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Differential regulation of C-type lectin expression on tolerogenic dendritic cell subsets
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Immunobiology 211 (2006) 577–585 www.elsevier.de/imbio
Differential regulation of C-type lectin expression on tolerogenic dendritic cell subsets Sandra J. van Vliet1, Ellis van Liempt1, Teunis B.H. Geijtenbeek, Yvette van Kooyk Department of Molecular Cell Biology and Immunology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands
Abstract Antigen presenting cells (APC) express high levels of C-type lectins, which play a major role in cellular interactions as well as pathogen recognition and antigen presentation. The C-type lectin macrophage galactose-type lectin (MGL), expressed by dendritic cells (DC) and macrophages, mediates binding to glycoproteins and lipids that contain terminal GalNAc moieties. To investigate MGL expression patterns in more detail, we generated two new monoclonal antibodies and set up a quantitative real-time PCR analysis to determine MGL mRNA levels. MGL is not expressed by blood-resident plasmacytoid DC and thus represents an exclusive marker for myeloid-type APC. Dexamethasone treatment upregulated MGL expression on DC both at the protein and mRNA level in a time- and dose-dependent manner. In contrast, DC generated in the presence of IL-10 did not display enhanced MGL levels. Furthermore, dexamethasone and IL-10 also differentially regulated expression of other C-type lectins, such as DC-SIGN and Mannose Receptor. Our results demonstrate that depending on the local microenvironment, DC can adopt different C-type lectin profiles, which could have major influences on cell–cell interactions, antigen uptake and presentation. r 2006 Elsevier GmbH. All rights reserved. Keywords: C-type lectins; Dendritic cells; Macrophages; Plasmacytoid DC
Introduction Professional antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages (Mj), are seeded throughout all peripheral tissues where they scan their surroundings for incoming pathogens or local environmental changes. Mj mainly represent traditional tissueAbbreviations: APC, antigen presenting cell; ASGP-R, asialoglycoprotein receptor; DC, dendritic cell; GILZ, glucocorticoid-induced leucine zipper; GC, glucocorticoids; mAbs, monoclonal antibodies; MGL, macrophage galactose-type lectin; MR, mannose receptor; Mj, macrophage; pDC, plasmacytoid DC Corresponding author. Tel.: +31 204448080; fax: +31 204448081. E-mail address: [email protected] (Y. van Kooyk). 1 These authors contributed equally to this paper. 0171-2985/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2006.05.022
resident scavenging cells important in the effector phase of the immune response. Similar to Mj, DC also play an essential role in uptake of self- or pathogenic antigens. DC, once activated by proinflammatory stimuli or infectious pathogens, migrate towards the draining lymph node, where they initiate adaptive immunity (Taylor et al., 2005; Mellman and Steinman, 2001). Recently, Pozzi et al. (2005) demonstrated that also Mj can migrate to draining lymph nodes and activate naı¨ ve CD8+ T cells, although with lower efficiencies than DC. Next to immunity, DC contribute to tolerance via the induction of T cell unresponsiveness or apoptosis or via the induction of regulatory T cells. These processes can be mimicked in vitro by the addition of glucocorticoids (GC), such as dexametha-
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sone, or by adding IL-10 to DC cultures (Xia et al., 2005; Steinman et al., 2003). Different DC lineages can develop from separate precursors or represent various activation states of a single subtype. DC clearly possess a unique plasticity to adapt to environmental stimuli, leading to different functional phenotypes based on cell surface markers and production of cytokines and/or reactive metabolites (Janeway and Medzhitov, 2002). Furthermore, recent evidence indicates that mouse splenic DC and Mj renew from a common bone marrow progenitor that is able to develop into both subtypes depending on differential cytokine signaling (Fogg et al., 2006). In humans, several pathways exist for the development of the different DC subtypes, such as Langerhans cells, plamacytoid DC (pDC) and interstitial DC, each requiring their own set of growth factors and/or cytokines (Shortman and Liu, 2002). As only a few surface proteins are expressed exclusively by DC; new potential markers are required that can distinguish between the different phenotypic DC subtypes. One family of proteins, known to be differentially expressed by the various DC subsets, are the C-type lectins (Figdor et al., 2002). C-type lectins recognize specific carbohydrate moieties in a Ca2+dependent manner. They function as cell–cell adhesion molecules (Geijtenbeek et al., 2000) and as pattern recognition receptors for pathogens (Gordon, 2002). Moreover, C-type lectins can internalize ligands, such as pathogens, but also self-glycoproteins for processing and presentation to T cells (Engering et al., 2002). The C-type lectin macrophage galactose-type lectin (MGL) is expressed on in vitro cultured monocyte-derived DC and Mj (Suzuki et al., 1996). The carbohydrate recognition domain of MGL facilitates binding of terminal GalNAc-residues on glycoproteins, glyolipids or pathogens, in contrast to the well-known mannose/ fucose-specific lectins DC-SIGN and mannose receptor (MR) (van Vliet et al., 2005). Although MGL was originally described to be a specific marker for cells at an intermediate stage of differentiation from monocytes to Mj (Higashi et al., 2002b), other reports demonstrate MGL to be expressed by dendritic cells and alternatively activated macrophages (Higashi et al., 2002a; Raes et al., 2005). To further analyze the expression pattern of MGL on human DC subtypes, two new monoclonal antibodies (mAbs) directed against the C-type lectin MGL were generated. Our findings extend the knowledge on MGL expression patterns both at RNA and protein level on functionally different immature DC subsets. We demonstrate that MGL is exclusively expressed by myeloid DC and not by blood-resident pDC. Only dexamethasone treatment, and not IL-10, can enhance MGL expression on tolerogenic DC at both the protein and mRNA level. Furthermore, we show that expression of the C-type
lectins MGL, DC-SIGN and MR is differentially regulated, suggesting that depending on the cellular environment, DC can adopt various phenotypes with variable C-type lectin expression profiles.
Materials and methods Cells and reagents The cell lines CHO and CHO-MGL were maintained in RPMI containing 10% fetal calf’s serum (Invitrogen, Carlsbad, CA). Immature DC were cultured for 3–7 days from monocytes obtained from buffy coats of healthy donors (Sanquin, Amsterdam, The Netherlands) in the presence of IL-4 (500 U/ml) and GM-CSF (800 U/ml, both from Biosource, Camarillo, CA). pDC were isolated from buffy coats using the BDCA-4 cell isolation kit according to the manufacturer’s protocol (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). To some DC cultures, dexamethasone (at concentrations indicated in the figures, Sigma-Aldrich), IL-10 (10 ng/ml, Biosource) or LPS (100 ng/ml, SigmaAldrich, St. Louis, MO) was added. Monocytes were stimulated with IL-2 (200 U/ml, Biosource), IL-4 (500 U/ml), IL-10 (10 ng/ml), dexamethasone (106 M) or GM-CSF (1000 U/ml).
Generation of anti-MGL mAbs Balb/c mice were immunized three times with recombinant MGL-Fc (van Vliet et al., 2005). After the final boost, spleen cells were fused with SP2/0 cells at a 1:1 ratio using PEG. Hybridoma supernatants were screened for the presence of anti-MGL mAbs on CHOMGL transfectants. After two rounds of cloning, two hybridomas (1G6.6 and 18E4) were obtained that specifically recognize MGL.
Flow cytometry Cells were incubated with primary antibody (5 mg/ml), followed by staining with a secondary FITC-labeled goat anti-mouse antibody (Zymed, San Francisco, CA) and analyzed on FACScalibur (BD Pharmingen, San Diego, CA). The following mAbs were used: isotype control 28-14-8 (mouse anti-mouse H2 Db), AZN-D1 (DC-SIGN) and 3.29.B1 (MR) (Engering et al., 2002). The anti-MGL antibody MLD-1 was kindly provided by Dr. T. Irimura.
Immunohistochemistry Cryosections of healthy humans tissues (7 mm) were fixed with 100% acetone and stained with primary
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antibodies (10 mg/ml) for 1 h at 37 1C. Sections were counterstained with goat anti-mouse IgG2A-specific Alexa488 antibodies (Molecular probes, Eugene, OR) and analyzed by fluorescence microscopy.
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15 s at 95 1C and 1 min 60 1C. The fluorescence monitoring occurred at the end of each cycle. The Ct value is defined as the number of PCR cycles where the fluorescence signal exceeds the threshold value, which is
mRNA isolation and cDNA synthesis mRNA was isolated by capturing of poly(A+) RNA in streptavidin-coated tubes with an mRNA Capture kit (Roche, Basel, Switzerland) and cDNA was synthesized with the Reverse Transcription Sytem kit (Promega, Madison, WI) following manufacturer’s guidelines. Cells (0.1 106/well) were washed twice with ice-cold PBS and harvested with 100 ml lysis buffer. Lysates were incubated with biotin-labeled oligo(dT)20 for 5 min at 37 1C. The mix was transferred to streptavidin-coated tubes and incubated for 5 min at 37 1C. After washing three times with 200 ml washing buffer, 30 ml of the reverse transcription mix (5 mM MgCl2, 1 reverse transcription buffer, 1 mM dNTP, 0.4 U recombinant RNasin ribonuclease inhibitor, 0.4 U AMV reverse transcriptase, 0.5 mg random hexamers in nuclease-free water) was added to the tubes and incubated for 10 min at room temperature followed by 45 min at 42 1C. To inactivate AMV reverse transcriptase and separate mRNA from the streptavidin–biotin complex, samples were heated at 99 1C for 5 min, transferred to microcentrifuge tubes and incubated on ice for 5 min, diluted 1:2 in nuclease-free water, and stored at 20 1C.
Quantitative real-time PCR Oligonucleotides have been designed by using computer software Primer Express 2.0 (Applied Biosystems, Foster City, CA). Primers were synthesized by Invitrogen (Invitrogen). Primer specificity was computer tested (BLAST, National center for Biotechnology Information) by homology search with the human genome and later confirmed by dissociation curve analysis. Primers used in this study: GAPDH Fwd 50 -CCA TGT TCG TCA TGG GTG TG and Rev 50 - GGT GCT AAG CAG TTG GTG GTG; MGL Fwd 5’- TAC ACC TGG ATG GGC CTC AG, MGL Rev 50 - TGT TCC ATC CAC CCA CTT CC, DC-SIGN Fwd 5- AAC AGC TGA GAG GCC TTG GA and DC-SIGN Rev 50 GGG ACC ATG GCC AAG ACA. PCR reactions were performed with SYBR green method in an ABI 7900HT sequence detection system (Applied Biosystems). The reactions were set on a 96-well plate by mixing 4 ml of 2 SYBR Green Master Mix (Applied Biosystems) with 2 ml of the primer solution containing 5 nmol/ml of both primers and 2 ml of a cDNA solution. The cDNA synthesis product was diluted 1:2 in nuclease-free water. The thermal profile for all the reactions was 2 min at 50 1C, followed by 10 min at 95 1C and then 40 cycles of
Fig. 1. Generation of hybridomas producing MGL-specific mAbs. (A) Stable transfectants of CHO-MGL express high levels of MGL as assessed by flow cytometric analysis with a previously described anti-MGL antibody (kindly provided by Dr. T. Irimura). Open histograms represent isotype control staining, filled histograms represent MGL staining. (B) After two rounds of cloning, two MGL-specific hybridomas (1G6.6 and 18E4) were obtained that specifically stain CHO-MGL transfectants. Open histograms represent isotype control staining, filled histograms represent MGL staining on CHOMGL by the hybridomas 1G6.6 and 18E4. (C) New MGLmAbs recognize MGLpos cells in skin and lymph node, whereas no crossreactivity was observed with the highly homologous liver-specific molecule ASGP-R. Human tissue sections of skin, lymph node and liver were stained with 18E4 mAbs. ED, epidermis; D, dermis; OZ, outer zones of the paracortex. Arrows indicate MGL+ cells. Original magnification 400 . One representative experiment out of three is shown.
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fixed above 10 times the standard deviation of the fluorescence during the first 15 cycles and typically corresponds to 0.2 relative fluorescence units. This threshold is set constant throughout the study and corresponds to the log linear range of the amplification curve. The normalized amount of target, or relative abundance, reflects the relative amount of target transcripts with respect to the expression of the endogenous reference gene. The endogenous reference gene chosen was GAPDH.
Results To generate specific mAbs reactive with the C-type lectin MGL, Balb/c mice were immunized three times with purified MGL-Fc. After the final boost, spleen cells were fused with the SP2/0 myeloma. Hybridoma supernatants were screened by FACS for positive staining on CHO-MGL transfectants and negative reactivity with the parental CHO cells. CHO-MGL transfectants expressed high levels of MGL, as determined by flow cytometric analysis with a previously described antihuman MGL mAb (Fig. 1A and Higashi et al., 2002b). After two rounds of cloning, two MGL-reactive hybridomas, 1G6.6 and 18E4, of the IgG2A isotype were obtained (Fig. 1B). To further confirm the specificity of these hybridomas, human skin, lymph node and liver sections were stained with the new mAbs (Fig. 1C). In agreement with earlier reports (Higashi et al., 2002b), MGLpos cells were readily detected in the dermis of human skin. No crossreactivity with the most closely related human homolog of MGL, the liverspecific C-type lectin ASGP-R, was observed, confirming the exclusive specificity of the generated mAbs for MGL (Fig. 1C). In addition, human lymph node contained numerous MGLpos cells within the outer zones of the paracortex. Similar results were obtained for both anti-MGL mAbs. The availability of the anti-MGL mAbs allowed us to investigate MGL expression levels in various human DC
Fig. 2. MGL is not expressed by plasmacytoid DC. Highly enriched plasmacytoid DC (84% pure) were stained for BDCA-2 and MGL by flow cytometry. Open histograms indicate isotype control staining, filled histograms represent BDCA-2 or MGL staining. One representative experiment is shown.
Table 1.
CLR expression on stimulated monocytes
Stimuli
DC-SIGNa
MGL
Mannose receptor
IL-2 IL-4 IL-10 GM-CSF IL-4/GM-CSF IL-4/GM-CSF/LPS Dexamethasone
+ + +
+b + + +
+ + + + +
a Monocytes were cultured for 3 days in the presence of the indicated cytokines, glucocorticoids or combinations, after which C-type lectin expression was determined by flow cytometry (DC-SIGN, MGL and MR) and real-time PCR (DC-SIGN and MGL). + indicates induction of C-type lectin expression compared to unstimulated monocytes, no expression. b Upregulation only observed at the RNA level.
subsets. pDC represent a distinct DC population that is characterized by the markers BDCA-2 and BDCA-4 (Dzionek et al., 2001). To investigate whether next to myeloid-type DC, pDC also express MGL, we isolated pDC from human buffy coats. Although the isolated
Fig. 3. Dexamethasone treatment enhances MGL expression during DC differentiation. Immature DC were generated from monocytes by culturing for 4 days with IL-4 and GM-CSF in the presence or absence of IL-10 (A) or dexamethasone (B–D). (A) Cell surface expression of MGL, DC-SIGN and MR was determined by FACS analysis on immature DC (filled histograms) and IL10-cultured DC (10 ng/ml, solid line). The isotype control staining is indicated by the thin line. (B) Cell surface expression of MGL, DC-SIGN and MR was determined by FACS analysis on immature DC (filled histograms) and dexamethasone-cultured DC (106 M, solid line). The isotype control staining is indicated by the thin line. (C) Dose response curve of dexamethasone-induced MGL expression. Monocytes were cultured with IL-4/GM-CSF and dexamethasone at the indicated concentrations. At day 4, cells were harvested and MGL mRNA levels were determined by quantitative real-time PCR. The dashed line indicates the MGL mRNA level in untreated immature DC. (D) Dose response curve of dexamethasone-mediated inhibition of DC-SIGN expression. Monocytes were cultured with IL-4/GM-CSF and dexamethasone at the indicated concentrations. At day 4, cells were harvested and DC-SIGN mRNA levels were determined by quantitative real-time PCR. The dashed line indicates the DC-SIGN mRNA level in untreated immature DC. (E) Kinetics of dexamethasone-induced MGL expression. Monocytes were cultured with IL-4/GM-CSF in the presence or absence of dexamethasone (106 M) for 3–7 days. At indicated timepoints, cells were harvested and MGL mRNA levels were determined by quantitative real-time-PCR. One representative experiment out of three is shown.
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pDC clearly express the pDC marker BDCA-2, no MGL expression could be detected by either 18E4 or 1G6.6 mAbs (Fig. 2). No MGL mRNA could be detected in pDC by quantative real-time PCR analysis (data not shown). These results demonstrate that MGL is specifically expressed by myeloid DC and not by pDC. To determine the factors capable of inducing MGL expression, monocytes were treated with several cytokines and the GC dexamethasone. At day 3, monocytes
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were analyzed by FACS for the expression of MGL (Table 1). To control for the effectiveness of the cytokine treatment, DC-SIGN and MR were included in the analysis. DC-SIGN expression has been reported to be induced by IL-4 (Relloso et al., 2002), whereas MR expression is upregulated by IL-4, as well as GM-CSF and dexamethasone (Shepherd et al., 1994). In contrast, MGL expression is not observed on the cell surface after IL-4 treatment, although MGL mRNA was elevated
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(Table 1 and Raes et al., 2005). Mj generated by culturing monocytes in the presence of GM-CSF and IL-4/GM-CSF-cultured immature DC express high levels of MGL. In contrast, LPS-matured DC completely lacked MGL expression, both on the protein and on the mRNA level, whereas expression of DC-SIGN or the MR could still be observed (Table 1). Although the mouse homolog of MGL was originally cloned from IL2-treated monocytes (Suzuki et al., 1996), we could not detect enhanced MGL expression on IL-2-treated human monocytes. Strikingly, a 3-day treatment with a single dose of the GC dexamethasone could enhance MGL expression substantially (Table 1). In conclusion, the expression of the C-type lectins MGL, DC-SIGN and MR are differentially regulated, suggesting that depending on the cellular environment, DC can adopt various phenotypes with variable C-type lectin expression profiles. DC cultured in the presence of IL-10 or dexamethasone are generally considered to be tolerogenic DC capable of inducing tolerance instead of immunity (Steinman et al., 2003). As MGL expression seems to be differentially regulated by IL-10 or dexamethasone, a single dose of these stimuli was added at the start of DC differentiation and the generated DC were evaluated at day 4 for MGL, DC-SIGN and MR expression. Strikingly, IL-10- and dexamethasone-cultured immature DC display differential expression profiles of C-type lectin receptors (Fig. 3A and B). Whereas MGL expression is slightly decreased on immature DC treated with IL-10, it is highly upregulated by dexamethasone exposure. In contrast to MGL, DC-SIGN and MR are downregulated by the addition of dexamethasone. DC treated with IL-10 acquire higher levels of MR, where as the DC-SIGN expression remains unchanged. These results indicate that IL-10 tolerogenic DC are characterized as MGLlow, DC-SIGNhigh and MRhigh, whereas dexamethasone DC are MGLhigh, DC-SIGNlow and MRhigh. To investigate whether the enhanced MGL expression observed after dexamethasone treatment is due to enhanced cell surface expression from intracellular pools (Valladeau et al., 2001) or from enhanced MGL translation, immature DC were cultured in the presence of the indicated concentrations of dexamethasone. At day 4, DC were harvested and MGL and DC-SIGN mRNA levels were determined by quantitative real-time PCR. Compared to untreated DC, MGL mRNA levels are clearly upregulated in dexamethasone-exposed DC in a dose-dependent manner. Enhanced MGL mRNA levels were observed in immature dexamethasonetreated DC at dexamethasone concentrations higher than 108 M (Fig. 3C). In contrast, DC-SIGN expression was downregulated at the mRNA level by dexamethasone exposure at concentrations higher than 108 M (Fig. 3D). These results confirm that MGL
expression is enhanced at the transcriptional level and is not the result of enhanced cell surface expression from intracellular stores. To investigate how MGL expression is regulated in time after dexamethasone exposure, a single dose of dexamethasone (106 M) was added at the onset of DC differentiation and MGL expression was followed in time by real-time PCR analysis. At every timepoint measured, MGL mRNA levels were higher in the dexamethasone-exposed immature DC compared to the untreated immature DC (Fig. 3E). In the dexamethasone DC MGL, mRNA levels peaked at day 5 and slowly started to decline thereafter, confirming that a single dose of dexamethasone is sufficient to selectively upregulate MGL expression for at least 1 week. In summary, dexamethasone, but not IL-10, enhances MGL expression at the mRNA levels in a time- and dose-dependent manner.
Discussion MGL is a C-type lectin that participates in the binding and uptake of glycoproteins and lipids by immature DC and Mj (Denda-Nagai et al., 2002). Recently, the carbohydrate recognition profile of MGL was elucidated, consisting of unique specificity for terminal GalNAc-residues (van Vliet et al., 2005). As most other C-type lectins expressed on the cell surface of APC, such as DC-SIGN and MR, have mannose/fucose specificity (Appelmelk et al., 2003; Taylor et al., 1992), MGL confers the specialized function of GalNac recognition to the APC. Therefore, a more detailed examination of MGL expression patterns and specific stimuli controlling MGL expression are warranted, to establish under which environmental conditions MGL is specifically expressed and which APC may exhibit its function. mAbs are unsurpassed tools to study not only receptor expression but also allow the identification of receptor involvement in biological processes and subsequent pathways induced by receptor activation. Here, the generation of two new mAbs directed against human MGL is described (Fig. 1). With the use of these new mAbs, the expression pattern of MGL on different human DC subsets could be assessed. Human Langerhans cells and interstitial DC are derived from a common precursor, whereas human pDC develop along a separate pathway (Shortman and Liu, 2002). Although the expression of MGL on DC and Mj has been well documented (Higashi et al., 2002b; van Vliet et al., 2005), the presence of MGL on human pDC has not been investigated. Here, we show that MGL is not expressed either at the protein or the mRNA level by human pDC (Fig. 2). MGL thus represents an exclusive marker for myeloid-type APC, similar to DC-SIGN and the MR (Patterson et al., 2001).
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Factors capable of driving the acquisition of MGL on monocytes include GM-CSF and dexamethasone. IL-4 induces MGL expression only at the mRNA level (Table 1 and Raes et al., 2005). IL-4/GM-CSFgenerated immature DC express high levels of MGL, DC-SIGN and MR. In contrast, LPS-matured DC completely lack MGL expression, both on the protein as well as the mRNA level, whereas MR and DC-SIGN are still expressed albeit at slightly lower levels (Table 1 and Engering et al., 2002). Furthermore, immature DC cultured in the presence of dexamethasone display enhanced levels of MGL. By contrast, IL-10 was identified as a negative regulator of MGL expression on immature DC (Fig. 3A and B). Both IL-10 and dexamethasone inhibit the immunostimulatory capacities of antigen-presenting DC. IL-10-cultured DC have an impaired capacity to stimulate alloreactive T cells, due to lower expression of costimulatory molecules and a decreased production of IL-12 (Moore et al., 2001; De Smedt et al., 1997). Similarly, dexamethasone treatment blocks DC maturation at an early stage, resulting in a more immature phenotype. Dexamethasone DC express lower levels of costimulatory molecules, whereas the expression of molecules involved in antigen uptake and cell-adhesion is elevated (Piemonti et al., 1999b). Although the endocytic capacity of dexamethasone DC was increased, subsequent antigen presentation to T cells occurred with a much lower efficiency (Piemonti et al., 1999a). Therefore, IL-10- or dexamethasonetreated DC are considered to be tolerogenic DC (Steinman et al., 2003). GC, like dexamethasone, are widely used as antiinflammatory and immunosuppressive agents in the therapy of allergy or autoimmune diseases. In vivo, GC belong to the family of steroid hormones whose production by thymic epithelial cells and the adrenal gland is increased during a stress response (Jondal et al., 2004) and as a natural feedback loop during an immune response to avoid potential damage to the host (Besedovsky et al., 1975). GC exert their many immunoregulatory effects via the GC receptor, which is widely expressed in cells of the immune system. GC penetrate the cell and bind to the cytosolic GC receptor after which the complex translocates to nucleus for positive and negative gene regulation (Hayashi et al., 2004). The reduced ability of dexamethasone-cultured DC to stimulate T cells is contributed to the induction of the transcriptional regulator glucocorticoid-induced leucine zipper (GILZ) (Cohen et al., 2006). Inhibition of GILZ reverses the negative effects on cytokine production and costimulatory molecule expression. It seems unlikely that MGL expression is regulated via GILZ, as both IL10 and dexamethasone are capable of inducing GILZ expression (Cohen et al., 2006). Moreover, GILZ functions as a potent inhibitor of the transcription
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factor AP-1 (Mittelstadt and Ashwell, 2001). Although the MGL promoter contains two AP-1 sites (Tsuiji et al., 1999), MGL levels are enhanced by dexamethasone, suggesting that MGL expression is not driven by AP-1. Strikingly, for all the C-type lectins investigated in this report, namely MGL, DC-SIGN and MR, the expression is differentially regulated on in vitro cultured DC. These results indicate that also in vivo different APC subsets exist that exhibit unique C-type lectin expression patterns. C-type lectins participate in cell–cell communication, pattern recognition, as well as antigen uptake for presentation in MHC class I and II (Geijtenbeek et al., 2000; Gordon, 2002; Engering et al., 2002). Changes in the local environment and thus on C-type lectin profiles could therefore greatly influence many aspects of the immune response that are mediated by C-type lectins. Future studies on the mechanisms controlling MGL expression are required to determine whether the presence of MGL correlates with distinct stages of an immune response or with certain pathological conditions.
Acknowledgements We thank Dr. Estella A. Koppel for her assistance in the immunization of the mice and Dr. Juan Garcı´ aVallejo for his help in setting up the MGL real-time PCR analysis. SJvV is supported by the Netherlands Organization for Health Research and Development, Pionier Grant 900-02-002.
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Immunobiology 211 (2006) 577–585 www.elsevier.de/imbio
Differential regulation of C-type lectin expression on tolerogenic dendritic cell subsets Sandra J. van Vliet1, Ellis van Liempt1, Teunis B.H. Geijtenbeek, Yvette van Kooyk Department of Molecular Cell Biology and Immunology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands
Abstract Antigen presenting cells (APC) express high levels of C-type lectins, which play a major role in cellular interactions as well as pathogen recognition and antigen presentation. The C-type lectin macrophage galactose-type lectin (MGL), expressed by dendritic cells (DC) and macrophages, mediates binding to glycoproteins and lipids that contain terminal GalNAc moieties. To investigate MGL expression patterns in more detail, we generated two new monoclonal antibodies and set up a quantitative real-time PCR analysis to determine MGL mRNA levels. MGL is not expressed by blood-resident plasmacytoid DC and thus represents an exclusive marker for myeloid-type APC. Dexamethasone treatment upregulated MGL expression on DC both at the protein and mRNA level in a time- and dose-dependent manner. In contrast, DC generated in the presence of IL-10 did not display enhanced MGL levels. Furthermore, dexamethasone and IL-10 also differentially regulated expression of other C-type lectins, such as DC-SIGN and Mannose Receptor. Our results demonstrate that depending on the local microenvironment, DC can adopt different C-type lectin profiles, which could have major influences on cell–cell interactions, antigen uptake and presentation. r 2006 Elsevier GmbH. All rights reserved. Keywords: C-type lectins; Dendritic cells; Macrophages; Plasmacytoid DC
Introduction Professional antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages (Mj), are seeded throughout all peripheral tissues where they scan their surroundings for incoming pathogens or local environmental changes. Mj mainly represent traditional tissueAbbreviations: APC, antigen presenting cell; ASGP-R, asialoglycoprotein receptor; DC, dendritic cell; GILZ, glucocorticoid-induced leucine zipper; GC, glucocorticoids; mAbs, monoclonal antibodies; MGL, macrophage galactose-type lectin; MR, mannose receptor; Mj, macrophage; pDC, plasmacytoid DC Corresponding author. Tel.: +31 204448080; fax: +31 204448081. E-mail address: [email protected] (Y. van Kooyk). 1 These authors contributed equally to this paper. 0171-2985/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2006.05.022
resident scavenging cells important in the effector phase of the immune response. Similar to Mj, DC also play an essential role in uptake of self- or pathogenic antigens. DC, once activated by proinflammatory stimuli or infectious pathogens, migrate towards the draining lymph node, where they initiate adaptive immunity (Taylor et al., 2005; Mellman and Steinman, 2001). Recently, Pozzi et al. (2005) demonstrated that also Mj can migrate to draining lymph nodes and activate naı¨ ve CD8+ T cells, although with lower efficiencies than DC. Next to immunity, DC contribute to tolerance via the induction of T cell unresponsiveness or apoptosis or via the induction of regulatory T cells. These processes can be mimicked in vitro by the addition of glucocorticoids (GC), such as dexametha-
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sone, or by adding IL-10 to DC cultures (Xia et al., 2005; Steinman et al., 2003). Different DC lineages can develop from separate precursors or represent various activation states of a single subtype. DC clearly possess a unique plasticity to adapt to environmental stimuli, leading to different functional phenotypes based on cell surface markers and production of cytokines and/or reactive metabolites (Janeway and Medzhitov, 2002). Furthermore, recent evidence indicates that mouse splenic DC and Mj renew from a common bone marrow progenitor that is able to develop into both subtypes depending on differential cytokine signaling (Fogg et al., 2006). In humans, several pathways exist for the development of the different DC subtypes, such as Langerhans cells, plamacytoid DC (pDC) and interstitial DC, each requiring their own set of growth factors and/or cytokines (Shortman and Liu, 2002). As only a few surface proteins are expressed exclusively by DC; new potential markers are required that can distinguish between the different phenotypic DC subtypes. One family of proteins, known to be differentially expressed by the various DC subsets, are the C-type lectins (Figdor et al., 2002). C-type lectins recognize specific carbohydrate moieties in a Ca2+dependent manner. They function as cell–cell adhesion molecules (Geijtenbeek et al., 2000) and as pattern recognition receptors for pathogens (Gordon, 2002). Moreover, C-type lectins can internalize ligands, such as pathogens, but also self-glycoproteins for processing and presentation to T cells (Engering et al., 2002). The C-type lectin macrophage galactose-type lectin (MGL) is expressed on in vitro cultured monocyte-derived DC and Mj (Suzuki et al., 1996). The carbohydrate recognition domain of MGL facilitates binding of terminal GalNAc-residues on glycoproteins, glyolipids or pathogens, in contrast to the well-known mannose/ fucose-specific lectins DC-SIGN and mannose receptor (MR) (van Vliet et al., 2005). Although MGL was originally described to be a specific marker for cells at an intermediate stage of differentiation from monocytes to Mj (Higashi et al., 2002b), other reports demonstrate MGL to be expressed by dendritic cells and alternatively activated macrophages (Higashi et al., 2002a; Raes et al., 2005). To further analyze the expression pattern of MGL on human DC subtypes, two new monoclonal antibodies (mAbs) directed against the C-type lectin MGL were generated. Our findings extend the knowledge on MGL expression patterns both at RNA and protein level on functionally different immature DC subsets. We demonstrate that MGL is exclusively expressed by myeloid DC and not by blood-resident pDC. Only dexamethasone treatment, and not IL-10, can enhance MGL expression on tolerogenic DC at both the protein and mRNA level. Furthermore, we show that expression of the C-type
lectins MGL, DC-SIGN and MR is differentially regulated, suggesting that depending on the cellular environment, DC can adopt various phenotypes with variable C-type lectin expression profiles.
Materials and methods Cells and reagents The cell lines CHO and CHO-MGL were maintained in RPMI containing 10% fetal calf’s serum (Invitrogen, Carlsbad, CA). Immature DC were cultured for 3–7 days from monocytes obtained from buffy coats of healthy donors (Sanquin, Amsterdam, The Netherlands) in the presence of IL-4 (500 U/ml) and GM-CSF (800 U/ml, both from Biosource, Camarillo, CA). pDC were isolated from buffy coats using the BDCA-4 cell isolation kit according to the manufacturer’s protocol (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). To some DC cultures, dexamethasone (at concentrations indicated in the figures, Sigma-Aldrich), IL-10 (10 ng/ml, Biosource) or LPS (100 ng/ml, SigmaAldrich, St. Louis, MO) was added. Monocytes were stimulated with IL-2 (200 U/ml, Biosource), IL-4 (500 U/ml), IL-10 (10 ng/ml), dexamethasone (106 M) or GM-CSF (1000 U/ml).
Generation of anti-MGL mAbs Balb/c mice were immunized three times with recombinant MGL-Fc (van Vliet et al., 2005). After the final boost, spleen cells were fused with SP2/0 cells at a 1:1 ratio using PEG. Hybridoma supernatants were screened for the presence of anti-MGL mAbs on CHOMGL transfectants. After two rounds of cloning, two hybridomas (1G6.6 and 18E4) were obtained that specifically recognize MGL.
Flow cytometry Cells were incubated with primary antibody (5 mg/ml), followed by staining with a secondary FITC-labeled goat anti-mouse antibody (Zymed, San Francisco, CA) and analyzed on FACScalibur (BD Pharmingen, San Diego, CA). The following mAbs were used: isotype control 28-14-8 (mouse anti-mouse H2 Db), AZN-D1 (DC-SIGN) and 3.29.B1 (MR) (Engering et al., 2002). The anti-MGL antibody MLD-1 was kindly provided by Dr. T. Irimura.
Immunohistochemistry Cryosections of healthy humans tissues (7 mm) were fixed with 100% acetone and stained with primary
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antibodies (10 mg/ml) for 1 h at 37 1C. Sections were counterstained with goat anti-mouse IgG2A-specific Alexa488 antibodies (Molecular probes, Eugene, OR) and analyzed by fluorescence microscopy.
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15 s at 95 1C and 1 min 60 1C. The fluorescence monitoring occurred at the end of each cycle. The Ct value is defined as the number of PCR cycles where the fluorescence signal exceeds the threshold value, which is
mRNA isolation and cDNA synthesis mRNA was isolated by capturing of poly(A+) RNA in streptavidin-coated tubes with an mRNA Capture kit (Roche, Basel, Switzerland) and cDNA was synthesized with the Reverse Transcription Sytem kit (Promega, Madison, WI) following manufacturer’s guidelines. Cells (0.1 106/well) were washed twice with ice-cold PBS and harvested with 100 ml lysis buffer. Lysates were incubated with biotin-labeled oligo(dT)20 for 5 min at 37 1C. The mix was transferred to streptavidin-coated tubes and incubated for 5 min at 37 1C. After washing three times with 200 ml washing buffer, 30 ml of the reverse transcription mix (5 mM MgCl2, 1 reverse transcription buffer, 1 mM dNTP, 0.4 U recombinant RNasin ribonuclease inhibitor, 0.4 U AMV reverse transcriptase, 0.5 mg random hexamers in nuclease-free water) was added to the tubes and incubated for 10 min at room temperature followed by 45 min at 42 1C. To inactivate AMV reverse transcriptase and separate mRNA from the streptavidin–biotin complex, samples were heated at 99 1C for 5 min, transferred to microcentrifuge tubes and incubated on ice for 5 min, diluted 1:2 in nuclease-free water, and stored at 20 1C.
Quantitative real-time PCR Oligonucleotides have been designed by using computer software Primer Express 2.0 (Applied Biosystems, Foster City, CA). Primers were synthesized by Invitrogen (Invitrogen). Primer specificity was computer tested (BLAST, National center for Biotechnology Information) by homology search with the human genome and later confirmed by dissociation curve analysis. Primers used in this study: GAPDH Fwd 50 -CCA TGT TCG TCA TGG GTG TG and Rev 50 - GGT GCT AAG CAG TTG GTG GTG; MGL Fwd 5’- TAC ACC TGG ATG GGC CTC AG, MGL Rev 50 - TGT TCC ATC CAC CCA CTT CC, DC-SIGN Fwd 5- AAC AGC TGA GAG GCC TTG GA and DC-SIGN Rev 50 GGG ACC ATG GCC AAG ACA. PCR reactions were performed with SYBR green method in an ABI 7900HT sequence detection system (Applied Biosystems). The reactions were set on a 96-well plate by mixing 4 ml of 2 SYBR Green Master Mix (Applied Biosystems) with 2 ml of the primer solution containing 5 nmol/ml of both primers and 2 ml of a cDNA solution. The cDNA synthesis product was diluted 1:2 in nuclease-free water. The thermal profile for all the reactions was 2 min at 50 1C, followed by 10 min at 95 1C and then 40 cycles of
Fig. 1. Generation of hybridomas producing MGL-specific mAbs. (A) Stable transfectants of CHO-MGL express high levels of MGL as assessed by flow cytometric analysis with a previously described anti-MGL antibody (kindly provided by Dr. T. Irimura). Open histograms represent isotype control staining, filled histograms represent MGL staining. (B) After two rounds of cloning, two MGL-specific hybridomas (1G6.6 and 18E4) were obtained that specifically stain CHO-MGL transfectants. Open histograms represent isotype control staining, filled histograms represent MGL staining on CHOMGL by the hybridomas 1G6.6 and 18E4. (C) New MGLmAbs recognize MGLpos cells in skin and lymph node, whereas no crossreactivity was observed with the highly homologous liver-specific molecule ASGP-R. Human tissue sections of skin, lymph node and liver were stained with 18E4 mAbs. ED, epidermis; D, dermis; OZ, outer zones of the paracortex. Arrows indicate MGL+ cells. Original magnification 400 . One representative experiment out of three is shown.
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fixed above 10 times the standard deviation of the fluorescence during the first 15 cycles and typically corresponds to 0.2 relative fluorescence units. This threshold is set constant throughout the study and corresponds to the log linear range of the amplification curve. The normalized amount of target, or relative abundance, reflects the relative amount of target transcripts with respect to the expression of the endogenous reference gene. The endogenous reference gene chosen was GAPDH.
Results To generate specific mAbs reactive with the C-type lectin MGL, Balb/c mice were immunized three times with purified MGL-Fc. After the final boost, spleen cells were fused with the SP2/0 myeloma. Hybridoma supernatants were screened by FACS for positive staining on CHO-MGL transfectants and negative reactivity with the parental CHO cells. CHO-MGL transfectants expressed high levels of MGL, as determined by flow cytometric analysis with a previously described antihuman MGL mAb (Fig. 1A and Higashi et al., 2002b). After two rounds of cloning, two MGL-reactive hybridomas, 1G6.6 and 18E4, of the IgG2A isotype were obtained (Fig. 1B). To further confirm the specificity of these hybridomas, human skin, lymph node and liver sections were stained with the new mAbs (Fig. 1C). In agreement with earlier reports (Higashi et al., 2002b), MGLpos cells were readily detected in the dermis of human skin. No crossreactivity with the most closely related human homolog of MGL, the liverspecific C-type lectin ASGP-R, was observed, confirming the exclusive specificity of the generated mAbs for MGL (Fig. 1C). In addition, human lymph node contained numerous MGLpos cells within the outer zones of the paracortex. Similar results were obtained for both anti-MGL mAbs. The availability of the anti-MGL mAbs allowed us to investigate MGL expression levels in various human DC
Fig. 2. MGL is not expressed by plasmacytoid DC. Highly enriched plasmacytoid DC (84% pure) were stained for BDCA-2 and MGL by flow cytometry. Open histograms indicate isotype control staining, filled histograms represent BDCA-2 or MGL staining. One representative experiment is shown.
Table 1.
CLR expression on stimulated monocytes
Stimuli
DC-SIGNa
MGL
Mannose receptor
IL-2 IL-4 IL-10 GM-CSF IL-4/GM-CSF IL-4/GM-CSF/LPS Dexamethasone
+ + +
+b + + +
+ + + + +
a Monocytes were cultured for 3 days in the presence of the indicated cytokines, glucocorticoids or combinations, after which C-type lectin expression was determined by flow cytometry (DC-SIGN, MGL and MR) and real-time PCR (DC-SIGN and MGL). + indicates induction of C-type lectin expression compared to unstimulated monocytes, no expression. b Upregulation only observed at the RNA level.
subsets. pDC represent a distinct DC population that is characterized by the markers BDCA-2 and BDCA-4 (Dzionek et al., 2001). To investigate whether next to myeloid-type DC, pDC also express MGL, we isolated pDC from human buffy coats. Although the isolated
Fig. 3. Dexamethasone treatment enhances MGL expression during DC differentiation. Immature DC were generated from monocytes by culturing for 4 days with IL-4 and GM-CSF in the presence or absence of IL-10 (A) or dexamethasone (B–D). (A) Cell surface expression of MGL, DC-SIGN and MR was determined by FACS analysis on immature DC (filled histograms) and IL10-cultured DC (10 ng/ml, solid line). The isotype control staining is indicated by the thin line. (B) Cell surface expression of MGL, DC-SIGN and MR was determined by FACS analysis on immature DC (filled histograms) and dexamethasone-cultured DC (106 M, solid line). The isotype control staining is indicated by the thin line. (C) Dose response curve of dexamethasone-induced MGL expression. Monocytes were cultured with IL-4/GM-CSF and dexamethasone at the indicated concentrations. At day 4, cells were harvested and MGL mRNA levels were determined by quantitative real-time PCR. The dashed line indicates the MGL mRNA level in untreated immature DC. (D) Dose response curve of dexamethasone-mediated inhibition of DC-SIGN expression. Monocytes were cultured with IL-4/GM-CSF and dexamethasone at the indicated concentrations. At day 4, cells were harvested and DC-SIGN mRNA levels were determined by quantitative real-time PCR. The dashed line indicates the DC-SIGN mRNA level in untreated immature DC. (E) Kinetics of dexamethasone-induced MGL expression. Monocytes were cultured with IL-4/GM-CSF in the presence or absence of dexamethasone (106 M) for 3–7 days. At indicated timepoints, cells were harvested and MGL mRNA levels were determined by quantitative real-time-PCR. One representative experiment out of three is shown.
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pDC clearly express the pDC marker BDCA-2, no MGL expression could be detected by either 18E4 or 1G6.6 mAbs (Fig. 2). No MGL mRNA could be detected in pDC by quantative real-time PCR analysis (data not shown). These results demonstrate that MGL is specifically expressed by myeloid DC and not by pDC. To determine the factors capable of inducing MGL expression, monocytes were treated with several cytokines and the GC dexamethasone. At day 3, monocytes
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were analyzed by FACS for the expression of MGL (Table 1). To control for the effectiveness of the cytokine treatment, DC-SIGN and MR were included in the analysis. DC-SIGN expression has been reported to be induced by IL-4 (Relloso et al., 2002), whereas MR expression is upregulated by IL-4, as well as GM-CSF and dexamethasone (Shepherd et al., 1994). In contrast, MGL expression is not observed on the cell surface after IL-4 treatment, although MGL mRNA was elevated
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(Table 1 and Raes et al., 2005). Mj generated by culturing monocytes in the presence of GM-CSF and IL-4/GM-CSF-cultured immature DC express high levels of MGL. In contrast, LPS-matured DC completely lacked MGL expression, both on the protein and on the mRNA level, whereas expression of DC-SIGN or the MR could still be observed (Table 1). Although the mouse homolog of MGL was originally cloned from IL2-treated monocytes (Suzuki et al., 1996), we could not detect enhanced MGL expression on IL-2-treated human monocytes. Strikingly, a 3-day treatment with a single dose of the GC dexamethasone could enhance MGL expression substantially (Table 1). In conclusion, the expression of the C-type lectins MGL, DC-SIGN and MR are differentially regulated, suggesting that depending on the cellular environment, DC can adopt various phenotypes with variable C-type lectin expression profiles. DC cultured in the presence of IL-10 or dexamethasone are generally considered to be tolerogenic DC capable of inducing tolerance instead of immunity (Steinman et al., 2003). As MGL expression seems to be differentially regulated by IL-10 or dexamethasone, a single dose of these stimuli was added at the start of DC differentiation and the generated DC were evaluated at day 4 for MGL, DC-SIGN and MR expression. Strikingly, IL-10- and dexamethasone-cultured immature DC display differential expression profiles of C-type lectin receptors (Fig. 3A and B). Whereas MGL expression is slightly decreased on immature DC treated with IL-10, it is highly upregulated by dexamethasone exposure. In contrast to MGL, DC-SIGN and MR are downregulated by the addition of dexamethasone. DC treated with IL-10 acquire higher levels of MR, where as the DC-SIGN expression remains unchanged. These results indicate that IL-10 tolerogenic DC are characterized as MGLlow, DC-SIGNhigh and MRhigh, whereas dexamethasone DC are MGLhigh, DC-SIGNlow and MRhigh. To investigate whether the enhanced MGL expression observed after dexamethasone treatment is due to enhanced cell surface expression from intracellular pools (Valladeau et al., 2001) or from enhanced MGL translation, immature DC were cultured in the presence of the indicated concentrations of dexamethasone. At day 4, DC were harvested and MGL and DC-SIGN mRNA levels were determined by quantitative real-time PCR. Compared to untreated DC, MGL mRNA levels are clearly upregulated in dexamethasone-exposed DC in a dose-dependent manner. Enhanced MGL mRNA levels were observed in immature dexamethasonetreated DC at dexamethasone concentrations higher than 108 M (Fig. 3C). In contrast, DC-SIGN expression was downregulated at the mRNA level by dexamethasone exposure at concentrations higher than 108 M (Fig. 3D). These results confirm that MGL
expression is enhanced at the transcriptional level and is not the result of enhanced cell surface expression from intracellular stores. To investigate how MGL expression is regulated in time after dexamethasone exposure, a single dose of dexamethasone (106 M) was added at the onset of DC differentiation and MGL expression was followed in time by real-time PCR analysis. At every timepoint measured, MGL mRNA levels were higher in the dexamethasone-exposed immature DC compared to the untreated immature DC (Fig. 3E). In the dexamethasone DC MGL, mRNA levels peaked at day 5 and slowly started to decline thereafter, confirming that a single dose of dexamethasone is sufficient to selectively upregulate MGL expression for at least 1 week. In summary, dexamethasone, but not IL-10, enhances MGL expression at the mRNA levels in a time- and dose-dependent manner.
Discussion MGL is a C-type lectin that participates in the binding and uptake of glycoproteins and lipids by immature DC and Mj (Denda-Nagai et al., 2002). Recently, the carbohydrate recognition profile of MGL was elucidated, consisting of unique specificity for terminal GalNAc-residues (van Vliet et al., 2005). As most other C-type lectins expressed on the cell surface of APC, such as DC-SIGN and MR, have mannose/fucose specificity (Appelmelk et al., 2003; Taylor et al., 1992), MGL confers the specialized function of GalNac recognition to the APC. Therefore, a more detailed examination of MGL expression patterns and specific stimuli controlling MGL expression are warranted, to establish under which environmental conditions MGL is specifically expressed and which APC may exhibit its function. mAbs are unsurpassed tools to study not only receptor expression but also allow the identification of receptor involvement in biological processes and subsequent pathways induced by receptor activation. Here, the generation of two new mAbs directed against human MGL is described (Fig. 1). With the use of these new mAbs, the expression pattern of MGL on different human DC subsets could be assessed. Human Langerhans cells and interstitial DC are derived from a common precursor, whereas human pDC develop along a separate pathway (Shortman and Liu, 2002). Although the expression of MGL on DC and Mj has been well documented (Higashi et al., 2002b; van Vliet et al., 2005), the presence of MGL on human pDC has not been investigated. Here, we show that MGL is not expressed either at the protein or the mRNA level by human pDC (Fig. 2). MGL thus represents an exclusive marker for myeloid-type APC, similar to DC-SIGN and the MR (Patterson et al., 2001).
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Factors capable of driving the acquisition of MGL on monocytes include GM-CSF and dexamethasone. IL-4 induces MGL expression only at the mRNA level (Table 1 and Raes et al., 2005). IL-4/GM-CSFgenerated immature DC express high levels of MGL, DC-SIGN and MR. In contrast, LPS-matured DC completely lack MGL expression, both on the protein as well as the mRNA level, whereas MR and DC-SIGN are still expressed albeit at slightly lower levels (Table 1 and Engering et al., 2002). Furthermore, immature DC cultured in the presence of dexamethasone display enhanced levels of MGL. By contrast, IL-10 was identified as a negative regulator of MGL expression on immature DC (Fig. 3A and B). Both IL-10 and dexamethasone inhibit the immunostimulatory capacities of antigen-presenting DC. IL-10-cultured DC have an impaired capacity to stimulate alloreactive T cells, due to lower expression of costimulatory molecules and a decreased production of IL-12 (Moore et al., 2001; De Smedt et al., 1997). Similarly, dexamethasone treatment blocks DC maturation at an early stage, resulting in a more immature phenotype. Dexamethasone DC express lower levels of costimulatory molecules, whereas the expression of molecules involved in antigen uptake and cell-adhesion is elevated (Piemonti et al., 1999b). Although the endocytic capacity of dexamethasone DC was increased, subsequent antigen presentation to T cells occurred with a much lower efficiency (Piemonti et al., 1999a). Therefore, IL-10- or dexamethasonetreated DC are considered to be tolerogenic DC (Steinman et al., 2003). GC, like dexamethasone, are widely used as antiinflammatory and immunosuppressive agents in the therapy of allergy or autoimmune diseases. In vivo, GC belong to the family of steroid hormones whose production by thymic epithelial cells and the adrenal gland is increased during a stress response (Jondal et al., 2004) and as a natural feedback loop during an immune response to avoid potential damage to the host (Besedovsky et al., 1975). GC exert their many immunoregulatory effects via the GC receptor, which is widely expressed in cells of the immune system. GC penetrate the cell and bind to the cytosolic GC receptor after which the complex translocates to nucleus for positive and negative gene regulation (Hayashi et al., 2004). The reduced ability of dexamethasone-cultured DC to stimulate T cells is contributed to the induction of the transcriptional regulator glucocorticoid-induced leucine zipper (GILZ) (Cohen et al., 2006). Inhibition of GILZ reverses the negative effects on cytokine production and costimulatory molecule expression. It seems unlikely that MGL expression is regulated via GILZ, as both IL10 and dexamethasone are capable of inducing GILZ expression (Cohen et al., 2006). Moreover, GILZ functions as a potent inhibitor of the transcription
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factor AP-1 (Mittelstadt and Ashwell, 2001). Although the MGL promoter contains two AP-1 sites (Tsuiji et al., 1999), MGL levels are enhanced by dexamethasone, suggesting that MGL expression is not driven by AP-1. Strikingly, for all the C-type lectins investigated in this report, namely MGL, DC-SIGN and MR, the expression is differentially regulated on in vitro cultured DC. These results indicate that also in vivo different APC subsets exist that exhibit unique C-type lectin expression patterns. C-type lectins participate in cell–cell communication, pattern recognition, as well as antigen uptake for presentation in MHC class I and II (Geijtenbeek et al., 2000; Gordon, 2002; Engering et al., 2002). Changes in the local environment and thus on C-type lectin profiles could therefore greatly influence many aspects of the immune response that are mediated by C-type lectins. Future studies on the mechanisms controlling MGL expression are required to determine whether the presence of MGL correlates with distinct stages of an immune response or with certain pathological conditions.
Acknowledgements We thank Dr. Estella A. Koppel for her assistance in the immunization of the mice and Dr. Juan Garcı´ aVallejo for his help in setting up the MGL real-time PCR analysis. SJvV is supported by the Netherlands Organization for Health Research and Development, Pionier Grant 900-02-002.
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