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The Journal of Immunology, 2006, 177: 5517-5523.
Copyright © 2006 by The American Association of Immunologists, Inc.

Mannose-Binding Lectin Augments the Uptake of Lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer Cells through Increased Cell Surface Expression of Scavenger Receptor A1

Kei Ono*,{dagger}, Chiaki Nishitani*,#, Hiroaki Mitsuzawa*,#, Takeyuki Shimizu*,#, Hitomi Sano*,#, Hiroshi Suzuki§, Tatsuhiko Kodama, Nobuhiro Fujii{ddagger}, Koichi Fukase||, Koichi Hirata{dagger} and Yoshio Kuroki2,*,#

* Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan; {dagger} First Department of Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan; {ddagger} Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo, Japan; § National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan; Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; || Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan; and # Core Research for Engineering, Science, and Technology, Japan Science and Technology Agency, Kawaguchi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We investigated roles of scavenger receptor A (SR-A) and mannose-binding lectin (MBL) in the uptake of endotoxin and bacteria by Kupffer cells. When [3H]lipid A was injected into retro-orbital plexus of mice, significantly less accumulation of lipid A in the liver was observed in SR-A-deficient mice and wild-type mice coinjected with fucoidan or acetylated low-density lipoprotein, which are known ligands for SR-A. Isolated Kupffer cells were able to take up [3H]lipid A in a time-dependent manner. The amount of lipid A associated with nonadherent Kupffer cells derived from SR-A-deficient mice was reduced by ~80% when compared with wild-type cells, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. The lipid A uptake by Kupffer cells was significantly enhanced in the presence of rMBL. Coincubation of fucoidan with [3H]lipid A significantly inhibited the basal and the MBL-stimulated uptake of lipid A by Kupffer cells. Preincubation of MBL with Kupffer cells also increased the uptake of lipid A. These results indicate that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells. Consistently, the exposure of MBL to Kupffer cells increased cell surface SR-A expression. The phagocytosis of Staphylococcus aureus and Escherichia coli by Kupffer cells was also enhanced by preincubation of MBL with the cells. In addition, MBL bound to lipid A, LPS, and S. aureus, and precipitated S. aureus. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lipopolysaccharide is referred to as endotoxin. It is abundantly found on the outer membrane of all Gram-negative bacteria, and it possesses the ability to elicit inflammatory responses. LPS consists of O-Ag, core oligosaccharides, and lipid A, an active moiety of LPS (1). The existence of nanograms of LPS or lipid A in the bloodstream causes endotoxin shock in humans (2, 3). Endotoxin in the bloodstream is normally derived from the gastrointestinal tract and from sites of infection (4). The liver rapidly clears circulating endotoxin through scavenger receptor (5). Both Kupffer cells and sinusoidal endothelial cells in the liver exhibit high activity of scavenger receptor A (SR-A)3 (6, 7). These hepatic cells could be a critical barrier to prevent endotoxin and other bacterial products from disseminating in the circulation.

Mannose-binding lectin (MBL) is synthesized by the liver and appears in the circulation (8). MBL belongs to the collectin subgroup of the C-type lectin superfamily along with surfactant proteins A and D and conglutinin (9, 10). The collectins possess characteristic structures consisting of the N-terminal region containing intermolecular disulfide bonding, a collagen-like region, a coiled-coil motif neck, and a carbohydrate recognition domain. These lectins prefer binding to mannose, glucose, and N-acetylglucosamine and are now believed to be important components of the innate immune system. MBL directly interacts with sugars on microbial surfaces and induces complement activation, which is called the lectin pathway (11, 12, 13). Children with low MBL concentrations in sera exhibit a plasma-associated phagocytic defect, resulting in then being susceptible to recurrent infection (14, 15, 16).

In this study, we investigated the roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells. We found that Kupffer cells are capable of taking up lipid A through SR-A, that MBL augments SR-A-mediated uptake of lipid A and Staphylococcus aureus by Kupffer cells, that MBL induces cell surface SR-A expression on Kupffer cells, and that MBL binds to endotoxin and S. aureus, and precipitates S. aureus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Hepatic uptake of lipid A in mice

Hepatic uptake of [3H]lipid A in mice was examined using C57BL/6 and SR-A I/II knockout (SR-A–/–) mice by the method described previously (5). SR-A–/– mice are described in detail elsewhere (17). Two hundred microliters of saline containing 100 ng of [3H]lipid A (166 cpm/ng lipid A) was injected into the retro-orbital plexus of mice that were being anesthetized under diethyl ether. Thirty minutes after the injection, the mice were sacrificed and the liver was removed. The liver was then minced and solved by Solvable (Packard Instrument), and the radioactivity of the liver was measured by a scintillation counter. In some experiments, fucoidan from Fucus vesiculosus (5 mg; Sigma-Aldrich) or acetylated low-density lipoprotein (AcLDL; 200 mg; Biomedical Technologies) was coinjected to block the SR-A-mediated uptake of lipid A. Focoidan is a polysaccharide composed predominantly of sulfated fucose.

Isolation of Kupffer cells

Kupffer cells were isolated from Sprague-Dawley rats by a modified method based on that described by Lin et al. (18). Briefly, rats were anesthetized by i.m. injection of 60 mg of ketamine HCl and 13.5 mg of xylazine, and lapalotomy was performed and the liver was perfused through the portal vein with HBSS containing 700 mg of pronase and 20 mg of collagenase. The liver was then removed and minced, and nonparenchymal cells were separated from parenchymal cells and undigested tissue by centrifugation at 50 x g for 1 min. Kupffer cells were isolated from the nonparenchymal fraction by arabinogalactan (Larcoll; Sigma-Aldrich) density gradient centrifugation at 57,500 x gav (Hitachi RPS40T rotor; 21,400 rpm) for 40 min. The Kupffer cell-enriched fraction was collected from the interface between 1.045 and 1.058. The purity of Kupffer cells was assessed by phagocytosis assay of latex beads, and 97.16 ± 0.82% (mean ± SD; n = 3) of the cells were capable of phagocytosing the beads. The viability was also assessed by the trypan blue exclusion test and was 94.04 ± 1.03% (mean ± SD; n = 3).

Kupffer cells derived from C57BL/6 and SR-A–/– mice were also isolated by the method described above.

Mannose-binding lectin

Rat rMBL (mannose-binding protein A) was expressed by the baculovirus-insect cell expression system and purified, as described previously (19).

[3H]Lipid A uptake by Kupffer cells

Kupffer cells isolated from rat were seeded onto 24-well plate (5 x 105/well) and incubated with 1 µg of [3H]lipid A in 500 µl of DMEM (Sigma-Aldrich) for the indicated periods at 37°C or at 4°C. After the incubation, the cells were washed three times with PBS, and then lysed with PBS containing 2% (w/v) Triton X-100. The radioactivity of the cell lysate was measured by a scintillation counter. In some experiments, fucoidan, mannan or C1q, and/or MBL were coincubated with the cells.

Kupffer cells derived from wild-type (wt) and SR-A–/– mice were also examined for [3H]lipid A uptake. The cell suspension (106/tube) of mouse Kupffer cells was incubated with 2 µg/ml [3H]lipid A (500 µl/tube) at 37°C for 2 h in the presence or the absence of 2.5 mg/ml fucoidan. After the incubation, the cells were washed with cold PBS and lysed with 2% (w/v) Triton X-100, and the radioactivity of the cell lysate was measured by a scintillation counter, as described above.

Immunohistochemistry

Rat liver tissue was fixed by 3% (w/v) formaldehyde and paraffin embedded. Those sections were double stained with rabbit anti-rat MBL polyclonal Ab (10 µg/ml) and ED-1 mAb (5 µg/ml; Serotec), a marker for rat tissue macrophages, and then stained with hematoxylin.

Binding of MBL to LPS and lipid A

LPS or lipid A (500 ng/well) in 20 µl of ethanol was put onto microtiter wells, and the solvent evaporated in the ambient air. Nonspecific binding was blocked with 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl, 0.1% (w/v) BSA, and 5 mM CaCl2 (buffer A). The wells were then incubated with the indicated concentrations of MBL in the buffer A at 37°C for 5 h. After the incubation, the wells were washed with PBS containing 3% (w/v) skim milk and 0.1% (v/v) Triton X-100 (buffer B) and were incubated with rabbit anti-rat MBL Ab (20 µg/ml) in buffer B at room temperature for 2 h, followed by incubation with HRP-conjugated goat anti-rabbit IgG for 2 h. The amount of MBL binding to the solid-phase LPS or lipid A was determined by measuring the absorbance at 492 nm using o-phenylenediamine as a substrate for the peroxidase reaction.

Flow cytometric analysis

Kupffer cells (107) were suspended in DMEM and incubated with or without 20 µg/ml MBL at 37°C for 1 h. The cells were washed with PBS and fixed in 4% (w/v) paraformaldehyde at room temperature for 10 min. After fixation, Kupffer cells were washed again with PBS and then incubated with 1 µg/ml anti-SR-A Ab (Santa Cruz Biotechnology) or control goat IgG in PBS containing 5 mg/ml BSA and 10 mM sodium azide, followed by incubation with FITC-conjugated anti-goat IgG. The cells were finally washed and analyzed using FACSCalibur and CellQuest software (BD Biosciences).

Binding of MBL to S. aureus

MBL was biotinylated using Sulfo-NHS-biotin (Pierce), according to the manufacturer’s instructions. S. aureus (106 CFU) was incubated with 100 ng of biotinylated MBL in 50 µl of 5 mM Tris buffer (pH 7.4) containing 0.15 M NaCl, 5% (w/v) BSA, and 5 mM CaCl2 at 37°C for 1 h. After the incubation, the mixture of S. aureus and the protein was washed three times with PBS containing 0.1% (v/v) Triton X-100 by centrifugation. The bacterial pellet obtained by the final centrifugation was suspended with 20 µl of PBS and 5 µl of SDS sample buffer. The suspension of the pellet was boiled for 5 min and was centrifuged. The supernatant obtained was subjected to SDS-PAGE. Blotting analysis was next performed to detect MBL cosedimented with the bacteria. Proteins on the gels were transferred onto polyvinylidene difluoride membrane (Millipore). The membrane was then incubated with HRP-conjugated streptavidin at room temperature for 30 min. The protein bands were visualized by using a chemiluminescence reagent (SuperSignal; Pierce), according to the manufacturer’s instructions.

Precipitation of S. aureus by MBL

S. aureus (108 CFU) was mixed with MBL (20 µg/ml) in the cuvett containing 1 ml of 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl and 5 mM CaCl2 or 5 mM EDTA, and the cuvett was left at rest for the indicated length of time. At each time point, bacterial precipitation was determined by measuring the absorbance at 660 nm.

Phagocytosis of S. aureus and Escherichia coli by Kupffer cells

Kupffer cells were seeded onto a 24-well plate (5 x 105/well) and incubated with the indicated concentrations of MBL at 37°C for 1 h. After the incubation, the cells were washed with PBS and further incubated with 5 x 106 CFU of FITC-labeled S. aureus or FITC-labeled E. coli in HBSS at 37°C for 30 min in the dark. After washing the wells three times with ice-cold PBS, the extracellular FITC was quenched by the addition of ethidium bromide (40 µg/ml) in PBS. The number of macrophages with or without intracellular bacteria was counted up to at least 100 macrophages in duplicate samples using a fluorescence microscope at x400 magnification. The results are expressed as the percentage of Kupffer cells containing intracellular bacteria in total macrophage counted (phagocytosis (percentage)). In some experiments, the indicated concentrations of fucoidan or AcLDL were coincubated with the cells and the bacteria.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
SR-A is involved in the uptake of lipid A in the liver

We first examined the uptake of lipid A in mouse liver. Two hundred microliters of saline containing 100 ng of [3H]lipid A was injected into the retro-orbital plexus of C57BL/6 mice and SR-A-deficient mice, and the amounts of [3H]lipid A that accumulated in the liver were determined. More than 50% of the total of [3H]lipid A injected was found in the liver 30 min after injection. SR-A–/– mice exhibited significantly less accumulation of lipid A in the liver than wt mice (Fig. 1A). A 26% reduction of [3H]lipid A accumulation in the liver of SR-A–/– mice was observed when compared with wt mice, although the data indicate that there is some SR-A-independent uptake of lipid A. Coinjection of fucoidan with [3H]lipid A significantly decreased the hepatic uptake of [3H]lipid A by ~26% in wt mice (Fig. 1B). We also performed the experiments in which AcLDL was coinjected with lipid A using wt and SR-A–/– mice (Fig. 1C). Coinjection of AcLDL in wt mice reduced the hepatic uptake of [3H]lipid A by ~33%. In addition, the amount of lipid A accumulated in the liver of SR-A–/– mice was almost comparable to that in wt mice when AcLDL was coinjected. These results indicate that SR-A is, at least in part, involved in the uptake of lipid A by the liver.


Figure 1
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  		FIGURE 1. SR-A is involved in hepatic uptake of lipid A. A and B, Two hundred microliters of saline containing 100 ng of [3H]lipid A (166 cpm/ng lipid A) was injected into the retro-orbital plexus of C57BL/6 (wt) and SR-A-deficient (SR-A–/–) mice. Thirty minutes after the injection, the mice were sacrificed and the radioactivity of [3H]lipid A accumulating in the liver was determined, as described in Materials and Methods. B, Fucoidan (5 mg) was coinjected with [3H]lipid A in wt mice. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with wt mice (A) or with fucoidan (–) (B). C, AcLDL (200 mg) was coinjected with [3H]lipid A in wt and SR-A–/– mice, and the radioactivity of [3H]lipid A accumulating in the liver was determined. The data shown are the means from two experiments. D, Time-dependent uptake of [3H]lipid A by Kupffer cells. Kupffer cells (5 x 105/well) were incubated with 2 µg/ml [3H]lipid A for the indicated periods at 37°C ({circ}) or at 4°C (•). After the incubation, the cells were washed and lysed with PBS containing 2% Triton X-100. The radioactivity of the cell lysate was determined, as described in Materials and Methods. The triangle indicates the amount of [3H]lipid A, obtained by subtracting that associated with the cells at 4°C from that at 37°C. The data shown are the means ± SD from three separate experiments. E, Fucoidan decreases the amount of [3H]lipid A associated with Kupffer cells. Kupffer cells (5 x 105/well) were incubated with 2 µg/ml [3H]lipid A in the absence or the presence of the indicated concentrations of fucoidan for 120 min at 37°C ({circ}) or at 4°C (•). After the incubation, the cells were washed and lysed with PBS containing 2% Triton X-100. The radioactivity of the cell lysate was determined, as described in Materials and Methods. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with the experiments without fucoidan. F, Uptake of lipid A by Kupffer cells derived from wt ({blacksquare}) and SR-A-deficient ({square}) mice. The cell suspension (106/tube) of mouse Kupffer cells was incubated with 2 µg/ml [3H]lipid A (500 µl/tube) at 37°C for 2 h in the presence or the absence of 2.5 mg/ml fucoidan. After the incubation, the cells were washed and lysed with 2% (w/v) Triton X-100, and the radioactivity of the cell lysate was measured. The data shown are the means from two experiments.

 
Uptake of [3H]lipid A by Kupffer cells

We next determined whether Kupffer cells were able to take up lipid A. The amounts of [3H]lipid A associated with rat Kupffer cells increased in a time-dependent manner at 37°C to a greater extent than at 4°C (Fig. 1D). Because internalization does not occur at 4°C, [3H]lipid A associated at 4°C is presumed to be the binding of [3H]lipid A on the cell surface. The fraction of lipid A obtained by subtracting the amount of lipid A associated with the cells at 4°C from those at 37°C appeared as an internalized pool in Kupffer cells. These results indicate that Kupffer cells can take up lipid A.

We also determined whether the phagocytic receptor including C1q receptor, mannose receptor, or SR-A was involved in the uptake of [3H]lipid A by rat Kuppffer cells using known ligands for the receptors. Neither C1q nor mannan significantly decreased the amounts of [3H]lipid A associated with Kupffer cells at 4°C and 37°C (data not shown), indicating that C1q receptor or mannose receptor is not involved in the uptake of lipid A. When excess fucoidan was coincubated, the amounts of [3H]lipid A associated at 4°C and at 37°C were significantly decreased (Fig. 1E). In addition, coincubation with AcLDL (100 mg/ml) or polyinosinic acid (50 mg/ml) at 37°C decreased the amounts of [3H]lipid A associated with Kupffer cells by 26 ± 4.6 or 31 ± 6.4% (the means ± SD from three experiments), respectively. These results indicate that SR-A is involved in the uptake of lipid A by Kupffer cells.

To determine an important role of SR-A in the uptake of lipid A by Kupffer cells, we further performed the experiments with Kupffer cells derived from SR-A-deficient mice. Because adherence of Kupffer cells to tissue culture plastic has been shown to down-regulate SR-A-dependent phagocytosis (20), the cell suspension was used for the uptake experiments. Significant amounts of lipid A were associated with Kupffer cells derived from wt C57BL/6 mice, and its level was decreased to 20.8% by coincubation with fucoidan (Fig. 1F). The amount of lipid A associated with SR-A-deficient mice-derived Kupffer cells was only 20% of that associated with wt cells in the absence of fucoidan. Its level was unchangeable regardless of the presence of fucoidan, and was almost equivalent to that obtained from wt Kupffer cells in the presence of fucoidan. These results reveal an important role of SR-A in the uptake of lipid A by Kupffer cells.

Mannose-binding lectin

We examined immunohistochemical localization of Kupffer cells and MBL using ED-1, a marker for rat tissue macrophages, and anti-rat MBL polyclonal Ab. A significant number of Kupffer cells was observed among hepatocytes (Fig. 2, A and B), which synthesize and secrete MBL. Anti-MBL Ab diffusely stained the hepatocytes and the gaps among the cells (Fig. 2A). The results suggest that MBL could interact with Kupffer cells. Thus, using rat rMBL, we examined whether MBL modulated the function of Kupffer cells and interacted with endotoxin and the bacteria.


Figure 2
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  		FIGURE 2. Immunohistochemistry of Kupffer cells and electrophoretic analysis of rat rMBL. A and B, Rat liver tissue was fixed by 3% formaldehyde and paraffin embedded. The sections were double stained with ED-1 mAb (A and B) and anti-rat MBL polyclonal Ab (A) or control rabbit IgG (B), and then stained with hematoxylin, as described in Materials and Methods. Brown, Kupffer cells; pink, MBL. C, Rat rMBL (5 µg/lane) was electrophoresed under reducing (lane a) and nonreducing (lane b) conditions using a 7–15% gradient polyacrylamide gel. The protein bands were stained with Coomassie brilliant blue. St, molecular mass standards.

 
rMBL was analyzed by SDS-PAGE using a 7–15% gradient polyacrylamide gel. MBL exhibited a major band with an apparent molecular mass of 30 kDa under reducing conditions (Fig. 2C, lane a). A minor protein band with apparent molecular mass of 26 kDa appeared to be an unglycosylated form. When the protein was electrophoresed under nonreducing conditions, the major forms had molecular masses of ~50 and 90 kDa and minor forms were higher oligomers (Fig. 2C, lane b).

MBL binds to lipid A and LPS

We first examined the binding of MBL to lipid A and LPS coated onto microtiter wells using anti-MBL Ab to detect the MBL protein binding to the solid-phase lipid. MBL exhibited a concentration-dependent binding to lipid A and two different serotypes of LPS (Fig. 3A).


Figure 3
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  		FIGURE 3. MBL binds to lipid A, LPS, and S. aureus, and precipitates the bacteria. A, Lipid A ({circ}), O26:B6 LPS ({blacktriangleup}), and Re595 LPS (•) (500 ng/well) in 20 µl of ethanol were put onto microtiter wells, and the solvent evaporated in the ambient air. After nonspecific binding was blocked, the wells were incubated with the indicated concentrations of MBL in the presence of 5 mM CaCl2 at 37°C for 5 h. After the incubation, the wells were washed and the MBL binding to the solid-phase lipid A or LPS was detected by using anti-rat MBL Ab, as described in Materials and Methods. B, S. aureus (106 CFU) was incubated with or without 100 ng (50 µl) of biotinylated MBL in the presence of 5 mM CaCl2 at 37°C for 1 h. After the incubation, the mixture of S. aureus and the protein was washed and centrifuged. The bacterial pellet obtained was subjected to SDS-PAGE, and blotting analysis was performed to detect MBL cosedimented with the bacteria, as described in Materials and Methods. C, S. aureus (108 CFU) was mixed with or without MBL (20 µg/ml) in the cuvett containing 1 ml of 20 mM Tris buffer (pH 7.4) containing 0.15 M NaCl and 5 mM CaCl2 or 5 mM EDTA, and the cuvett was left at rest for the indicated length of time. At each time point, bacterial precipitation was determined by measuring the absorbance at 660 nm. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with the absorbance at time 0.

 
MBL augments SR-A-mediated uptake of lipid A by Kupffer cells

When various concentrations of MBL were coincubated with [3H]lipid A and Kupffer cells, MBL augmented the uptake of lipid A by the cells at 37°C in a concentration-dependent manner (Fig. 4A). MBL at 20 µg/ml increased the uptake of lipid A by 62%. However, MBL failed to increase the lipid A uptake at 4°C. This indicates that the MBL-stimulated uptake of lipid A is temperature dependent. When fucoidan was included in the binding buffer, the basal and the MBL-stimulated uptake of lipid A was significantly inhibited (Fig. 4B). These results clearly indicate that lipid A is taken up through SR-A and that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells.


Figure 4
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  		FIGURE 4. MBL enhances the uptake of [3H]lipid A by Kupffer cells. A, Kupffer cells (5 x 105/well) were incubated with 2 mg/ml [3H]lipid A in the absence or the presence of the indicated concentrations of MBL for 120 min at 37°C ({circ}) or at 4°C (•). After the incubation, the cells were washed and lysed with PBS containing 2% Triton X-100. The radioactivity of the cell lysate was determined, as described in Materials and Methods. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with the experiments in the absence of MBL. B, Kupffer cells (5 x 105/well) were incubated with 2 µg/ml [3H]lipid A in the absence or the presence of 2.5 mg/ml fucoidan and 20 µg/ml MBL for 120 min at 37°C. The radioactivity of [3H]lipid A associated with the cells was determined, as described above. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with fucoidan (–) and MBL (–). **, p < 0.05, when compared with fucoidan (–) and MBL (+). C, Kupffer cells (5 x 105/well) were incubated with 20 µg/ml MBL at 37°C for 60 min. After the incubation, the medium containing MBL was removed and the cells were washed. The fresh medium containing [3H]lipid A was then added and incubated with the cells at 37°C for 120 min. The radioactivity of [3H]lipid A associated with the cells was determined, as described above. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with MBL preincubation (–).

 
MBL at 20 µg/ml was preincubated with Kupffer cells, and the uptake of lipid A was examined after replacing the medium containing MBL (Fig. 4C). The uptake of lipid A by MBL-pretreated cells was significantly increased when compared with that by non-treated cells. This indicates that the direct interaction of MBL with Kupffer cells is important for the MBL-stimulated uptake of lipid A.

MBL increases cell surface expression of SR-A on Kupffer cells

We next assessed the cell surface expression of SR-A on Kupffer cells by flow cytometry. SR-A was constitutively expressed on the cell surface of Kupffer cells (Fig. 5A, dotted line). After MBL (20 µg/ml) was exposed to Kupffer cells at 37°C for 60 min, cell surface expression of SR-A was enhanced (Fig. 5A, solid line). When the mean fluorescence intensity was calculated as a ratio to control, MBL significantly increased the mean fluorescence intensity ratio (Fig. 5B). This result is consistent with that showing that preincubation of MBL with Kupffer cells increases the uptake of lipid A.


Figure 5
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  		FIGURE 5. MBL enhances cell surface expression of SR-A on Kupffer cells. A, Kupffer cells (107) were incubated with or without 20 µg/ml MBL in DMEM at 37°C for 1 h. The cells were washed with PBS and fixed in 4% (w/v) paraformaldehyde at room temperature for 10 min. After fixation, Kupffer cells were washed again with PBS and then incubated with 1 µg/ml anti-SR-A Ab or control goat IgG in PBS containing 5 mg/ml BSA and 10 mM sodium azide, followed by incubation with FITC-conjugated anti-goat IgG. The cells were finally washed and analyzed by using FACSCalibur and CellQuest software (BD Biosciences), as described in Materials and Methods. The histograms shown are representatives from three experiments. The solid line shows cytometric analysis of the MBL-treated cells, and the dotted line shows the untreated cells. The gray shadow shows the control without the first Ab. B, The mean fluorescence intensity ratio to the control. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with MBL (–).

 
MBL binds and precipitates S. aureus

We next examined the interaction of MBL with the bacteria. S. aureus was incubated with or without MBL in the presence of Ca2+ and sedimented, and it was determined whether MBL was cosedimented. MBL was cosedimented with S. aureus (Fig. 3B), indicating that MBL binds to S. aureus. We next examined whether MBL aggregated and precipitated the bacteria. When S. aureus and MBL were coincubated in the cuvett, which was then left at rest, and the turbidity was measured at 660 nm, coincubation of the bacteria with 20 µg/ml MBL decreased the tubidity (Fig. 3C). This indicates that MBL can aggregate and precipitate the bacteria, resulting in the increased transparency in the cuvett. When 20 µg/ml MBL was coincubated in the presence of EDTA, decreased turbidity was not observed, suggesting that aggregation of the bacteria by MBL is Ca2+ dependent. Taken together, these results show that MBL can bind S. aureus and precipitate the bacteria.

MBL augments SR-A-mediated phagocytosis of S. aureus and E. coli by Kupffer cells

We next examined whether MBL affected the phagocytosis of S. aureus by Kupffer cells. Because MBL enhances cell surface SR-A expression on Kupffer cells (see Fig. 5), we performed the phagocytosis assay after preincubation of MBL with Kupffer cells. MBL at 1–20 µg/ml was preincubated with Kupffer cells, the medium containing MBL was replaced with fresh medium, and FITC-labeled S. aureus was incubated with the cells. At all concentrations tested, MBL significantly increased the phagocytosis of S. aureus (Fig. 6A). Because Gram-negative bacteria including E. coli are also ligands for SR-A, we examined the phagocytosis of E. coli by Kupffer cells. Preincubation of MBL with Kupffer cells increased the phagocytosis of E. coli (Fig. 6C). Because fucoidan and AcLDL inhibited the phagocytosis of S. aureus and E. coli by Kupffer cells in a concentration-dependent manner (Fig. 6, B and D), the phagocytosis of these bacteria by Kupffer cells must be mediated through SR-A.


Figure 6
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  		FIGURE 6. MBL augments SR-A-mediated phagocytosis of S. aureus and E. coli by Kupffer cells. A and C, Kupffer cells (5 x 105/well) were incubated with the indicated concentrations of MBL at 37°C for 1 h. After the incubation, the cells were washed with PBS and were further incubated with 5 x 106 CFU of FITC-labeled S. aureus (A) or FITC-labeled E. coli (C) in HBSS at 37°C for 30 min. After washing the cells to remove unbound bacteria, the cells were suspended with ethidium bromide solution, and the numbers of Kupffer cells with or without intracellular bacteria were counted. The results are expressed as the percentage of Kupffer cells containing intracellular bacteria in total macrophages counted (phagocytosis (percentage)), as described in Materials and Methods. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with the experiments in the absence of MBL. B and D, The phagocytosis assay of S. aureus (B) and E. coli (D) by Kupffer cells was performed in the presence of the indicated concentrations of fucoidan or AcLDL. The data shown are the means ± SD from three separate experiments. *, p < 0.05, when compared with fucoidan (0) or AcLDL (0).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The liver plays pivotal roles in the clearance of endotoxin and bacteria derived from the gastrointestinal tract and from sites of infection (5). An in vivo study using gadolinium chloride indicates an important role of Kupffer cells in the clearance of endotoxin in endotoxemic rats (21). LPS clearance is regulated by apical-sinusoidal endocytic pathways, as well as canalicular routes, in the liver (22). The present results obtained from in vivo experiments showed that >50% of [3H]lipid A accumulated in the liver 30 min after the retro-orbital plexus injection of lipid A (see Fig. 1A). This is consistent with a previous study (5) showing that 79% of the total of [32P]lipid IVA injected is found in the liver in as little as 10 min after injection. In that study, coinjection of AcLDL or poly(I) significantly reduced hepatic uptake of [32P]lipid IVA, indicating the involvement of SR-A in the hepatic uptake of endotoxin. In this study, we confirmed the involvement of SR-A by using SR-A-deficient mice. However, the hepatic uptake of lipid A was reduced by only 26% in SR-A-deficient mice, suggesting that an SR-A-independent uptake of endotoxin in the liver also takes place. This is consistent with the results obtained from coinjection of SR-A ligands with endotoxin in this (see Fig. 1, B and C) and previous (5) studies.

This study demonstrates that Kupffer cells are capable of taking up lipid A through SR-A. MBL, which is synthesized in the liver, augments SR-A-mediated phagocytosis of lipid A, S. aureus, and E. coli by increasing cell surface SR-A expression on Kupffer cells. Because other collectins, surfactant proteins A and D, also augment the phagocytosis of Streptococcus pneumoniae, Mycobacterium avium, and Mycobacterium tuberculosis through increased cell surface expression of the phagocytic receptors, including SR-A and mannose receptor (23, 24, 25, 26), the up-regulation of cell surface expression of the phagocytic receptors is an important function common to the collectin group, although different specificities for cell types and phagocytic receptors appear to exist. Surfactant protein A, but not D, increases cell surface SR-A expression on alveolar macrophages (24), while both pulmonary collectins increase mannose receptor activities of macrophages, whereas MBL does not (23).

In this study, the phagocytosis assay was performed with rat Kupffer cells that had been seeded on a 24-well plastic plate. Under these conditions, coincubation with fucoidan decreased the uptake of lipid A by Kupffer cells by only 29% (see Fig. 1E), indicating weak contribution of SR-A to the uptake of lipid A by Kupffer cells that had been adhered on plastic. Because macrophages are activated by adherence to plastic and adherence to tissue culture plastic has been shown to down-regulate SR-A-dependent phagocytosis (20), the in vitro assay on plastic may not accurately reflect the activity of Kupffer cells. Thus, we further performed the lipid A-uptake experiments under nonadherent conditions using cell suspension of Kupffer cells derived from SR-A–/– mice. The amount of lipid A associated with SR-A–/– mice-derived Kupffer cells was reduced by ~80% when compared with wt Kupffer cells (see Fig. 1F). In addition, coincubation of fucoidan inhibited the uptake of lipid A by wt Kupffer cells by 80%, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. However, in vivo experiments have revealed that the hepatic uptake of lipid A was reduced by only 26–33% in SR-A–/– mice and in wt mice that were coinjected with fucoidan and AcLDL (see Fig. 1, A–C). Thus, it is possible to infer that other mechanisms besides SR-A-dependent uptake may also contribute to blood clearance of endotoxin in the liver in vivo.

Human MBL binds to Klebsiella O3 LPS (27), and it appears to exhibit a strong binding to LPS possessing the mannose homopolysaccharide, but not to LPS possessing the heteropolysaccharide. Because human MBL was isolated by binding to a Klebsiella O3 LPS affinity column in the presence of Ca2+ and by elution with EDTA in that study, the binding of MBL to Klebsiella O3 LPS is Ca2+ dependent. Human MBL has also been shown to bind lipoteichoic acids (28). Because lipoteichoic acids lacking terminal sugars or containing galactosyl substituents are poor ligands, human MBL binds lipoteichoic acids through the lectin property of MBL. In this study, we have shown that rat MBL binds to lipid A as well as Re595 LPS, O26:B6 LPS, and S. aureus. We have shown previously that rat MBL binds phospholipids in a Ca2+-dependent manner (29). Thus, rat MBL may bind endotoxin through the recognition of carbohydrate moiety and/or lipid moiety. In addition, this study has revealed that MBL can precipitate S. aureus. Taken together, these studies demonstrate the direct actions of MBL on microbes.

MBL has been shown to augment the uptake of lipid A by Kupffer cells when incubated at 37°C, but not at 4°C (see Fig. 4A), indicating that the MBL-mediated increase of lipid A uptake is temperature dependent. When Kupffer cells are incubated with MBL at 37°C, cell surface SR-A expression is enhanced (see Fig. 5). However, 4°C incubation of MBL with the cells does not increase cell surface SR-A expression (data not shown). Thus, it is possible to presume that the recruitment of SR-A to the plasma membrane is caused by a temperature-dependent process such as protein phosphorylation. This is consistent with a previous study showing that surfactant protein A stimulates cell surface SR-A expression on alveolar macrophages as a posttranslational event involved in the protein phosphorylation caused by casein kinase 2 (24).

This study shows that MBL binds LPS and S. aureus, but that the preincubation of MBL with Kupffer cells augments the uptake of these ligands, indicating that the process of the MBL-stimulated uptake of endotoxin and S. aureus is independent of the binding of MBL to the ligands. MBL-deficient mice have been shown to be susceptible to infection of S. aureus, demonstrating the key role of MBL in S. aureus infection (30). One recent study indicates that MBL also acts as an opsonin (31). Opsonization with MBL significantly increases the internalization of Neisseria meningitidis and reduces the survival of meningococci within macrophages. Thus, in addition to the direct effects on microbes, MBL may function as a stimulant of phagocytosis by two mechanisms, that is, by serving as an opsonin and by increasing cell surface expression of the phagocytic receptors on macrophages. Because Kupffer cells exist among hepatocytes that synthesize and secrete MBL, secreted MBL can directly interact with Kupffer cells. Thus, it is possible to suggest that MBL may function as a paracrine factor.

In conclusion, Kupffer cells are capable of taking up endotoxin and bacteria. MBL augments the uptake of endotoxin and bacteria by increasing cell surface expression of SR-A. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.


   Acknowledgments
 
We thank Dr. Hidekazu Tsukamoto (University of Southern California School of Medicine, Los Angeles, CA) for teaching us the procedure for isolating Kupffer cells from rats.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture. Back

2 Address correspondence and reprint requests to Dr. Yoshio Kuroki, Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. E-mail address: kurokiy{at}sapmed.ac.jp Back

3 Abbreviations used in this paper: SR-A, scavenger receptor A; AcLDL, acetylated low-density lipoprotein; MBL, mannose-binding lectin; wt, wild type. Back

Received for publication November 23, 2005. Accepted for publication July 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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