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Originally published online as doi:10.1189/jlb.1204705 on March 14, 2005

Published online before print March 14, 2005
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(Journal of Leukocyte Biology. 2005;77:934-943.)
© 2005 by Society for Leukocyte Biology

The human macrophage mannose receptor is not a professional phagocytic receptor

Véronique Le Cabec1, Laurent J. Emorine2, Isabelle Toesca, Céline Cougoule and Isabelle Maridonneau-Parini

Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, Toulouse, France

1Correspondence: I.P.B.S., CNRS UMR 5089, Department "Mécanismes moléculaires des infections mycobactériennes" 205, route de Narbonne 31077 Toulouse, France. E-mail: Veronique.Le-Cabec{at}ipbs.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The macrophage mannose receptor (MR) appears to play an important role in the binding and phagocytosis of several human pathogens, but its phagocytic property and signaling pathways have been poorly defined. The general strategy to explore such topics is to express the protein of interest in nonphagocytic cells, but in the case of MR, there are few reports using the full-length MR cDNA. When we searched to clone de novo the human MR (hMR) cDNA, problems were encountered, and full-length hMR cDNA was only obtained after devising a complex cloning strategy. Chinese hamster ovary cells, which have a fully functional phagocytic machinery when expressing professional phagocytic receptors, were stably transfected, and cell clones expressing hMR at quantitatively comparable levels than human macrophages or J774E cells were obtained. They exhibited a functional hMR-mediated endocytic capacity of a soluble ligand but failed to ingest classical particulate ligands of MR such as zymosan, Mycobacterium kansasii, or trimannoside bovine serum albumin-coated latex beads. Transient expression of hMR in two human cell lines did not provide a phagocytic capacity either. In conclusion, we show that MR is not a professional phagocytic receptor, as it does not possess the ability to promote particle ingestion in nonphagocytic cells on its own. We propose that MR is a binding receptor, which requires a partner to trigger phagocytosis in some specialized cells such as macrophages. Our new expression vector could represent a useful tool to study the receptor and its partnership further.

Key Words: phagocytosis • endocytosis • de novo cloning


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are central actors of the innate and adaptive immune responses. They are disseminated throughout most organs to protect against entry of infectious agents by internalizing and most of the time, killing them. Among the surface receptors present on macrophages, the mannose receptor (MR; see refs. [1 2 3 4 ] for reviews) recognizes a variety of molecular patterns generic to microorganisms [5 ].

The MR is a 180-kDa type I transmembrane protein, first identified in mammalian tissue macrophages [6 ] and later in dendritic cells [7 , 8 ] and a variety of endothelial and epithelial cells [9 10 11 12 13 14 ]. It is composed of a single subunit with N- and O-linked glycosylations and consists of five domains [15 , 16 ]: an N-terminal cysteine-rich region, which recognizes terminal sulfated sugar residues [17 ]; a fibronectin type II domain with unclear function; a series of eight C-type, lectin-like carbohydrate recognition domains (CRDs) involved in Ca2+-dependent recognition of mannose, fucose, or N-acetylglucosamine residues on the envelop of pathogens or on endogenous glycoproteins [18 , 19 ] with CRDs 4–8 showing affinity for ligands comparable with that of intact MR [20 ]; a single transmembrane domain; and a 45 residue-long cytoplasmic tail that contains motifs critical for MR-mediated endocytosis [16 , 21 ] and sorting in endosomes [22 ].

The MR has been recognized early as an endocytic scavenger molecule that binds and internalizes a variety of glycoconjugated ligands [23 ] and thus, participates in the clearance by endocytosis of soluble macromolecules such as lysosomal enzymes [24 25 26 ], neutrophil granulocyte-derived myeloperoxidase [27 ], tissue plasminogen activator [28 29 30 ], C-terminal propeptide of type I procollagen [13 ], and pituitary hormones lutropin and thyrotropin [17 , 31 ]. These molecules are normally released into the body fluids or during inflammatory processes. Their accumulation may be damaging to the organism. The regulation of serum glycoprotein homeostasis seems to be the main function of the MR, as recently demonstrated in vivo in MR–/– knockout mice [32 ].

Besides, the MR has important immune-related functions in innate and adaptive immunity. As part of the innate immune response, the MR has been implicated in binding and internalization of a wide range of microorganisms, among them, protozoa [33 , 34 ], viruses [35 36 37 ], fungi [16 , 38 ], and bacteria [4 , 39 40 41 ]. It is quite surprising that internalization of particles through the MR can be dissociated from activation of human macrophage bactericidal responses such as superoxide anion production or fusion of Hck-positive lysosomes with phagosomes [42 ]. It was therefore suspected to be used by intracellular pathogens as a safe portal of entry into host cells.

The purpose of our study was to gain additional knowledge about the role of the MR in the phagocytosis of microorganisms. Our main attempt was to elucidate the signaling pathways operating downstream of the receptor, a research field that has been poorly studied. It has been reported that macrophages ingesting latex beads coated with a ligand of the MR form pseudopodia [43 ]. Two types of phagocytic processes have been described to date [44 ]: type I phagocytosis, which involves formation of pseudopodia around the particles, and type II, which involves invagination of the plasma membrane. In both cases, reorganization of the actin cytoskeleton is taking place but through distinct signaling pathways [44 ]. We have reported that the professional phagocytic receptor, complement receptor 3 (CR3), has the particularity, through its distinct binding sites, to elicit type I or type II phagocytosis [45 ]. As the MR also has several binding sites, we hypothesized that it could signal several pathways as well, depending on its particulate ligand. Our previous study about CR3 was carried out in Chinese hamster ovary (CHO) cells; hence, we decided to express the MR in the same cells.

When CHO cells were transfected using de novo-cloned human MR (hMR) cDNA, we observed that the MR efficiently mediated endocytosis of mannosylated glycoproteins but surprisingly, did not support phagocytosis of three of its known particulate ligands: zymosan, Mycobacterium kansasii, and mannosylated latex beads. We therefore propose that the MR is not a phagocytic receptor per se but may require interaction with signaling molecules present in some specialized cells such as macrophages to promote particle ingestion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Polystyrene microspheres were purchased from Polysciences Inc. (Warrington, PA), zymosan particles were from Sigma Chemical Co. (St. Louis, MO), and {alpha}-minimum essential medium ({alpha}-MEM), Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, and antibiotics were from Gibco (Cergy Pontoise, France). Mouse monoclonal antibodies (mAb) against hMR were from BD Biosciences PharMingen (San Diego, CA). Goat polyclonal antibodies against the hMR were a generous gift from Philip D. Stahl (Washington University School of Medicine, St. Louis, MO), and anti-Fc receptor for immunoglobulin G (IgG; Fc{gamma}R)IIa IV.3 mAb were from Catherine Sautes-Fridman (Institut Curie, France). Antimycobacterium rabbit antibodies (camelia) were obtained by immunizing rabbits with heat-killed mycobacteria (Mycobacterium smegmatis, Mycobacterium phlei, Mycobacterium avium, and M. kansasii) as described previously [46 ]. Fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit and anti-mouse antibodies and rabbit anti-goat antibodies were purchased from Sigma Chemical Co.

DNA constructs
C. Sautes-Fridman [47 ] nicely supplied expression vector encoding Fc{gamma}RIIa. MR full-length cDNA were gifts from Dr. R. Alan Ezekowitz (Pediatric Service, Harvard Medical School, Massachusetts General Hospital, Boston, MA) and Kurt Drickamer (Glycobiology Institute, Department of Biochemistry, University of Oxford, UK).

For hMR cDNA cloning, total RNA was extracted from human placenta using RNAzol B (Celbio, Milan, Italy) and enriched in polyadenylated mRNA. This was used to amplify four fragments by reverse transcriptase-polymerase chain reaction (RT–PCR) with primers hMR-S and AS 1–4 (Table 1 ). Two substitutions (AA->TC) were introduced in hMR-S1 to create an EcoRV site upstream from the ATG initiation codon. The full-length hMR cDNA was then reconstituted from these fragments into pBSK plasmids (Stratagen Europe, Amsterdam, The Netherlands) by using internal restriction sites (see Fig. 1A ).


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  		Table 1. Oligonucleotides Sequences (5' to 3')

 


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  		Figure 1. Human MR cloning strategy. (A) Scheme illustrating the different steps of hMR cDNA cloning described in Results (SP, Signal peptide; CR, cysteine-rich domain; FN, fibronectin domain; TM, transmembrane domain; IC, intracellular domain; TAG, stop codon). (B) Scheme illustrating the site of insertion of the composite intron from the pBK-CMV phagemid in the coding region of the hMR. Insertion was realized between the G and T residues of the AGT/Ser1138 codon. The intron was amplified by PCR using primers that contained at their extremity the hMR sequences comprised between the BamHI and NsiI sites that naturally occur in the receptor-coding region.

 
The composite intron from the pBK-CMV phagemid (nucleotides 730–1012, Stratagene, La Jolla, CA) was amplified using primers int-S and int-AS, which contained, at their extremities, the hMR sequences comprised between the BamHI and NsiI sites in the receptor coding region (Table 1) . These sites were used to introduce the resulting fragment into the hMR cDNA, inserting the intron between the G and T residues of the AGT/Ser1138 codon (see Fig. 1B ).

The pACYC184 plasmid (New England Biolabs, Beverly, MA) was cut with Tth111I and ClaI and self-ligated to delete the tetracycline-resistance gene. The resulting plasmid, containing the p15A origin of replication and chloramphenicol resistance, was open with BstZ17I and cloned between the SspI and Bst1107I sites of pcDNA3.1 (Invitrogen BV, Groningen, The Netherlands) in place of the ColE1 origin and ampicillin gene. The full-length hMR cDNA was cloned into the KpnI and NotI restriction sites. DNA sequencing was performed by Genomex (Grenoble, France).

Mycobacteria, zymosan particles, and Man3-coated latex beads
M. kansasii (ATCC 124478) were grown, prepared, and stained with FITC as described [48 ]. Zymosan particles isolated from Saccharomyces cerevisiae were stained with FITC [48 ]. In some experiments, mycobacteria and zymosan were serum-opsonized by incubation in pooled human sera [48 , 49 ]. Man3 bovine serum albumin (BSA)-coated latex beads were obtained as described: The trimannoside {methoxycarbonyl octyl 2-O-[({alpha}-D-mannopyranosyl)-2-O {alpha}-D-mannopyranosyl]-({alpha}-D-mannopyranoside)} was produced, conjugated to BSA, and adsorbed on latex microparticles [42 ]. The number of particles or bacteria was counted in a Thoma’s chamber.

Cells and transfection
CHO cells were transfected with hMR cDNA by the DNA/calcium precipitation. After 3 days, G418 (Geneticin) was added (50 µg/ml), and the cells were grown under selection for 2 weeks. Subclones of stably transfected cells (CHO-hMR) were obtained by limiting dilution in 96-well plates. Wild-type (WT), CHO-hMR, and CHO cells stably expressing human CR3 (CHO-CR3) [50 ] were cultured in {alpha}-MEM supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine, and 0.1 µM methotrexate for CHO-CR3 cells [2 ] or 50 µg/ml G418 for CHO-hMR cells. In some experiments, transient transfections of hMR or Fc{gamma}RIIa were performed in CHO-CR3 and/or CHO-hMR cells using the calcium phosphate method [51 ]. Cell viability has been examined by Trypan blue exclusion; the viability of stably transfected cells (hMR-positive or -negative) was identical to control cells. Leukocytes were isolated from healthy donor blood by dextran sedimentation and centrifugation through Ficoll-Hypaque [49 ], and macrophages were prepared as described [42 ]. J774E, HeLa, and Hep2 cells were cultured in {alpha}-MEM or DMEM supplemented with L-glutamine, antibiotics, and 10% heat-inactivated fetal calf serum.

Membrane fractionation
Subconfluent cells in 12-cm petri dishes were washed three times with phosphate-buffered saline (PBS), gently scraped using a rubber policeman, and resuspended in 1 ml PBS. After centrifugation (10 min, 200 g, 4°C), cell pellet was resuspended in Tris 50 mM, pH 7.5, supplemented with 1 mM EDTA, 1 mM EGTA, 3 µg/ml leupeptine, 1 µg/ml pepstatine, 1 µM aprotinine, and 0.5 mM phenylmethylsulfonyl fluoride. After Potter homogenization (20 strokes, 4°C) and centrifugation (10 min, 1000 g, 4°C), the pellet was homogenized in the same buffer and centrifuged as above. Supernatants were pooled, and membranes were pelleted (20,000 g, 4°C for 30 min) and resuspended in Laemmli sample buffer.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis
Human monocyte-derived macrophages (MDMs), CHO, and CHO-hMR cells were washed and lysed in boiling Laemmli sample buffer. Solubilized proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes that were probed with anti-hMR immune serum (1:200 dilution) and revealed by using an enhanced chemiluminescence system [42 ]. Quantification of hMR expression in the different cell types was performed on Western blots using the Quantity-One 4.5.0TM software.

Fluorescein-activated cell sorter (FACS) analysis
1x105 MDMs, CHO, or CHO-hMR cells grown in six-well plates for 24 h were washed with PBS, fixed in 3.7% paraformaldehyde (30 min, room temperature), and neutralized in 50 mM NH4Cl for 2 min. After washing and saturation for 15 min in PBS supplemented with 1% BSA (PBS-BSA), fixed but not permeabilized cells were incubated for 1 h at 4°C with PBS-BSA (control) or 0.5 µg anti-hMR mAb diluted in 1 ml PBS-BSA so that only surface-exposed hMR antigens were detected. Cells were washed three times in PBS-BSA at 4°C, incubated for 1 h with FITC-coupled goat anti-mouse IgG, washed three times in PBS-BSA, gently scraped, resuspended in 500 µl PBS, and analyzed on FACScan (Becton Dickinson, San Jose, CA) as described [46 ]. The specific fluorescence (expressed in arbitrary units) was obtained by substraction of the control value from the fluorescence measured with anti-hMR antibodies. The specific fluorescence was measured on the whole cell population.

Infection of cells and phagocytosis assay
Three types of particles known to bind to the MR were used: M. kansasii [42 ], zymosan particles from S. cerevisiae [52 , 53 ], and Man3-coated particles [42 ]. Prior to the phagocytosis assay, cells were washed three times to remove serum proteins and incubated for 30 min in serum-free medium. FITC-labeled zymosan or mycobacteria or Man3 BSA-coated latex beads were then added at a multiplicity of infection (MOI) of 50:1. Contact between cells and nonopsonized particles was maintained for 3–16 h. Cells were then washed extensively with {alpha}-MEM medium and fixed in paraformaldehyde as described previously [46 ]. To exclusively quantify genuine phagocytosis and not binding, extracellular zymosan and mycobacteria were stained as described [46 ]. Briefly, extracellular mycobacteria were stained with Camelia antimycobacterium antibodies revealed by TRITC-conjugated anti-rabbit antibodies. As the cells were not permeabilized, antimycobacterium antibodies did not reach intracellular bacteria, which thus appeared as green fluorescent particles (bacteria are prestained with FITC prior to infection experiments). Extracellular mycobacteria were stained with antibodies and were therefore fluorescent in red and green. Extracellular zymosan particles were stained using the same protocol, and extracellular Man3-BSA latex beads were visualized using an anti-BSA serum revealed by TRITC-conjugated anti-rabbit antibodies. At least 100 transfected cells per coverslip were counted for zymosan, M. kansasii, or Man3-BSA latex bead phagocytosis. In transient transfection experiments, phagocytosis of particle cells was measured in transfected cells and compared with mock-transfected cells. Coverslips were viewed using a Leica DM-RB fluorescence microscope.

Data are presented as the mean ± SEM of the indicated number of experiments performed in duplicates. Statistical significance was determined using unpaired Student’s t-test (*P<0.05; **P<0.01; ***P<0.005).

Measurement of MR-mediated endocytosis
MR-mediated endocytosis was measured by uptake of horseradish peroxidase (HRP) in the absence or in the presence of mannan [54 55 56 57 ]. J774E cells, a subclone of J774 expressing the MR endogenously, were used as a positive control [55 , 58 ]. J774E and CHO cells were seeded in 12-well plates at a density of 5 x 105 cells/well. The next day, the medium was discarded, and cells were incubated for 30 min in 0.5 ml Hanks’ balanced saline solution supplemented with 1% BSA. HRP (0.5 ml) was then added to the indicated final concentrations, alone or together with yeast mannan (2 mg/ml) to determine nonspecific uptake. Cells were incubated for 60 min at 37°C, washed three times in PBS, 10 mM EGTA, and solubilized in 200 µl PBS, 10 mM EGTA, and 1% Triton-X 100. Cell-associated HRP activity was quantified by adding o-dianisidine and H2O2 in 50 mM sodium acetate, pH 5.5 [57 ]. HRP uptake was normalized to total cellular protein. Specific uptake of HRP by hMR was calculated (HRP uptake in the absence of mannan minus HRP uptake in the presence of mannan) and is expressed in arbitrary units. Results represent mean ± SEM of three independent experiments performed in quadruplicate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of the hMR cDNA
The two hMR cDNA provided by the groups who originally isolated them [15 , 16 ] were first transformed in Escherichia coli and amplified. CHO cells transfected with one of these cDNA clones [16 ] secreted in the culture medium a 110-kDa polypeptide recognized by anti-MR antibodies in place of the expected 180-kDA plasma membrane-anchored protein [6 ]. Sequencing of the cDNA showed that it differed from published sequences by the deletion of an A residue from the AAT/Asn969 codon. This resulted in a frame-shift stop codon 10 amino acid residues further down, yielding a truncated protein lacking almost 490 residues comprising the COOH-terminal transmembrane and intracellular domains. Restriction mapping of the second cDNA clone [15 ] revealed that a transposon was inserted in the coding sequence, close to the BamHI restriction site.

We thus decided to clone the hMR cDNA again by RT-PCR. Four fragments were obtained (Fig. 1A ) and cloned in pBSK vectors. The two first fragments and the two last fragments were joined together to yield fragments A and B (Fig. 1A) . Upon reconstitution of the full-length cDNA, few transformants were obtained from each ligation reaction and corresponded to side-products. Screening of hundreds of such transformants yielded a single, positive clone. Its sequencing revealed the deletion of the C residue from the ACG/Thr1306 codon, yielding a protein truncated from ~150 COOH-terminal residues. Bacteria containing either of these cDNA grew very slowly and yielded low amounts of plasmid, which upon sequence analysis, proved to contain several mutations in the ColE1 origin as compared with the initial vector. This suggested that the presence of the cDNA interfered with bacterial growth or plasmid replication.

Two strategies were devised to bypass these problems. First, an intron was introduced in the coding region of the hMR cDNA obtained by RT-PCR. This mimicked the insertion of a transposon in the above cDNA [15 ] and introduced several downstream stop codons in all three reading frames of the receptor, as occurring in the two other hMR clones. Second, the resulting cDNA was introduced into an expression vector derived from pcDNA3 by replacing the ColE1-replicative origin by the p15A origin. This origin is compatible with ColE1 and thus functions differently. In this way, we were able to propagate the full-length hMR in bacteria. When compared with the sequence published by Taylor and co-workers [15 ], seven substitutions occurred in our full-length cDNA, and only one of them induced an amino acid change, Thr521Pro (a->c). Similarly to Taylor and co-workers [15 ], we found an Arg1415His (g->a substitution) when compared with the sequence published by Ezekowitz’s group [16 ]. Sequencing of 10 independent clones of the 1.2-kb BamHI to XbaI fragment (Fig. 1) showed that six had an Arg, and four had a His at position 1415. It is noteworthy that a His is also present at the homologous position of the mouse MR, suggesting that this variation (Arg1415His) is likely to represent an allelic polymorphism of the hMR locus.

hMR stably expressed in CHO cells mediates HRP endocytosis
CHO cells were tranfected with this construct, and stable transfectants were selected by FACS analysis. Expression levels varied between the different initial clones and were lower than in MDMs (Fig. 2A ). The protein detected in transfected CHO cells was associated to the membrane fraction and had a similar apparent molecular weight as that in human MDMs (Fig. 2B) or J774E cells (a MR-positive murine macrophage cell line; Fig. 2C , right) [58 ].



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  		Figure 2. Characterization of stably transfected CHO-hMR cells. (A) Surface expression of hMR in different clones of CHO-hMR cells compared with human MDMs (hatched bar) and WT-CHO cells (solid bar) was analyzed by flow cytometry. Cells were stained with anti-hMR antibodies and FITC-coupled antibodies as described in Materials and Methods. (Inset) The histogram shows the specific fluorescence measured on the complete cell population for WT-CHO cells (black) and CHO-hMR clone 2.5 (shaded). Results are expressed as mean ± SD of three experiments (B) Western blot analysis of hMR expression on membrane fractions isolated from three clones of CHO-hMR cells compared with human MDMs and WT-CHO cells. Cell equivalents (5x105) are loaded per lane, and the presence of hMR was detected using the goat anti-hMR antibodies. Positions of molecular weight standards are shown on the right in thousands. One experiment representative of three is shown. (C) Western blot analysis of CHO-hMR subclones. (Left) Five subclones obtained from clone 2.5 were isolated. Whole cell lysates were prepared, and 5 x 105 cell equivalents were loaded per lane. (Right) Membrane (lane 2) and cytosolic (lane 3) fractions from clone 65 were loaded at 5 x 105 cell equivalents per lane and compared with the membrane fraction of J774E cells (lane 1). (D) CHO-hMR mediates endocytosis of HRP. Cells were incubated for 60 min at 37°C with increasing concentrations of HRP in the presence or absence of inhibitory concentrations of mannan. HRP uptake was positive in CHO-hMR cells and J774E cells (used as a positive control) but not in WT-CHO cells.

 
Subclones were isolated from clone 2.5, which showed the most homogenous cell population (Fig. 2A , inset) and the highest level of hMR expression (Fig. 2A) . hMR expression level in clone 2.5 was quantified and shown to be 3.9 ± 0.6-fold lower than in MDMs (analysis of three independent Western blots). To isolate cells with a higher hMR expression level, subclones were selected and characterized by Western blot. As shown in Figure 2C , subclones 65 and 83 expressed a higher level of hMR than clone 2.5. Therefore, clone 65, referred to as CHO-hMR, was used for all subsequent experiments shown in the figures. Clone 83 was also used to control the results obtained with clone 65. In these clones, hMR expression levels were stable along cell cultures for at least 14 days (not shown). Clone 54, negative for hMR expression, was used as control and referred to as WT-CHO cells.

Upon fractionation of CHO-hMR cells, the receptor was mostly found in the membrane fraction (Fig. 2C) . Its binding and endocytic functionality were assessed using a classical hMR ligand HRP [54 55 56 57 ]. Endocytosis of HRP was measured in the presence and the absence of mannan to distinguish receptor-mediated endocytosis from fluid-phase endocytosis. In contrast to WT-CHO cells, a significant MR-mediated HRP endocytosis was observed in CHO-hMR cells (Fig. 2D) . It was quantitatively comparable with J774E cells, as the expression level of MR was two times higher in these cells, as determined by quantifying two Western blots including that shown in Figure 2C , right. These data demonstrate that de novo-cloned hMR has an efficient binding and endocytic capacity.

CHO-hMR cells do not display phagocytic properties
Zymosan particles and mycobacteria have been described to be internalized through the MR in human macrophages or MR-transfected COS cells [41 , 42 , 52 ]. It is surprising that phagocytosis of zymosan (Fig. 3A ) or M. kansasii (Fig. 3B) in CHO-hMR cells did not exceed that measured in WT-CHO cells, even when the time of contact between cells and particles was prolonged until 16 h (Fig. 3) or when different MOIs (100:1, 50:1, and 20:1) were used (not shown). Even when the high-affinity hMR ligand Man3-BSA [42 ] was coated on latex beads, no phagocytosis was obtained (Fig. 3C) . Subclone 83 gave identical results as clone 65 (data not shown).



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  		Figure 3. MR does not mediate phagocytosis when stably expressed in CHO cells. CHO-hMR cells were incubated with zymosan (A), M. kansasii (B), or Man3 BSA-coated latex beads (C) for 3 or 16 h. Where indicated, particles were opsonized in human serum (Opsonization) and incubated with cells transiently expressing the human Fc{gamma}RIIA receptor (FcR). The percentage of phagocytic cells having ingested at least one particle was measured by fluorescence microscopy by counting at least 100 cells per slide. Results are expressed as the mean ± SD of three independent experiments performed in duplicate.

 
Next, WT-CHO cells and CHO-hMR clone 65 were transfected with Fc{gamma}RIIA, a professional phagocytic receptor. Under these conditions, they ingested Ig-coated zymosan (Fig. 3A) or M. Kansasii (Fig. 3B) , indicating that they are able to accomplish a phagocytic process. In addition, two human cell lines, HeLa and Hep2 cells, were used to test the phagocytic activity of the hMR. These cells transiently expressing hMR cDNA did not perform phagocytosis of M. kansasii or zymosan, whereas they efficiently internalized IgG-opsonized zymosan when transfected with Fc{gamma}RIIa in parallel experiments (data not shown). These data indicate that the hMR is not a professional phagocytic receptor.

As the MR of human macrophages is able to ingest mannosylated particles [42 ], we hypothesized that it could collaborate with a partner to mediate phagocytosis. A large pattern of receptors is operating at the surface of macrophages to ingest microorganisms [5 ]. CR3 (CD11b/CD18) is a good candidate to cooperate with MR, as it has been described to participate in phagocytosis of mycobacteria [46 , 59 , 60 ], and it cooperates with multiple receptors [61 ]. To study its potential collaboration with MR, CHO cells stably expressing the human CR3 were transiently transfected with hMR. Phagocytosis of zymosan and mycobacteria by CR3 expressed in CHO cells requires overnight incubation [45 , 46 ]. To examine a potential synergistic effect of the two receptors, we thus reduced the time of contact between cells and particles to 3 h. Expression of hMR in CR3-expressing CHO cells did not potentiate the rate of CR3-mediated phagocytosis of zymosan or M. kansasii (Fig. 4 ), indicating that CR3 is unable to link hMR to the phagocytic machinery in CHO cells. Potentiation of phagocytosis was not detected when cells and particles were left in contact for 16 h (not shown) either. When phagocytosis was triggered by complement-coated particles (opsonization in human serum), phagocytosis mediated by CR3 was, as expected, more active than under nonopsonic conditions (Fig. 4) but was not improved by the presence of hMR either.



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  		Figure 4. CR3 does not cooperate with MR to mediate particles phagocytosis. CHO cells stably expressing CR3 (CHO-CR3) were transiently transfected with hMR cDNA (CHO-CR3-hMR) and were incubated with nonopsonized or opsonized zymosan or M. kansasii for 3 h. The percentage of phagocytic cells having ingested at least one particle was measured by fluorescence microscopy by counting at least 100 cells per slide. Results are expressed as the mean ± SD of three independent experiments performed in duplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our present purpose was to gain additional knowledge about the role and signaling pathways of the MR during phagocytosis of mycobacteria and other particles. Professional phagocytic receptors are able to ingest particles when expressed in nonphagocytic cells. It is surprising that although hMR endocytic activity was functional, expression of de novo-cloned hMR had no phagocytic activity in the three cell types studied, which, however, acquired a full phagocytic capacity when they expressed a professional phagocytic receptor. Although there are substantial evidences that MR has a role to play in phagocytosis (see ref. [1 ] for review) and thereby, is an actor of immunity, the data presented here demonstrate that it is not a professional phagocytic receptor but rather a binding receptor that needs a coeffector to trigger the phagocytic machinery.

Difficulties were experienced when manipulating the full-length hMR cDNA. This may explain why most of the studies about the structure/function relationships have used chimeric constructs containing extracellular, transmembrane, or intracellular domains of hMR in fusion with parts of other receptors [21 , 22 , 62 63 64 65 66 67 68 ]. The problems encountered when using the full-length hMR cDNA were overcome by interrupting the coding frame with an intron in which stop codons were present and by propagating the cDNA in a plasmid containing a p15A-replicative origin instead of ColE1. A Thr521Pro substitution and an allelic Arg1415His polymorphism, which yielded a BspLU11 I restriction fragment-length polymorphism, were detected in our cDNA. The Thr521Pro substitution is located upstream of CRDs 4–8, which possess identical affinity for ligands as the intact MR [20 ]. The ability of the hMR in CHO cells to mediate endocytosis of HRP, with apparently a similar efficiency as in J774E cells, confirms that none of the variations affect the ligand-binding or endocytosis capacities of the hMR.

Only two studies documenting expression of the hMR have been published contemporary to the initial cloning of the cDNA. They report that the MR is able to internalize Candida albicans or Pneumocystis carinii when expressed in COS cells [16 , 33 ], but whether cells other than COS can mediate MR-dependent phagocytosis or whether this is a feature of COS cells that expresses accessory molecule(s) required to complement the receptor in the phagocytic event was discussed [33 ]. In the present paper, we decided to examine this point using CHO, HeLa, and Hep2 cells. We report here that CHO cells stably expressing hMR are unable to promote phagocytosis of zymosan, M. kansasii, or Man3 BSA-coated latex beads, all known to be internalized by the MR in human macrophages [41 , 42 , 52 ]. Actually, despite the capacity of CHO cells to perform type I and type II phagocytosis when expressing CR3 [44 , 45 ] and of HeLa and Hep2 cells to ingest IgG-coated particles when expressing Fc{gamma}RIIa, none of these cells internalized mannosylated particles upon hMR expression. It indicates that although these cells possess the signaling machinery and the cytoskeleton dynamics required for phagocytosis, expression of hMR was not sufficient to trigger a phagocytic process. Actually, none of the receptors belonging to the MR family, the phospholipase A2 receptors [69 ], DEC-205 [70 , 71 ], and Endo180 [72 ], is able to mediate phagocytosis, despite their high sequence homology (see ref. [4 ] for review). Also, MR is expressed in various reticuloendothelial tissues [9 10 11 12 13 14 , 73 ], where MR-mediated phagocytosis of microorganisms has never been reported. Therefore, contrary to Fc{gamma}RIIa and CR3, which possess on their own the ability to bind particles and trigger their ingestion in every kind of cell in which they are expressed, the MR is not a professional phagocytic receptor.

Actually, the MR could play a binding function. Fusion of human MDMs to form giant cells [74 ] and binding of lymphocytes to L-selectin on lymphatic endothelium involve the MR [75 ]. So, the MR may function to bind particles with appropriate saccharide residues, triggering its interaction with plasma membrane partners that promote the cytoskeleton reorganization required for phagocytosis. For example, uptake of apoptotic cells by macrophages and nonprofessional phagocytes, e.g., epithelial cells, uses a combination of binding activities (phosphatidyl serine and/or scavenger receptors, mannose-binding activity, thrombospondin) coupled to cytoskeleton rearrangement mediated by integrins and {alpha}-2-macroglobulin receptors [76 77 78 79 ]. Other multimolecular complexes implicated in phagocytic processes often involved CR3 (see ref. [80 ] for review). Although in macrophages, CR3 was a potential partner of binding receptors to link them to the cytoskeleton and in fine, mediate phagocytosis, we could not demonstrate that CR3 is functionally associated with MR in CHO cells. The involvement of other "interactors" has to be further investigated, possibly by coexpression in CHO-hMR cells. A cellular model to identify these hMR interactors would be to use COS cells, which may have, like macrophages, the particularity to express a (set of) protein(s) required for hMR to mediate phagocytosis, as previously discussed by Ezekowitz and coauthors [16 ].

In conclusion, by generating a new expression vector, we have been able to consistantly produce hMR in different cell lines. It constitutes a critical tool to study the function and signaling of the full-length receptor. In addition, we propose to reconsider the MR as a binding, rather than a phagocytic receptor. It only mediates ingestion of microorganisms in macrophages and dendritic cells, which specifically express its phagocytic partners. In the other cell types, it might rather work as an endocytic receptor for hormones and glycoproteins, thus participating in serum protein homeostasis. In fact, the phenotype exhibited by MR–/– knockout mice is not a phagocytic defect [81 , 82 ] but a strong modification of serum glycoproteins homeostasis [32 ].


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Centre National de la Recherche Scientifique (Action Incitative 0693 and Action Incitative Microbiologie MIC 0336-040032/CNRS). We gratefully acknowledge Philip D. Stahl and Elizabeth Peters (Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO) for the generous gift of anti-hMR antibodies and for HRP endocytosis experiments. We thank Jacques Prandi (I.P.B.S. CNRS UMR 5089, Toulouse, France) for the generous gift of the trimannoside and T. A. Springer for the generous gift of CHO-CR3 cells. We thank P. D. Stahl, D. Zerbib, and D. Hodzig for critical reading of the manuscript. We thank A. Ezekowitz (Pediatric Service, Harvard Medical School, Massachusetts General Hospital, Boston, USA) and K. Drickamer (Glycobiology Institute, Department of Biochemistry, University of Oxford, UK) for the generous gift of their respective hMR cDNA.


   FOOTNOTES
 
2 Current address: CNRS UMR 5088, 118 route de Narbonne, Université Paul Sabatier Toulouse III, 31000 Toulouse, France. Back

Received December 7, 2004; revised January 31, 2005; accepted February 11, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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