* Sir William Dunn School of Pathology, Oxford, United Kingdom; and
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
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Key Words: macrophage • shedding
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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-
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.
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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).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
Received September 11, 2002; revised December 20, 2002; accepted January 13, 2003.
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