPA400, SEIKO Instruments Inc., Chiba, Japan) with a 100-µm scanner under ambient conditions. The measurements were carried out in a tapping mode using an aluminum-coated silicon tip (SI-DF3, SEIKO Instruments Inc.) on a cantilever with a spring constant of 1.6 N/m. AFM images were presented by deflection mode.
Quantitative analysis of cell-associated fluorescent particles by fluorospectrometry and flow cytometry.
Five hours after transfection, the MARCO- or empty vector–ransfected cells were plated at 4.5 x 105 cells per 35-mm dish and incubated overnight. The cells were incubated with 20-nm, 200-nm, or 1-µm yellow-green fluorescent particles for periods ranging from 0 to 5 h. Following treatment with the 20- or 200-nm particles, the cells were lysed in 100 µl 0.2% Triton X-100 after washing with PBS three times. The fluorescence intensity of the lysate was measured using a spectrofluorometer (RF1500, Shimadzu, Kyoto, Japan) using an excitation wavelength of 480 nm and an emission wavelength of 512 nm. Protein concentrations were determined using a BCA protein assay kit. For 1-µm particles, after three washes with PBS the cells were detached by trypsinization. The cells were then washed once with PBS and suspended in PBS containing 0.3% BSA. The intensity of cell-associated fluorescence was analyzed by flow cytometry (FACS Calibur; BD Biosciences), with 1 x 104 cells measured in each sample.
Inhibition of ingestion.
Nocodazole and cytochalasin D are drugs that disrupt microtubules and destabilize actin filaments, respectively. PolyG, but not polyC, is a MARCO ligand. Exoenzyme C3 is a bacterial toxin that specifically inactivates a small guanosine triphosphate-binding protein, Rho, through adenosine diphosphate ribosylation of Asn41, and Y-27632 is a novel and specific inhibitor of Rho-associated kinases.
Six hours after transfection, the MARCO- or empty vector–transfected cells were plated at 4.5 x 105 cells per 35-mm dish and incubated overnight. The cells were preincubated with 10 µg/ml cytochalasin D or 5 µg/ml nocodazole for 1 h, 400 µg/ml polyG (ligand) or polyC (control) for 30 min, and 30µM Y-27632 for 1 h or 2.5 µg/ml exoenzyme C3 overnight. The cells were incubated with 200-nm yellow-green particles for 30 min and were then lysed in 100 µl 0.2% Triton X-100 after washing with PBS three times. The fluorescence intensity of the lysate was measured.
Statistical analysis.
Data are presented as means ± SE of three experiments. Statistical analyses were performed by ANOVA with Bonferroni/Dunn's post hoc analysis. The statistical significance of difference was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of MARCO in MARCO- and Empty Vector–Transfected Cells
To confirm the expression of MARCO in MARCO vector–transfected cells, total RNA was extracted from both MARCO- and empty vector–transfected cells and analyzed by RT-PCR. Figure 1A shows that MARCO-transfected cells expressed MARCO mRNA (lane 2) but the empty vector–transfected cells did not (lane 1).
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Western blot analysis showed that MARCO-transfected cells expressed MARCO proteins with molecular masses of approximately 60 and 55 kDa (Fig. 1B).
Fluorescence Microscopy
The association of 20-nm (Fig. 2A), 200-nm (Fig. 2B), or 1-µm (Fig. 2C) fluorescent particles with MARCO- or empty vector–transfected cells was visualized using fluorescent microscopy. The cell viability was not changed by those particles in either empty vector– or MARCO-transfected cells (data not shown). MARCO-transfected cells associated with all three sizes of fluorescent particles within 1 h, with association increasing over a period of 18 h. The MARCO-mediated association of particles was more prominent with 200-nm and 1-µm particles than with 20-nm particles. The association of fluorescent particles with empty vector–transfected cells was not obvious even after 3 h; however, after 18 h, a slight uptake of the 200- nm and 1-µm (but not the 20 nm) fluorescent particles was observed with empty vector–transfected cells.
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Atomic force microscopy
It is shown that carboxylate-modified polystyrene nanobeads increase slightly in size due to agglomeration in cell culture medium (Xia et al., 2006
). The 1-µm particles, but not 20- and 200-nm particles, were observed as individual spheres by fluorescence microscopy. Thus, we confirmed the cell-associated particles in MARCO-transfected cells by AFM. The deflection images demonstrated that most of 1-µm (Fig. 3A) and 200-nm (Fig. 3B) particles were associated with the cells as individual particles. On the other hand, 20-nm particles form small agglomerates (Figs. 3C and 3D).
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Quantitative Analysis of Fluorescent Particles in MARCO- and Empty Vector–Transfected Cells
The cell-associated fluorescent particles were quantified by flow cytometry and fluorescence spectroscopy. Flow cytometry was used to analyze the fluorescence intensity of the 1-µm particles, and fluorescence spectroscopy was used for the 20- and 200-nm particles (the fluorescence intensity of a single 20- or 200-nm particle is too weak to allow differentiation of particle-associated from nonparticle-associated cells). Fluorescence intensity resulting from the 20-nm, 200-nm, and 1-µm particles in MARCO-transfected cells increased in a time-dependent manner significantly (Figs. 4A, 4B, and 5C, respectively), whereas no increase in fluorescence intensity was observed after 5 h in the empty vector–transfected cells.
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Effects of Cytochalasin D and Nocodazole on Cellular-Associated Fluorescence Intensity in MARCO-Transfected Cells
We examined whether the MARCO-mediated association is inhibited by nocodazole (microtubules disruptor) or cytochalasin D (inhibitor of actin assembling). Nocodazole and cytochalasin D slightly but significantly reduced particle-MARCO association when the cells were treated with 20-nm (Fig. 6A) or 200-nm (Fig. 6B) particles for 120 or 30 min, respectively.
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Effects of PolyG, PolyC, or Rho Inhibitor on Cellular Association of the Fluorescent Particles in MARCO-Transfected Cells
We confirmed that MARCO-mediated particle uptake was inhibited by the MARCO ligand polyG significantly, but not by polyC (a control) (Fig. 7). A higher dose of polyG reduced the association of particles more efficiently (data not shown).
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To examine whether MARCO-mediated association of particles interact with Rho protein, MARCO-transfected cells were incubated with 200-nm fluorescent particles for 2 h in the presence of either Rho inhibitor, Y-27632, or exoenzyme C3. Neither exoenzyme C3 nor Y-27632 reduced the fluorescence intensity in MARCO-transfected cells (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MARCO is a scavenger receptor expressed on the cell surface of macrophages (Elomaa et al., 1998
). MARCO mRNA is known to be highly upregulated in dendritic cells and in mouse macrophage cell lines following LPS (van der Laan et al., 1999) or bacterial activation (Granucci et al., 2003). LPS is known to be a MARCO ligand (Sankala et al., 2002). To obtain MARCO mRNA, J774.1 cells were stimulated with 10 µg/ml LPS (Escherichia coli) for 6 h. MARCO was strongly upregulated 6 h after LPS treatment (data not shown). The polyribonucleotides polyG and polyI are also ligands and competitive inhibitors of the scavenger receptor, whereas polyC, polyU, and polyA are not (Pearson et al., 1993). This is because polyG and polyI form quadruplexes on whose surfaces the stereospecific distribution of phosphate groups generate a negative charge that is complementary to the lysine cluster of the collagenous domain of scavenger receptors (Pearson et al., 1993). MARCO-mediated uptake of bacteria is also inhibited by polyG, but not by polyC (van der Laan et al., 1999). In this study, cellular association of particles was inhibited by MARCO ligand polyG but not by polyC (Fig. 7), suggesting that this association is mediated by MARCO. However, polyG did not inhibit cellular association completely. Polystyrene particles and polyG may competitively associate with MARCO or there may be other receptors that recognize polystyrene particles in cells. The molecular structure of the MARCO receptor resembles that of scavenger receptor-AI, which contains a triple-helix collagenous domain and a scavenger receptor cysteine-rich domain at the C terminus (van der Laan et al., 1999). Mutagenesis studies with human MARCO have revealed that the N-terminal side of the cysteine-rich domain is important for ligand binding (Elomaa et al., 1998).
MARCO has been reported to bind unopsonized environmental particles with a median diameter of about 1 µm (Palecanda et al., 1999). However, very little is known about whether MARCO mediates the cellular uptake of UFP. According to an in vitro study, the uptake of UFP by macrophages is mediated by nonspecific ligand receptors (Peters et al., 2006). We first hypothesized that MARCO recognizes large particles (median diameter of 1 µm), whereas UFP associate passively with both MARCO- and empty vector–transfected cells through a receptor-independent pathway. Thus, we examined whether MARCO associates with particles (20 nm, 200 nm, and 1 µm) and compared differences in the number of fluorescent particles found in MARCO- and empty vector–transfected COS-7 cells.
Contrary to our hypothesis, MARCO-transfected cells associated with not only 1-µm particles, but also with 20- and 200-nm particles. The cells started to associate with all three sizes of particles within 1 h, and fluorescence intensity increased in a time-dependent manner as shown Figures 4 and 5. The MARCO-mediated uptake of all three sizes of particles occurred similarly (Figs. 2A, 2B, and 2C). On the other hand, none of the fluorescent particles bound with empty vector–transfected cells after 3 h, although there was slight uptake of the 200-nm and 1-µm particles after 18 h. It is likely that fluorescent particles can bind slowly to cells by means of MARCO-independent pathways. No morphological changes were seen between MARCO- and empty vector–transfected cells (Figs. 2A, 2B, and 2C). Some variation in the morphological effects of MARCO among MARCO-expressing Chinese hamster ovary, HeLa, NIH3T3, and 293 cells have been reported (Pikkarainen et al., 1999), with typical morphological changes including the formation of large lamellipodia-like structures and long dendritic growths. However, MARCO-expressing COS-7 cells do not change morphologically (Pikkarainen et al., 1999). It has been suggested that morphological changes are related to the strength of adhesion between cells and serum-coated surfaces (Pikkarainen et al., 1999).
Particle association with MARCO-transfected cells pretreated with cytochalasin D was analyzed by fluorospectrometry to determine whether MARCO-mediated association requires rearrangement of the actin cytoskeleton. In an in vitro study, Geiser et al. (2005) reported that cytochalasin D inhibited the uptake of 1-µm particles by macrophages, although the uptake of 7.8- or 200-nm particles was not inhibited. Phagocytosis of large particles (greater than 0.5 µm diameter) requires an active actin cytoskeleton. In the present study, MARCO-mediated association with particles was only slightly affected by cytochalasin D regardless of the particle size (Fig. 6), although cellular spreading was completely perturbed by cytochalasin D (data not shown). Nocodazole, which is known to disrupt microtubule structure, perturbed internalization of small particles (50 and 100 nm) in nonphagocytic eukaryotic cells (Rejman et al., 2004). In the present study, nocodazole slightly reduced MARCO-mediated association like cytochalasin D (Fig. 6). It is reported that cytochalasin D inhibited internalization of bacteria but did not inhibit binding to bacteria in MARCO+/+ AMs (Arredouani et al., 2004). These findings indicate that most of the MARCO-mediated cellular association with particles appears to be due to binding to the particles rather than internalization in MARCO-transfected COS-7 cells.
Cells internalize various particulate materials by endocytosis, and particle size may be important in controlling particle entry into cells (Rejman et al., 2004). We confirmed by AFM and fluorescence microscopy that 20-nm particles agglomerated when associated with the cells and those agglomerates, most of which were within nano size (< 100 nm), were taken up by the cells via MARCO. The uptake of UFP by macrophages is mediated by nonspecific ligand receptors, whereas the uptake of fine particles is mediated by a specific ligand receptor (Peters et al., 2006). In an in vitro study, small particles (78 and 200 nm) passively and rapidly entered erythrocytes, which do not possess receptors at their outer surface, whereas 1-µm particles did not enter (Geiser et al., 2005). If small particles can be taken up passively without ligand-receptor mediation, empty vector–transfected cells should also be capable of internalizing fluorescent 20-nm particles, just as MARCO-transfected cells did in this study. However, only MARCO-transfected cells internalized 20-nm particles immediately, whereas empty vector–transfected cell did not internalize particles significantly. These results indicate that the internalization mechanism for UFP is not only passive uptake, but also receptor-mediated, and MARCO is one of the receptors which recognizes and internalizes UFP.
In conclusion, we have shown that the rapid uptake of polystyrene particles, including nanoparticles, is mediated by MARCO in MARCO-transfected cells. These results suggest that the cellular association of UFP is mediated at least in part through MARCO and that MARCO plays a role in clearing deposited UFP by macrophages in the alveolar region.
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