Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martinez-Pomares, L.
Right arrow Articles by Wong, S. Y. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martinez-Pomares, L.
Right arrow Articles by Wong, S. Y. C.
(Journal of Leukocyte Biology. 2003;73:604-613.)
© 2003 by Society for Leukocyte Biology

Analysis of mannose receptor regulation by IL-4, IL-10, and proteolytic processing using novel monoclonal antibodies

Luisa Martinez-Pomares*, Delyth M. Reid{dagger}, Gordon D. Brown*, Philip R. Taylor*, Richard J. Stillion*, Sheena A. Linehan*, Susanne Zamze{dagger}, Siamon Gordon* and Simon Y. C. Wong{dagger}

* Sir William Dunn School of Pathology, Oxford, United Kingdom; and
{dagger} The Edward Jenner Institute for Vaccine Research, Compton, Newbury, United Kingdom

Correspondence: Luisa Martinez-Pomares, Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK. E-mail: Luisa.Martinez-Pomares{at}pathology.ox.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The study of the murine macrophage mannose receptor (MR) has been hampered by the lack of specific reagents. We have generated and characterized novel anti-MR monoclonal antibodies and used them to analyze MR expression in primary mouse macrophages (MØ). In BioGel- and thioglycollate-elicited MØ, interleukin (IL)-4 up-regulated total cell-associated MR (cMR), correlating with enhanced surface expression. We investigated the influence of IL-10, a well-characterized deactivator of MØ function, on MR levels and observed that it had a similar effect to IL-4. In both cases, enhanced cMR levels translated into increased production of the soluble form of the receptor (sMR). We have demonstrated the presence of sMR in cultures of stable non-MØ transductants expressing full-length MR, indicating that the proteolytic activity responsible for cMR cleavage is not MØ-restricted. These data support a role for the MR in T helper cell type 2 cytokine-driven, immune responses and suggest a non-MØ contribution to sMR production in vivo.

Key Words: macrophage • shedding


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mannose receptor (MR) recognizes sugar moieties present on endogenous as well as foreign molecules. It was first described by its ability to mediate clearance of lysosomal hydrolases that share the presence of branched sugars with exposed mannose residues [1 ]. A role for the MR in this homeostatic process has been confirmed through the generation of MR-deficient mice [2 ]. These animals exhibited increased levels of multiple lysosomal enzymes under steady-state and inflammatory conditions. Therefore, this endocytic receptor seems to provide a clearance system for molecules that are up-regulated during inflammation, including ß-glucuronidase, tissue plasminogen activator, and neutrophil-derived myeloperoxidase [1 ]. This binding is mediated by some of the eight C-type lectin domains (CRD) arranged in tandem, which are present in the extracellular domain of the MR [3 ]. Abundant MR–CRD-binding activity has been found in salivary glands, exocrine pancreas, and thyroid [4 ]. Further analysis led to the identification of the prohormone thyroglobulin as a novel MR ligand [4 ]. These results suggested a wider role for the MR in clearance of self-molecules. Because of its ability to interact through its CRD with molecular structures associated with multiple microbes, such as Candida albicans [5 ], Pneumocystis carinii, [6 , 7 ], Mycobacterium tuberculosis [8 , 9 ], Klebsiella pneumoniae, and Streptococcus pneumoniae [10 ], the MR is also considered a pattern-recognition receptor [11 ].

A second lectin activity is mediated through the MR cysteine-rich (CR) domain. This extracellular domain recognizes sulfated N-acetyl-glucosamine or galactose residues present on glycoprotein hormones synthesized by the anterior pituitary [12 13 14 ], chondroitin sulfate, and sulfated Lewisx [15 ]. Sulfated glycoforms of sialoadhesin and CD45 expressed by specialized myeloid cells in secondary lymphoid organs [16 ] have also been described as CR domain ligands (CRL) [17 ]. No microbe-associated sugars have been found to interact with this region of the receptor [10 ].

The in situ distribution of the MR in the murine system has been analyzed using a rabbit polyclonal antiserum, which required the use of protease treatment [18 ] or prewetting of tissues [19 ]. Under these conditions, this reagent detected MR in tissue macrophages (MØ), sinusoidal and lymphatic endothelia, perivascular microglia, and mesangial cells [19 ]. Although no MR was observed on dendritic cells (DC) in naïve mice [19 ], limited expression on recruited or activated DC has been detected in inflamed human skin [20 ] and tonsils [21 ], indicating that the presence of MR in these antigen-presenting cells can occur under some circumstances in vivo. CRL+ cells do not themselves express the MR [19 ]. We hypothesized that these cells could interact with cell-associated MR (cMR) on MR+ cells and/or with a soluble form of the MR (sMR) [22 ] present in circulation [23 ], whose origin is ill-defined.

MR expression by cultured murine MØ is well-documented. The T helper cell type 1 (Th1)–Th2 paradigm correlated with down- and up-modulation, respectively, of this receptor. Interferon-{gamma} reduced MR synthesis [24 ], and interleukin (IL)-4 and IL-13, inducers of an alternative activation state in MØ [25 ], have opposite effects [25 , 26 ]. As a result of the limited availability of rabbit anti-MR polyclonal antiserum, these studies have been performed using biochemical approaches that could not provide information of the cellular heterogeneity in cultures or the subcellular distribution of the receptor in a quantitative manner.

Functional sMR has been detected in conditioned media from BioGel-elicited MØ [23 ]. Pulse-chase experiments demonstrated that this form of the receptor is released through proteolytic cleavage of the cMR by a metalloprotease [23 ]. Human monocyte-derived DC also produce sMR [27 ].

To date, no information is available regarding MR levels in murine cells in the presence of exogenous IL-10, a cytokine produced by, among others, Th2 cells, subsets of regulatory T cells [28 ], and stimulated MØ [29 ]. IL-10 induces a deactivated phenotype on MØ characterized by inhibition of proinflammatory cytokines [30 , 31 ]. Analysis of MR protein expression under these conditions will expand our understanding of the physiological role of MØ found during repair processes and parasitic infections [32 ] or that could originate in the periphery through the action of regulatory T cells [28 ].

In this report, we describe the generation and characterization of anti-mouse MR monoclonal antibodies (mAb) that could be used as MR-specific, detection reagents and as surrogate ligands to modulate MR activity. These unique reagents were used to assess MR regulation by IL-4 and IL-10 in two distinct populations of elicited, primary MØ, using a combination of flow cytometry and Western blotting. The effect of a metalloprotease inhibitor on MR processing and surface expression under these conditions has also been analyzed. Furthermore, we showed that sMR production is not restricted to cells of the MØ lineage, as stable cell lines expressing full-length MR also shed sMR. The implications of these findings for MR biology will be discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Murine IL-4 and IL-10 were obtained from R&D Systems (Minneapolis, MN). Fluorescein isothiocyanate (FITC)-labeled mannose bovine serum albumin (BSA) and FITC-labeled galactose BSA were from Sigma (Poole, Dorset, UK) or EY Laboratories (San Mateo, CA). Mannan was from Sigma. Metalloprotease inhibitor BB2116 was a kind gift of Dr. Andrew Gearing of British Biotech (Oxford, UK). DiI(1,1'-Dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled)-acetylated, low-density lipoprotein (AcLDL) was from Intracell (Rockville, MD).

Animals
BALB/c and C57BL/6J mice were kept under specific, pathogen-free conditions at the Sir William Dunn School of Pathology (Oxford, UK) and were used at 10–12 weeks of age. All animals were handled in accordance with institutional guidelines.

Production of Fc chimaeric proteins
The chimaeric proteins CR-Fc [16 ], CR-FNII-Fc [16 ], CRD4-7-Fc [4 ], and CD33-Fc (a generous gift from Paul R. Crocker, University of Dundee) were generated as described [4 , 16 ].

Generation of stable transductants
NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) containing 10% fetal calf serum (FCS), 50 units/ml penicillin, and 50 µg/ml streptomycin. Chinese hamster ovary (CHO) cells were maintained in F12 medium (Invitrogen) containing 10% FCS, 50 units/ml penicillin, and 50 µg/ml streptomycin. Full-length MR was generated as follows: a HindIII fragment encoding the amino terminal end of MR, derived from the pIG-derived plasmid encoding the fusion protein CR-Fc [16 ], was subcloned into the HindIII site of the pSH plasmid to introduce a SalI site at the 5' end. A SalI–Eco47III fragment was cloned between the SalI–Eco47III sites of the pUC18-derived plasmid containing MR cDNA starting at nucleotide 175 of the full-length clone (kindly provided by Dr. R. Alan B. Ezekowitz). To obtain stable cell lines, the cDNA clone was subcloned into the retroviral vector pFB(neo); (Stratagene, San Diego, CA) and was transfected into Phoenix ecotropic packaging cells using Fugene transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) following the manufacturer’s instructions. After 48 h, retroviral supernatants were harvested and used to transduce NIH3T3 cells in the presence of 5 µg/ml Polybrene (Sigma). CHO cells were transduced after treatment with tunicamycin as described [33 ]. Stable transductants were selected using 0.6 mg/ml geneticin (Sigma). NIH3T3-derived cells expressing MR were clonally selected by Western blot analysis using a rabbit polyclonal anti-mouse MR antiserum (kindly provided by Dr. Philip Stahl, Washington University Medical School, St. Louis, MO). CHO-derived cells were enriched by fluorescein-activated cell sorting (FACS), based on their ability to endocytose FITC-labeled, mannosylated BSA. NIH3T3-derived dectin-1 transductants are described elsewhere [34 ].

Endocytosis assays
Using stable transductants
Cells plated on tissue culture-treated plastic were incubated with FITC-labeled mannose BSA, galactose BSA (5 µg/ml), or Alexa 488-labeled rat mAb (5 µg/ml) and were incubated at 37°C for 60–120 min. After incubation, cells were washed with phosphate-buffered saline (PBS), harvested using trypsin-EDTA, and fixed in 2% formaldehyde in PBS. Uptake was quantified on a FACScalibur flow cytometer, using CellQuest software.

Using primary MØ
Thioglycollate-elicited, peritoneal MØ were cultured overnight in poly-D-lysine-treated tissue-culture plastic (Becton Dickinson Labware, Bedford, MA) containing 100 µg/ml purified rat mAb cross-linked to the plastic as described [35 ]. After washing cells with Opti-MEM (Invitrogen), FITC-labeled galactose BSA or mannose BSA (5 µg/ml) in Opti-MEM was added in the presence or absence of DiI-AcLDL (5 µg/ml). After 30 min, plates were transferred to 4°C and washed with ice-cold PBS. MØ were suspended in PBS containing 10 mM EDTA by scraping, fixed using 2% formaldehyde, and analyzed as above.

Generation of anti-MR mAb
Fischer rats were immunized and boosted with 50 µg CRD4-7-Fc in complete Freund’s adjuvant or incomplete Freund’s adjuvant, respectively. Four days before fusion, animals were boosted with 30 µg CRD4-7-Fc in PBS. Spleens were collected, and splenocytes were fused to the Y3 myeloma cell line [36 ]. Selection of anti-MR Ab-producing hybridomas was performed by immunohistochemistry on mouse tissues as described below and confirmed by Western blot analysis of 3T3-MR, MØ cell lysates, and Fc-chimaeric proteins.

Antibody purification and labeling
Rat antibodies were purified from hybridoma supernatants using GammaBind Plus sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden). Antibodieswere labeled using an Alexa 488-labeling kit from Molecular Probes (Eugene, OR) and were biotinylated using Sulfo-NHS-biotin (Pierce, Rockford, IL), according to the manufacturers’ instructions.

Immunohistochemistry
Fresh mouse tissues were embedded in Tissue-Tek OCT compound (Bayer Diagnostics) and were frozen on dry ice-cooled isopentane. Sections (5 µm) were cut, air dried, and frozen. Sections were thawed at room temperature, fixed in ethanol or 2% paraformaldehyde in Hepes-buffered saline, permeabilized in 0.1% Triton X-100 in PBS, quenched using glucose oxidase or 3% H2O2, and blocked in 5% normal rabbit serum in PBS. Primary rat antibodies diluted in blocking buffer were added and incubated for 1 h at room temperature. Binding was detected using biotinylated rabbit anti-rat immunoglobluin (Ig; Vector, Peterborough, UK) and the HRP-ABC detection system (Vector). To test for MR expression in stable transfectants, cells were plated on coverslips and after overnight culture, were fixed using 2% paraformaldehyde in Hepes-buffered saline. MR detection was performed as before but using normal goat serum for blocking and horseradish peroxidase (HRP)-conjugated goat anti-rat Ig (Chemicon, Harrow, UK) for detection.

Macrophage cultures
BioGel- and thioglycollate-elicited peritoneal cells were collected by lavage 4 days after intraperitoneal injection of each stimulus. MØ were selected by adherence for 2 h in Opti-MEM and were cultured in RPMI medium containing 10% FCS, 50 units/ml penicillin, and 50 µg/ml streptomycin.

Detection of MR by Western blot
After removing the media, cells were washed in PBS and lysed in ice-cold lysis buffer (2% Triton X-100, 10 mM Tris-HCl, pH 8, 150 mM NaCl, 2 mM NaN3, 2 mM EDTA) containing protease inhibitors (Roche Molecular Biochemicals) for 45 min at 4°C. Lysates were harvested and centrifuged at 2000 rpm in a tabletop centrifuge to eliminate nuclei and were stored at -20°C. Protein concentration was determined using the bicinchoninic acid assay (Pierce). Cell lysates and supernatants were electrophoresed in a 6% sodium dodecyl sulfate-polyacrylamide gel under nonreducing conditions and were transferred to nitrocellulose using standard procedures. MR was visualized using a combination of anti-MR mAb and HRP-conjugated anti-rat IgG (Chemicon) or rabbit polyclonal antiserum and HRP-conjugated anti-rabbit IgG (Jackson Laboratories, Bar Harbor, ME). Binding was detected using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Bucks, UK).

Surface biotinylation
Cells plated in 9 cm2 dishes were washed extensively in ice-cold PBS containing 10 mM CaCl2 and 10 mM MgCl2 (PBS/Ca2+/Mg2+). Biotin solution (Sulfo-NHS-biotin, 0.5 mg/ml in PBS/Ca2+/Mg2+, Pierce) was then added (750 µl/dish) and left for 30 min at 4°C. To stop the reaction, cells were incubated at 4°C with RPMI medium and washed with ice-cold PBS/Ca2+/Mg2+. Cell lysates were prepared as before but with further centrifugation at 100,000 g for 30 min. Total MR was immunoprecipitated using MR5D3 (10 µg/ml) and Gammabind Plus sepharose. Detection of total MR was performed by Western blot analysis, using rabbit polyclonal antiserum against murine MR. Biotinylated protein was detected using streptavidin-HRP (Sigma).

FACS analysis
As there were no differences regarding MR expression between cells plated on bacteriologic or tissue culture-treated plastic (data not shown), the first substrate, which facilitates cell recovery with minimal cell damage, was adopted for flow cytometry. MØ, plated on bacteriologic plastic, were harvested using 5 mM EDTA and lidocaine (4 mg/ml) in PBS. To analyze surface MR expression, cells were washed in washing buffer (PBS containing 0.5% BSA, 5 mM EDTA, and 2 mM NaN3), blocked in blocking buffer (washing buffer containing 5% heat-inactivated normal rabbit serum and 2.4G2, a rat anti-FcRI/II mAb, at 10 µg/ml) for 45 min at 4°C, and incubated with primary rat Ab (10 µg/ml in blocking buffer) for 1 h. After this incubation, cells were washed three times in washing buffer and fixed in 2% formalin in PBS. If the primary Ab was not labeled, 2.4G2 treatment was omitted, and FITC-conjugated donkey anti-rat IgG (Jackson Laboratories) diluted 1:100 in blocking buffer was added after the washes. After 45 min, samples were processed as before. If biotinylated Ab were used, detection was performed using streptavidin–allophycocyanin (APC; PharMingen, San Diego, CA). Control, biotinylated IgG2a was from PharMingen. Phycoerythrin (PE)-labeled F4/80 was from Serotec (Oxford, UK).

For total MR labeling, cells were fixed in 2% paraformaldehyde in PBS for 30 min at 4°C, and all incubations were performed in the presence of 0.5% saponin (Sigma).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of stable transductants expressing functional MR
Two stable cell lines, 3T3-MR and CHO-MR, expressing full-length, functional, murine MR, were generated by transduction of NIH3T3 and CHO cells, respectively. Both transduced cell lines selectively endocytosed FITC-labeled, mannosylated BSA (Fig. 1 ) and biotinylated D-mannose coupled to a soluble polyacrylamide support (data not shown). Uptake was inhibited by mannan (0.5 mg/ml). This activity correlated with the presence in cell lysates from 3T3-MR of a 175-kDa molecule that cross-reacted with a rabbit polyclonal serum raised against mouse MR (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 1. Presence of MR induces specific uptake of mannose BSA by NIH3T3 and CHO cells. Wild-type (wt) and MR-expressing NIH3T3 and CHO cells were incubated with FITC-labeled mannose BSA (Man-BSA) or galactose BSA (Gal-BSA; 5 µg/ml) for 90 min at 37°C, and uptake was quantified by flow cytometry as described in Materials and Methods.

 
Generation of antimurine MR mAb
Hybridomas were screened by immunohistochemistry, and antibodies that labeled tissues following a MR-staining pattern similar to that described by Linehan et al. [19 ] were selected, cloned, and expanded. Three mAb (MR6F3, MR6C3, and MR5D3), all IgG2a, were purified and tested by flow cytometry for binding to MR-transduced and nontransduced (wt) cells. None showed binding to the wt cells (data not shown) but specifically labeled MR-expressing cells (Fig. 2A ). An apparent binding hierarchy that correlated with Western blot analysis (data not shown) was observed, indicating that MR5D3 probably recognized murine MR with highest affinity. As shown in Figure 2B , the CRD4-7 region of the receptor contained the MR5D3 epitope, as assessed by its recognition of cell-associated and soluble MR in cell lysates and supernatants from 3T3-MR and of the recombinant protein CRD4-7-Fc. As expected, based on the selection procedure, these reagents labeled transduced cells by immunocytochemistry (Fig. 2C ; and data not shown).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 2. Anti-MR mAb specifically recognize MR transductants. (A) Flow cytometric analysis of CHO-MR and 3T3-MR using anti-MR mAb. Cells were harvested using trypsin-EDTA and fixed using 2% paraformaldehyde. To perform intracellular staining, fixed cells were permeabilized using 0.5% saponin, which was maintained throughout the procedure. Cells were labeled with an isotype control (IgG 2a) mAb, MR6F3, MR6C3, or MR5D3 at 10 µg/ml. Binding was detected using FITC-labeled donkey-anti-rat IgG. No staining of nontransduced cells was observed (data not shown). (B) The epitope recognized by MR5D3 is within the CRD4-7 fragment of MR. Cell lysates from wt and 3T3-MR (20 µg/line), 3T3-MR supernatant (20 µl from 10x concentrated), and 30 ng CRD4-7-Fc, CR-FNII-Fc, CR-Fc, and CD33-Fc were electrophoresed under nonreducing conditions and transferred to nitrocellulose. MR5D3 binding was assessed using neat hybridoma supernatant and HRP-conjugated goat-anti-rat IgG. Molecular weight markers are shown. (C) MR5D3 binds specifically to 3T3-MR cells by immunocytochemistry. wt (3T3) and MR-expressing cells (3T3-MR) plated on coverslips were stained with purified MR5D3 (10 µg/ml) and HRP-conjugated goat-anti-rat Ig as described in Materials and Methods.

 
J774E, a murine MØ cell line selected for high expression of MR [37 ], was chosen to assess the specificity of these reagents in MØ-like cells. As shown in Figure 3A , MR6C3 and MR5D3 labeled J774E cells but failed to recognize J774 cells, which in our hands, do not express MR (data not shown). These results indicate that there was neither cross-reactivity with other MØ molecules nor high background as a result of Fc receptors. The expression pattern of the MR in mouse tissues was reassessed using purified MR5D3. The pattern observed with the polyclonal reagent [19 ] was confirmed, but in this case, there was no requirement for prewetting the tissues before fixation or protease treatment for detection. MR expression detected by mAb MR5D3 in lung (alveolar MØ), spleen (red pulp), and liver (Kupffer cells and liver endothelium) is shown in Figure 3B .



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Anti-MR mAb recognize native mouse MR. (A) Anti-MR mAb specifically recognize J774E cells by flow cytometry. J774 (MR-) and J774E (MR+) were detached using lidocaine-EDTA—fixed, permeabilized, and labeled with Alexa 488-conjugated IgG2a, MR6C3, or MR5D3 and F4/80-PE as described in Materials and Methods. Although both cell lines bound the F4/80 mAb, only the J774E clone interacted with the anti-MR reagents. (B) MR5D3 labeling of alveolar MØ in lung (B, panel A), splenic red pulp (B, panel B), and Kupffer cells and endothelium in liver (B, panel C). (B, panels D–F) Negative controls (secondary reagents only) for B, panels A–C, respectively.

 
Use of anti-mouse MR mAb as tools to analyze MR function
To determine if anti-MR mAb could be used as MR-specific endocytic tracers, wt NIH3T3 and NIH3T3-derived, stable transductants expressing the MR or the ß-glucan receptor (dectin-1) were incubated with several Alexa 488-labeled mAb. As shown in Figure 4A , only cells expressing MR showed specific uptake of anti-MR mAb regardless of the relatively elevated uptake of the IgG2a control mAb observed for all cell types. Unexpectedly, internalization of MR6F3 and MR6C3 was more efficient than that of MR5D3. These differences could not be explained by differences in the conjugation levels of the mAb or by enhanced binding properties upon labeling, as they all labeled unfixed, freshly isolated thioglycollate-elicited MØ in a similar manner (data not shown).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. Use of anti-MR mAb as tools to analyze MR function. (A) Anti-MR mAb behave as MR-specific endocytic tracers. wt NIH3T3 (dotted line) and NIH3T3-derived transductants expressing MR (bold line) or dectin-1 (thin line) were incubated with the indicated Alexa 488-labeled mAb (5 µg/ml) for 1 h. After harvesting, the amount of protein internalized by the cells was quantified by FACS as described in Materials and Methods. (B) Plating MØ on anti-MR mAb reduces their capacity to internalize mannose BSA. Thioglycollate-elicited, peritoneal MØ plated on anti-MR mAb, MR5D3 (bold line) and MR6F3 (dotted line), or an isotype-control antibody (IgG2a, thin line) were incubated with FITC-labeled mannose BSA or galactose BSA for 50 min, and internalized protein was quantified by FACS. (C) Plating MØ on anti-MR mAb does not affect their capacity to internalize DiI-AcLDL. Thioglycollate-elicited, peritoneal MØ, treated as above, were incubated with DiI-AcLDL for 50 min. Internalized DiI-AcLDL was quantified by FACS. The dotted histogram appearing at the left of the graph corresponds to MØ plated on the IgG2a control mAb and incubated in the absence of tracers.

 
To determine if anti-MR mAb could be used to modulate MR activity in primary MØ, thioglycollate-elicited MØ were plated on anti-MR or control mAb, and their ability to take up FITC-labeled mannose BSA was assessed. As shown in Figure 4B , a substantial decrease in the amount of internalized mannose BSA was observed in MØ plated on the MR-specific mAb MR5D3 and MR6F3. Under these conditions, uptake of DiI-AcLDL, an endocytic tracer specific for scavenger receptors, remained largely unaffected.

MR in primary MØ cultures is up-regulated by IL-4 and IL-10
MR expression on BioGel-elicited, peritoneal MØ was analyzed by Western blot using MR5D3 (Fig. 5A ). At the concentration tested, IL-10 enhanced MR expression in cell lysates and supernatants. This increase was comparable with that observed with IL-4. These conclusions were further supported by FACS analysis of thioglycollate- and BioGel-elicited MØ. Detailed analysis of total cMR expression in permeabilized cells at different times is shown in Figure 5B . A steady increase in the number of high MR expressing cells up to day 3 can be observed.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 5. Up-regulation of MR expression in IL-4- and IL-10-treated MØ. (A) Western blot analysis of cMR and sMR expression in cell lysates (Cells) and supernatants (Sups) from BioGel-elicited, peritoneal MØ. Cells cultured as described in Materials and Methods were left untreated (Unt) or treated with IL-4 (10 ng/ml) or IL-10 (100 U/ml). After 2 days, cell lysates and supernatants were harvested and analyzed for MR expression using purified MR5D3 (2 µg/ml) and HRP-conjugated goat anti-rat Ig. (B) FACS analysis of total cMR expression in BioGel (Bg) and thioglycollate (Thio)-elicited MØ at days 1–3 of culture in the absence of cytokines or in the presence of IL-4 (10 ng/ml) or IL-10 (100 U/ml). Cells, plated on bacteriologic plastic, were harvested using 5 mM EDTA in PBS, fixed with 2% paraformaldehyde, and permeabilized. Alexa 488-conjugated isotype-control IgG2a (dotted line), MR5D3, and MR6C3 were used in these assays. Similar binding of the isotype-control mAb in all conditions was observed (data not shown). Only MR5D3 labeling of MR is shown, but similar labeling results were obtained for MR6C3. Although a slight time-dependent cMR up-regulation was detected in untreated cultures (thick line), an enhanced cMR expression occurred in the presence of IL-4 (dotted and dashed lines) and IL-10 (thin line), and most cells expressed high levels of the receptor by day 3.

 
Surface MR expression is not affected by proteolytic cleavage
Although most MR is located intracellularly in early endosomal compartments [1 ], surface staining can be achieved in nonfixed cells by FACS analysis using biotinylated antibodies and APC-conjugated streptavidin. As shown in Figure 6 , increased, total cMR expression translated into enhanced surface labeling, indicating that surface expression was not specifically influenced by the cytokines tested.



View larger version (35K):
[in this window]
[in a new window]
  		Figure 6. Enhanced, total cMR levels correlate with increased cMR expression at the cell surface. Thioglycollate-elicited MØ, plated on bacteriologic plastic and maintained in the absence [untreated (Unt)] or presence of IL-4 or IL-10, were analyzed for total and surface MR expression using biotinylated MR5D3 and streptavidin APC as described in Materials and Methods.

 
Shedding has been shown to be an effective mechanism to regulate surface expression [38 ]. To determine if the metalloprotease-mediated cleavage of cMR regulated the cell-surface expression of MR, cells cultured overnight with the metalloprotease inhibitor BB2116 were tested for total and surface cMR levels. As shown in Figure 7A , no major differences in MR surface levels were observed in the presence of this compound, indicating that cleavage per se did not control the amount of cMR at the cell surface. These results were confirmed by surface biotinylation and immunoprecipitation studies (Fig. 7B) . Although BB2116 dramatically reduced the amount of sMR in MØ-conditioned media in the absence or presence of IL-4 (Fig. 7B , upper), it did not substantially increase MR surface expression (Fig. 7B , lower). Under these conditions, IL-4 treatment increased the amount of biotinylated receptor, which is consistent with the FACS analysis data shown in Figure 5 .



View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. cMR surface expression in MØ is not affected by shedding. (A) Similar levels of cMR were detected at the cell surface in the presence and absence of the metalloprotease inhibitor BB2116. Cultures (2 days old) of thioglycollate-elicited MØ, plated on bacteriologic plastic and maintained in the absence (Untreated) or presence of IL-4 or IL-10, were treated with BB2116 (50 µg/ml) overnight. On the next day, cells were harvested, and surface cMR levels were assessed as above. Dotted lines: isotype control; continuous lines: MR5D3. (B) Inhibition of sMR production does not result in enhanced cMR expression at the cell surface. (Upper) Media from 2-day-old cultures of thioglycollate-elicited MØ, plated in bacteriological plastic and maintained in the absence (untreated) or presence of IL-4, were replaced with fresh media, which for half of the cultures, included BB2116 (50 µg/ml). On the following day, cell lysates (Cells) and supernatants (Sup) were harvested and assessed for the presence of MR. Although cMR levels in cell lysates were not affected by the presence of BB2116, there was a major reduction of the levels of sMR in supernatants in the presence of this compound. (Lower) Thioglycollate-elicited MØ cultured for 3 days in the absence (-) or presence (+) of IL-4 and treated overnight (+) with BB2116 (50 µg/ml) were washed, and surface proteins were biotinylated as described in Materials and Methods. Total cell lysates were prepared, and MR was immunoprecipitated using MR5D3 (10 µg/ml) and GammaBind Plus sepharose. Immunoprecipitated material was electrophoresed and transferred to nitrocellulose. Detection of total cMR was achieved using a rabbit polyclonal antiserum against MR (anti-MR) and of biotinylated protein, using HRP-conjugated Extravidin.

 
Production of sMR by MR stable transductants
Cell lysates and supernatants from CHO and 3T3 (wt and MR transductants) cells were analyzed for the presence of MR (Fig. 8 ). A cleavage product that comigrated with the form produced by BioGel-elicited MØ was detected in the conditioned media from the transductants.



View larger version (83K):
[in this window]
[in a new window]
  		Figure 8. sMR is produced by MR transductants. Cell lysates and supernatants from BioGel-elicited MØ, CHO cells (wt and MR transductants), and supernatants from NIH3T3-MR transductants were analyzed by Western blot for the presence of MR as described in Materials and Methods. Different volumes of supernatants from CHO-MR transductants (as indicated by the triangle) were analyzed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The MR is a well-characterized, endocytic receptor involved in homeostasis and immunity. Its recognition of self-derived [1 , 2 , 4 , 12 ] and foreign molecules [11 ] together with its regulated expression by MØ [24 25 26 ] and the presence of MR–CR domain ligands in secondary lymphoid organs [16 , 39 , 40 ] make it a suitable model to analyze the interplay between the immunological and physiological functions performed by MØ. In the murine system, the study of MR biology has been limited by the lack of specific reagents. We are first to report the generation of antimurine MR mAb, MR5D3, MR6F3, and MR6C3. These reagents detect recombinant and native receptors by Western blotting, flow cytometry, immunoprecipitation, and immunohistochemistry. Anti-MR mAb would be valuable for the analysis of MR regulation in situ and in vitro under normal and pathological conditions and the characterization of MR+ cells ex vivo. Additionally, these reagents could be used as highly specific MR surrogate ligands, an approach that has been successfully used in other systems [21 , 41 , 42 ] and in the identification of signaling pathways that are triggered upon MR ligation. Results from previous attempts have been inconclusive as a result of the complex nature of the ligand(s) used [43 ] and the existence of additional mannose-binding lectins [44 , 45 ]. No blocking activity was observed for MR6F3, MR6C3, or MR5D6 in in vitro binding assays in which the interaction of sMR with immobilized MR-specific ligands was analyzed [10 ]. Approximately 50% inhibition of mannose–BSA uptake by MR-3T3 was observed when high concentrations (40 µg/ml) of MR6F3 were used (data not shown). Nevertheless, these reagents have proved to be useful in down-modulating MR activity in primary MØ cultures by sequestrating the receptor from the cell surface.

We used the anti-MR mAb to assess MR regulation in cultured MØ by two Th2 cytokines, IL-4 and IL-10. Our results confirmed the reported up-regulation of cMR and sMR levels in response to IL-4 in BioGel-elicited MØ [25 ] and provided additional data showing enhanced surface cMR expression under these conditions and heterogeneity in primary MØ cultures in regard to MR expression. Furthermore, we demonstrated that these effects are not restricted to this particular MØ population, as peritoneal MØ elicited by the sterile stimulus thioglycollate broth responded in a similar manner. sMR production, enhanced by IL-4 treatment, was also detected in conditioned media from thioglycollate-elicited MØ.

We have detected increased MR expression in response to IL-10 in the two murine peritoneal MØ populations tested. These results increase the number of overlapping effects on MØ between IL-4 and IL-10. Both cytokines have been shown to increase arginase activity in murine MØ [46 ]. Although enhanced MR expression normally correlates with enhanced endocytic capacity, further analysis of MR function in IL-10-treated MØ is required to clarify the role of this receptor on these cells. In agreement with our results, IL-10 has been reported to increase MR expression in human monocyte-derived DC [47 ], but reduced endocytic activity has been described in human monocyte-derived MØ treated with IL-10 [48 ].

Cell-surface expression of cMR did not appear to be regulated by metalloprotease activity, as levels of cMR at the cell surface remained unchanged in the presence of BB2116, a major inhibitor of cMR cleavage. In contrast, the production of sMR in the presence or absence of IL-4 was almost totally abolished in the presence of BB2116. Therefore, sMR production doesn’t seem to represent a byproduct of MR synthesis involved in the control of cMR surface expression. The detection of ligands for the MR–CR domain in secondary organs, cells that surround B cell follicles in naïve animals, which can be found within B cell areas upon stimulation (CRL+ cells) [16 , 39 , 40 ], inspired the "sMR-mediated antigen-delivery hypothesis." According to this hypothesis, sMR could act as a bifunctional molecule and transport CRD-bound ligands to CRL+ cells [22 ]. Purified sMR from 3T3-MR cells fulfilled the functional requirements for such a role, as it bound SO4-3-galactose (a CR domain ligand) and bacterial capsular polysaccharides (CRD ligands) simultaneously [10 ]. The results presented in this work strengthen the case for a specific function for the sMR in vivo.

Stable transductants expressing full-length MR offered a suitable system to analyze the requirement for a MØ-restricted protease for the proteolytic cleavage of cMR to produce sMR. As sMR could be readily detected in the conditioned media of MR transductants, we conclude that the ability to produce sMR through cleavage of cMR is widely distributed. In vivo [23 ], sMR could be derived from lymphatic and hepatic endothelia, among others [19 ], in addition to MØ.

In summary, we describe the generation of highly specific mAb against murine MR. These mAb have proved useful in a wide range of techniques, such as Western blotting, immunohistochemistry, flow cytometry, immunoprecipitation, and affinity chromatography, and hence, are powerful tools for the analysis of MR regulation. We demonstrated that up-regulation of MR by IL-4 is not restricted to BioGel-elicited MØ and that IL-10 also has a positive effect on the expression of this molecule. Finally, our results indicate that sMR production does not affect surface cMR levels and that circulating sMR can derive from all MR+ cells.


   ACKNOWLEDGEMENTS
 
This work has been funded by the Wellcome Trust, the Edward Jenner Institute for Vaccine Research, the Arthritis Research Campaign, and the Medical Research Council. L. M-P. is a Yamanouchi Research Fellow at Wolfson College, Oxford. We thank Liz Darley for her expert help in immunohistochemistry.


   FOOTNOTES
 
Current address of Sheena A. Linehan, Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, The Flowers Building, Armstrong Road, London, SW7 2AZ, UK.

Received September 11, 2002; revised December 20, 2002; accepted January 13, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pontow, S. E., Kery, V., Stahl, P. D. (1992) Mannose receptor Int. Rev. Cytol. 137B,221-244
  2. Lee, S. J., Evers, S., Roeder, D., Parlow, A. F., Risteli, J., Risteli, L., Lee, Y. C., Feizi, T., Langen, H., Nussenzweig, M. C. (2002) Mannose receptor-mediated regulation of serum glycoprotein homeostasis Science 295,1898-1901[Abstract/Free Full Text]
  3. Taylor, M. E., Bezouska, K., Drickamer, K. (1992) Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor J. Biol. Chem. 267,1719-1726[Abstract/Free Full Text]
  4. Linehan, S. A., Martinez-Pomares, L., da Silva, R. P., Gordon, S. (2001) Endogenous ligands of carbohydrate recognition domains of the mannose receptor in murine macrophages, endothelial cells and secretory cells; potential relevance to inflammation and immunity Eur. J. Immunol. 31,1857-1866[CrossRef][Medline]
  5. Marodi, L., Korchak, H. M., Johnston, R. B., Jr (1991) Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages J. Immunol. 146,2783-2789[Abstract]
  6. Ezekowitz, R. A., Williams, D. J., Koziel, H., Armstrong, M. Y., Warner, A., Richards, F. F., Rose, R. M. (1991) Uptake of Pneumocystis carinii mediated by the macrophage mannose receptor Nature 351,155-158[CrossRef][Medline]
  7. O’Riordan, D. M., Standing, J. E., Limper, A. H. (1995) Pneumocystis carinii glycoprotein A binds macrophage mannose receptors Infect. Immun. 63,779-784[Abstract]
  8. Kang, B. K., Schlesinger, L. S. (1998) Characterization of mannose receptor-dependent phagocytosis mediated by Mycobacterium tuberculosis lipoarabinomannan Infect. Immun. 66,2769-2777[Abstract/Free Full Text]
  9. Bernardo, J., Billingslea, A. M., Blumenthal, R. L., Seetoo, K. F., Simons, E., Fenton, M. J. (1998) Differential responses of human mononuclear phagocytes to mycobacterial lipoarabinomannans: role of CD14 and the mannose receptor Infect. Immun. 66,28-35[Abstract/Free Full Text]
  10. Zamze, S., Martinez-Pomares, L., Jones, H., Taylor, P. R., Stillion, R. J., Gordon, S., Wong, S. Y. (2002) Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor J. Biol. Chem. 277,41613-41623[Abstract/Free Full Text]
  11. Stahl, P. D., Ezekowitz, R. A. (1998) The mannose receptor is a pattern recognition receptor involved in host defense Curr. Opin. Immunol. 10,50-55[CrossRef][Medline]
  12. Fiete, D., Baenziger, J. U. (1997) Isolation of the SO4-4-GalNAcbeta1,4GlcNAcbeta1,2Manalpha-specific receptor from rat liver J. Biol. Chem. 272,14629-14637[Abstract/Free Full Text]
  13. Fiete, D., Beranek, M. C., Baenziger, J. U. (1997) The macrophage/endothelial cell mannose receptor cDNA encodes a protein that binds oligosaccharides terminating with SO4-4-GalNAcbeta1,4GlcNAcbeta or Man at independent sites Proc. Natl. Acad. Sci. USA 94,11256-11261[Abstract/Free Full Text]
  14. Fiete, D. J., Beranek, M. C., Baenziger, J. U. (1998) A cysteine-rich domain of the "mannose" receptor mediates GalNAc-4-SO4 binding Proc. Natl. Acad. Sci. USA 95,2089-2093[Abstract/Free Full Text]
  15. Leteux, C., Chai, W., Loveless, R. W., Yuen, C-T., Uhlin-Hansen, L., Combarnous, Y., Jankovic, M., Maric, S. C., Misulovin, Z., Nussenzweig, M. C., Feizi, T. (2000) The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewisa and Lewisx types in addition to the sulfated N-glycans of lutropin J. Exp. Med. 191,1117-1126[Abstract/Free Full Text]
  16. Martinez-Pomares, L., Kosco-Vilbois, M., Darley, E., Tree, P., Herren, S., Bonnefoy, J. Y., Gordon, S. (1996) Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers J. Exp. Med. 184,1927-1937[Abstract/Free Full Text]
  17. Martínez-Pomares, L., Crocker, P. R., Da Silva, R., Holmes, N., Gordon, S. (1999) Cell-specific glycoforms of sialoadhesin and CD45 are counter receptors for the cysteine-rich domain of the mannose receptor J. Biol. Chem. 274,35211-35218[Abstract/Free Full Text]
  18. Takahashi, K., Donovan, M. J., Rogers, R. A., Ezekowitz, R. A. (1998) Distribution of murine mannose receptor expression from early embryogenesis through to adulthood Cell Tissue Res. 292,311-323[CrossRef][Medline]
  19. Linehan, S. A., Martinez-Pomares, L., Stahl, P. D., Gordon, S. (1999) Mannose receptor and its putative ligands in normal murine lymphoid and nonlymphoid organs: in situ expression of mannose receptor by selected macrophages, endothelial cells, perivascular microglia, and mesangial cells, but not dendritic cells J. Exp. Med. 189,1961-1972[Abstract/Free Full Text]
  20. Wollenberg, A., Mommaas, M., Oppel, T., Schottdorf, E. M., Gunther, S., Moderer, M. (2002) Expression and function of the mannose receptor CD206 on epidermal dendritic cells in inflammatory skin diseases J. Invest. Dermatol. 118,327-334[CrossRef][Medline]
  21. Engering, A., Geijtenbeek, T. B. H., van Vliet, S. J., Wijers, M., van Liempt, E., Demaurex, N., Lanzavecchia, A., Fransen, J., Figdor, C. G., Piguet, V., van Kooyk, Y. (2002) The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells J. Immunol. 168,2118-2126[Abstract/Free Full Text]
  22. Martinez-Pomares, L., Gordon, S. (1999) The mannose receptor and its role in antigen presentation Immunol. Lett. 65,9-13[CrossRef][Medline]
  23. Martinez-Pomares, L., Mahoney, J. A., Kaposzta, R., Linehan, S. A., Stahl, P. D., Gordon, S. (1998) A functional soluble form of the murine mannose receptor is produced by macrophages in vitro and is present in mouse serum J. Biol. Chem. 273,23376-23380[Abstract/Free Full Text]
  24. Harris, N., Super, M., Rits, M., Chang, G., Ezekowitz, R. A. (1992) Characterization of the murine macrophage mannose receptor: demonstration that the downregulation of receptor expression mediated by interferon-gamma occurs at the level of transcription Blood 80,2363-2373[Abstract/Free Full Text]
  25. Stein, M., Keshav, S., Harris, N., Gordon, S. (1992) Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation J. Exp. Med. 176,287-292[Abstract/Free Full Text]
  26. Doyle, A. G., Herbein, G., Montaner, L. J., Minty, A. J., Caput, D., Ferrara, P., Gordon, S. (1994) Interleukin-13 alters the activation state of murine macrophages in vitro: comparison with interleukin-4 and interferon-gamma Eur. J. Immunol. 24,1441-1445[Medline]
  27. Jordens, R., Thompson, A., Amons, R., Koning, F. (1999) Human dendritic cells shed a functional, soluble form of the mannose receptor Int. Immunol. 11,1775-1780[Abstract/Free Full Text]
  28. Maloy, K. J., Powrie, F. (2001) Regulatory T cells in the control of immune pathology Nat. Immunol. 2,816-822[CrossRef][Medline]
  29. Sutterwala, F. S., Noel, G. J., Salgame, P., Mosser, D. M. (1998) Reversal of proinflammatory responses by ligating the macrophage Fcgamma receptor type I J. Exp. Med. 188,217-222[Abstract/Free Full Text]
  30. Bogdan, C., Vodovotz, Y., Nathan, C. (1991) Macrophage deactivation by interleukin 10 J. Exp. Med. 174,1549-1555[Abstract/Free Full Text]
  31. D’Andrea, A., Aste-Amezaga, M., Valiante, N. M., Ma, X., Kubin, M., Trinchieri, G. (1993) Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells J. Exp. Med. 178,1041-1048[Abstract/Free Full Text]
  32. Hesse, M., Modolell, M., La Flamme, A. C., Schito, M., Fuentes, J. M., Cheever, A. W., Pearce, E. J., Wynn, T. A. (2001) Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism J. Immunol. 167,6533-6544[Abstract/Free Full Text]
  33. Miller, A. D., Miller, D. G., Garcia, J. V., Lynch, C. M. (1993) Use of retroviral vectors for gene transfer and expression Methods Enzymol. 217,581-599[Medline]
  34. Brown, G. D., Gordon, S. (2001) Immune recognition. A new receptor for beta-glucans Nature 413,36-37[Medline]
  35. Michl, J., Pieczonka, M. M., Unkeless, J. C., Silverstein, S. C. (1979) Effects of immobilized immune complexes on Fc- and complement-receptor function in resident and thioglycollate-elicited mouse peritoneal macrophages J. Exp. Med. 150,607-621[Abstract/Free Full Text]
  36. Galfre, G., Milstein, C., Wright, B. (1979) Rat x rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG Nature 277,131-133[CrossRef][Medline]
  37. Diment, S., Leech, M. S., Stahl, P. D. (1987) Generation of macrophage variants with 5-azacytidine: selection for mannose receptor expression J. Leukoc. Biol. 42,485-490[Abstract]
  38. Ludwig, A., Berkohut, T., Moores, K., Groot, P., Chapman, G. (2002) Fractalkine is expressed by smooth muscle cells in response to IFN-{gamma} and TNF-{alpha} and is modulated by metalloproteinase activity J. Immunol. 168,604-612[Abstract/Free Full Text]
  39. Berney, C., Herren, S., Power, C. A., Gordon, S., Martinez-Pomares, L., Kosco-Vilbois, M. H. (1999) A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein J. Exp. Med. 190,851-860[Abstract/Free Full Text]
  40. Yu, P., Wang, Y., Chin, R. K., Martinez-Pomares, L., Gordon, S., Kosco-Vibois, M. H., Cyster, J., Fu, Y. X. (2002) B cells control the migration of a subset of dendritic cells into B cell follicles via CXC chemokine ligand 13 in a lymphotoxin-dependent fashion J. Immunol. 168,5117-5123[Abstract/Free Full Text]
  41. Jiang, W., Swiggard, W. J., Heufler, C., Peng, M., Mirza, A., Steinman, R. M., Nussenzweig, M. C. (1995) The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing Nature 375,151-155[CrossRef][Medline]
  42. Howard, M. J., Isacke, C. M. (2002) The C-type lectin receptor Endo180 displays internalization and recycling properties distinct from other members of the mannose receptor family J. Biol. Chem. 277,32320-32331[Abstract/Free Full Text]
  43. Linehan, S. A., Martinez-Pomares, L., Gordon, S. (2000) Macrophage lectins in host defense Microbes Infect. 2,279-288[CrossRef][Medline]
  44. Valladeau, J., Ravel, O., Dezutter-Dambuyant, C., Moore, K., Kleijmeer, M., Liu, Y., Duvert-Frances, V., Vincent, C., Schmitt, D., Davoust, J., Caux, C., Lebecque, S., Saeland, S. (2000) Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules Immunity 12,71-81[CrossRef][Medline]
  45. Mitchell, D. A., Fadden, A. J., Drickamer, K. (2001) A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands J. Biol. Chem. 276,28939-28945[Abstract/Free Full Text]
  46. Munder, M., Eichmann, K., Moran, J. M., Centeno, F., Soler, G., Modolell, M. (1999) Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells J. Immunol. 163,3771-3777[Abstract/Free Full Text]
  47. Longoni, D., Piemonti, L., Bernasconi, S., Mantovani, A., Allavena, P. (1998) Interleukin-10 increases mannose receptor expression and endocytic activity in monocyte-derived dendritic cells Int. J. Clin. Lab. Res. 28,162-169[CrossRef][Medline]
  48. Montaner, L. J., da Silva, R. P., Sun, J., Sutterwala, S., Hollinshead, M., Vaux, D., Gordon, S. (1999) Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10 J. Immunol. 162,4606-4613[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
F. Marttila-Ichihara, R. Turja, M. Miiluniemi, M. Karikoski, M. Maksimow, J. Niemela, L. Martinez-Pomares, M. Salmi, and S. Jalkanen
Macrophage mannose receptor on lymphatics controls cell trafficking
Blood, July 1, 2008; 112(1): 64 - 72.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. J. McKenzie, P. R. Taylor, R. J. Stillion, A. D. Lucas, J. Harris, S. Gordon, and L. Martinez-Pomares
Mannose Receptor Expression and Function Define a New Population of Murine Dendritic Cells
J. Immunol., April 15, 2007; 178(8): 4975 - 4983.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. O. Martinez, S. Gordon, M. Locati, and A. Mantovani
Transcriptional Profiling of the Human Monocyte-to-Macrophage Differentiation and Polarization: New Molecules and Patterns of Gene Expression
J. Immunol., November 15, 2006; 177(10): 7303 - 7311.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
M. Dupasquier, P. Stoitzner, H. Wan, D. Cerqueira, A. van Oudenaren, J. S. A. Voerman, K. Denda-Nagai, T. Irimura, G. Raes, N. Romani, et al.
The dermal microenvironment induces the expression of the alternative activation marker CD301/mMGL in mononuclear phagocytes, independent of IL-4/IL-13 signaling
J. Leukoc. Biol., October 1, 2006; 80(4): 838 - 849.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
K. M. Jansen and G. K. Pavlath
Mannose receptor regulates myoblast motility and muscle growth
J. Cell Biol., July 31, 2006; 174(3): 403 - 413.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
G. Hassanzadeh Ghassabeh, P. De Baetselier, L. Brys, W. Noel, J. A. Van Ginderachter, S. Meerschaut, A. Beschin, F. Brombacher, and G. Raes
Identification of a common gene signature for type II cytokine-associated myeloid cells elicited in vivo in different pathologic conditions
Blood, July 15, 2006; 108(2): 575 - 583.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. E. M. Heinsbroek, P. R. Taylor, M. Rosas, J. A. Willment, D. L. Williams, S. Gordon, and G. D. Brown
Expression of Functionally Different Dectin-1 Isoforms by Murine Macrophages
J. Immunol., May 1, 2006; 176(9): 5513 - 5518.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Boskovic, J. N. Arnold, R. Stilion, S. Gordon, R. B. Sim, A. Rivera-Calzada, D. Wienke, C. M. Isacke, L. Martinez-Pomares, and O. Llorca
Structural Model for the Mannose Receptor Family Uncovered by Electron Microscopy of Endo180 and the Mannose Receptor
J. Biol. Chem., March 31, 2006; 281(13): 8780 - 8787.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Su, T. Bakker, J. Harris, C. Tsang, G. D. Brown, M. R. Wormald, S. Gordon, R. A. Dwek, P. M. Rudd, and L. Martinez-Pomares
Glycosylation Influences the Lectin Activities of the Macrophage Mannose Receptor
J. Biol. Chem., September 23, 2005; 280(38): 32811 - 32820.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
M. C. Gagliardi, R. Teloni, F. Giannoni, M. Pardini, V. Sargentini, L. Brunori, L. Fattorini, and R. Nisini
Mycobacterium bovis Bacillus Calmette-Guerin infects DC-SIGN- dendritic cell and causes the inhibition of IL-12 and the enhancement of IL-10 production
J. Leukoc. Biol., July 1, 2005; 78(1): 106 - 113.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Chaitidis, E. E. Billett, V. B. O'Donnell, A. B. Fajardo, J. Fitzgerald, R. J. Kuban, U. Ungethuem, and H. Kuhn
Th2 Response of Human Peripheral Monocytes Involves Isoform-Specific Induction of Monoamine Oxidase-A
J. Immunol., October 15, 2004; 173(8): 4821 - 4827.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
P. R. Taylor, S. Zamze, R. J. Stillion, S. Y. C. Wong, S. Gordon, and L. Martinez-Pomares
Development of a specific system for targeting protein to metallophilic macrophages
PNAS, February 17, 2004; 101(7): 1963 - 1968.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
J. Chehimi, Q. Luo, L. Azzoni, L. Shawver, N. Ngoubilly, R. June, G. Jerandi, M. Farabaugh, and L. J. Montaner
HIV-1 transmission and cytokine-induced expression of DC-SIGN in human monocyte-derived macrophages
J. Leukoc. Biol., November 1, 2003; 74(5): 757 - 763.
[Abstract] [Full Text] [PDF]

This Article