DCIR is endocytosed into human dendritic cells and ... - CiteSeerX

Uncorrected Version. Published on November 21, 2008 as DOI:10.1189/jlb.0608352

DCIR is endocytosed into human dendritic cells and inhibits TLR8-mediated cytokine production Friederike Meyer-Wentrup, Alessandra Cambi, Ben Joosten, Maaike W. Looman, I. Jolanda M. de Vries, Carl G. Figdor, and Gosse J. Adema1 Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands

Abstract: C-type lectin receptors (CLRs) expressed on APCs play a pivotal role in the immune system as pattern-recognition and antigen-uptake receptors. In addition, they may signal directly, leading to cytokine production and immune modulation. To this end, some CLRs, like dectin-1 and dendritic cell immunoreceptor (DCIR), contain intracellular ITIMs or ITAMs. In this study, we explored expression and function of the ITIM-containing CLR DCIR on professional APCs. DCIR is expressed on immature and mature monocyte-derived DCs (moDC) but also on monocytes, macrophages, B cells, and freshly isolated myeloid and plasmacytoid DCs. We show that endogenous DCIR is internalized efficiently into human moDC after triggering with DCIR-specific mAb. DCIR internalization is clathrin-dependent and leads to its localization in the endo-/lysosomal compartment, including lysosome-associated membrane protein-1ⴙ lysosomes. DCIR triggering affected neither TLR4- nor TLR8-mediated CD80 and CD86 up-regulation. Interestingly, it did inhibit TLR8mediated IL-12 and TNF-␣ production significantly, and TLR2-, TLR3-, or TLR4-induced cytokine production was not affected. Collectively, the data presented characterize DCIR as an APC receptor that is endocytosed efficiently in a clathrin-dependent manner and negatively affects TLR8-mediated cytokine production. These data provide further support to the concept of CLR/ TLR cross-talk in modulating immune responses. J. Leukoc. Biol. 85: 000 – 000; 2009. Key Words: cell surface molecule 䡠 C-type lectin 䡠 receptor cross-talk

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pattern recognition receptor

INTRODUCTION APCs recognize intruding pathogens through pattern-recognition receptors (PRRs). These receptors bind to conserved pathogen-associated molecular patterns that are essential for pathogen survival. Among the PRRs, two receptor families stand out: on the one hand, the TLRs, recognizing a broad array of ligands ranging from single-stranded RNA to bacterial cell wall components [1], and on the other hand, the C-type lectin receptors (CLRs), binding exclusively to specific carbohydrate 0741-5400/09/0085-0001 © Society for Leukocyte Biology

moieties through their carbohydrate-recognition domain [2, 3]. Although TLR triggering induces immune activation via MyD88- or Toll/IL-1R domain-containing adapter-inducing IFN-␤-dependent signaling pathways, CLRs function primarily as antigen-uptake receptors. C-type lectin-mediated antigen uptake can lead to efficient antigen presentation and immune stimulation [4, 5]. However, pathogens may also use binding to CLRs to evade an immune response [3]. Some C-type lectins can signal intracellularly directly via ITAM and ITIM [6], present in their cytoplasmic domains. For the C-type lectin dectin-1, direct signaling through association of its cytoplasmic ITAM motif with the tyrosine kinase Syk has been demonstrated [7, 8]. Furthermore, C-type lectins and TLRs may cross-talk, as demonstrated for dectin-1 and TLR2. Coexpression of both molecules on the APC surface enables cooperative, synergistic signaling by TLR2 and dectin-1 in response to the common ligand zymosan [9, 10]. Recently, we have shown that human myeloid APCs such as monocytes, macrophages, and immature dendritic cells (DCs) express the ITAM-containing CLR dectin-1, whose function is regulated by tetraspanin CD37 [11]. It has been suggested that ITAM- and ITIM-containing receptors function as receptor pairs to balance immune activation and inhibition [6]. We were therefore interested in expression and function of the ITIMcontaining CLRs in human APCs. DC immunoreceptor (DCIR), next to DC-associated C-type lectin 2 (DCAL-2) [12], is the only known human CLR containing an ITIM motif expressed on APCs. The DCIR (or CLECSF6) was first described as an ITIM-containing C-type lectin found on the APC surface and differentially expressed on DCs, depending on their maturation status [13]. In line with these findings, DCIR expression in neutrophils was shown to be down-regulated by neutrophilactivating agents such bacterial LPS or TNF-␣ [14]. A possible immune inhibitory role based on its ITIM motif was substantiated by reduced BCR-mediated Ca2⫹ mobilization and protein tyrosinase phosphorylation after coligation with chimeric molecules consisting of the extracellular domain of Fc␥RIIB and the cytoplasmic portion of murine DCIR [15]. Furthermore, the tyrosine phosphatases Src homology 2-containing tyrosine

1 Correspondence: Department of Tumor Immunology, Radboud UMC Nijmegen, NCMLS/278 TIL, Post Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: [email protected] Received June 13, 2008; revised October 17, 2008; accepted October 23, 2008. doi: 10.1189/jlb.0608352

Journal of Leukocyte Biology Volume 85, March 2009

Copyright 2008 by The Society for Leukocyte Biology.

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phosphatase 1 (SHP-1) and SHP-2, implicated in immune inhibition, were immunoprecipitated with peptides comprising the phosphorylated DCIR ITIM domain [16]. So far, neither endogenous nor exogenous DCIR ligands have been identified. The present study demonstrates that APCs coexpress the ITAM-containing dectin-1 and the ITIM-containing DCIR, and DCIR is not only expressed on immature and mature monocytederived DCs (moDC) but also on macrophages, B cells, and freshly isolated myeloid and plasmacytoid DCs. This is the first report showing that DCIR is internalized into human myeloid DCs after triggering. Our data demonstrate that in these DCs, internalization is clathrin-dependent and leads to receptor trafficking to lysosomal compartments. Furthermore, DCIR triggering selectively inhibits TLR8-meditated IL-12 and TNF-␣ production, thereby representing a novel example of CLR/TLR cross-talk in immune regulation.

GAA ATC ACT TAT G-3⬘, reverse primer: 5⬘-CTT GGG GAA TCC GGT ATT AC-3⬘). The DCIR coding sequence was cloned into a plasmid enhanced green fluorescence protein (pEGFP)-C1 vector placing the EGFP moiety on the N terminus of the protein or a plasmid encoding fluorescent fusion proteins (pECFP)-N1 vector, in which ECFP had been replaced by a hemagglutinin tag. Both vectors allow selection of stably transfected cells with geneticin (BD Biosciences Clontech, Fremont, CA, USA). Constructs were checked for correct reading frame and mutations at the Department of Anthropogenetics [Nijmegen Centre for Molecular Life Sciences (NCMLS), Nijmegen, The Netherlands]. CHO cells were transfected with lipofectamine (Sigma Chemical Co.).

Immunoprecipitation and Western blot analysis

MATERIALS AND METHODS

Cells were lysed in lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 1% Triton X-100, 2 ␮g/ml aprotinin, 2 ␮g/ml leupeptin, and 1 mM PMSF. Lysates were then incubated with murine anti-DCIR antibodies bound to protein G sepharose beads. After incubating for 2 h at 4°C, beads were washed three times in lysis buffer. SDS sample buffer containing 5% ␤-ME was added. Samples were boiled, separated by PAGE, and blotted onto nitrocellulose membranes, which were blocked in PBS, 3% BSA, and 1% skim milk powder at 4°C overnight and stained with specific antibodies. Antibody signals were detected with HRP-coupled secondary antibodies using an ECL detection kit (Pierce, Rockford, IL, USA).

Cell lines and reagents

DCIR internalization assay

All reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless indicated otherwise. Human PBMCs were isolated from buffy coats and monocytes enriched by plastic adherence. Monocytes were cultured with 450 U/ml GM-CSF (Strathmann, Germany) and 300 U/ml IL-4 (Strathmann) for 6 days to generate immature DCs; DC maturation was induced by addition of TNF-␣ (10 ␮g/ml), IL-6 (15 ng/ml; both from CellGenix, Germany), IL-1␤ (5 ␮g/ml; ImmunoTools, Germany), and PGE2 (10 ␮g/ml; Pharmacia Upjohn, Bridgewater, NJ, USA) for 24 h [17]. R848 was purchased from Pharmatec (Pakistan); tripalmitoyl-S-glyceryl cysteine (Pam3Cys) from EMC Microcollections (Germany). Human macrophages were generated by culturing monocytes in complete RPMI 1640 (Life Technologies, Gibco-BRL, Grand Island, NY, USA), supplemented with 10% human serum for 4 – 6 days. Myeloid DCs were enriched from PBMCs by depletion of CD19- and CD3-positive cells with antibody-coated magnetic beads (Becton Dickinson, San Jose, CA, USA). Chinese hamster ovary (CHO) cells were transfected with lipofectamine (Invitrogen, Carlsbad, CA, USA) and cultured in complete HamF12 (Life Technologies, Gibco-BRL) supplemented with 10% FCS.

Cells were incubated with anti-DCIR, anti-DC-SIGN, or anti-␤2-integrin mAb (10 ␮g/ml) in PBS, supplemented with 1% human serum, 3% BSA, and 10 mM glycine for 30 min on ice. Unbound antibodies were washed away, and cells were incubated at 37°C or 4°C for various time-points. The amount of antibody complexes remaining on the cell surface was then determined by FACS analysis after staining cells with anti-mouse IgG-PE secondary antibodies. Isotype control mouse IgG1 did not show any staining. To detect internalized DCIR protein, cells were first incubated for 30 min on ice with mouse anti-DCIR mAb, followed by incubation with goat anti-mouse PE or goat anti-mouse Alexa488 antibodies for 30 min on ice. Cells were then incubated at 37°C and 4°C for various time-points and fixed with 1% paraformaldehyde in PBS. Noninternalized antibodies were stripped off the cell surface by shifting the pH to 2.5 for 5 min on ice. In some experiments, clathrin-mediated receptor endocytosis was inhibited by preincubating cells in 450 mM sucrose in PBS. Antibody labeling was then also performed in the presence of 450 mM sucrose. Endocytic capacity was restored by washing in buffer without sucrose. In addition, internalization experiments were performed with CHO cells stably expressing DCIR-EGFP. Cells were then analyzed by FACS or mounted on poly-L-lysine-coated coverslips, permeabilized with 0.1% Triton X-100, and counterstained with anti-MHC class I (10 ␮g/ml) antibodies for analysis by CLSM (MRC1024, Bio-Rad, Hercules, CA, USA). In some experiments, cells were fixed after DCIR internalization, permeabilized with 0.1% saponin, and labeled with rabbit polyclonal anti-LAMP-1 (10 ␮g/ml) or matched isotype control antibodies for CLSM analysis.

Antibodies Murine anti-human DCIR mAb (clone 216110, R&D Systems, Minneapolis, MN, USA) were used for FACS analysis and confocal laser-scanning microscopy (CLSM). FACS analysis was performed with a FACSCalibur (Becton Dickinson). FACS data were analyzed with WinMDI (http://facs.scripps.edu/ software.html) and CellQuest (Becton Dickinson). MHC class I molecules, clathrin, and ␤2-integrins were stained with mouse mAb (clone W6/32, American Type Culture Collection, Manassas, VA, USA; clone CON.1, Sigma Chemical Co.; and clone NKI-L19, respectively). Human DC-specific ICAMgrabbing nonintegrin (SIGN) was detected with murine anti-DC-SIGN antibodies (clone AZND1, Beckman Coulter, Fullerton, CA, USA). Human lysosomeassociated membrane protein (LAMP)-1 was detected with rabbit polyclonal antibodies (Sigma Chemical Co.). Secondary antibodies for FACS analysis were PE-labeled (Becton Dickinson). To detect human CD80, CD83, and CD86, PE-labeled antibodies were used (clone 340294, Becton Dicksinon; clone 2218, Immunotech, France; and clone 555658, Becton Dickinson, respectively). To control for unspecific binding, samples were also stained with PE-labeled mouse IgG1 and mouse IgG2b. For confocal imaging, isotypespecific goat anti-mouse Alexa568- and goat anti-mouse Alexa488-labeled or goat anti-rabbit Alexa647-labled secondary antibodies were used (Molecular Probes, Eugene, OR, USA). Immunoprecipitations were performed with mouse anti-DCIR mAb (R&D Systems). Western blots were labeled with goat polyclonal anti-DCIR antibodies (R&D Systems).

cDNA constructs cDNA was generated from mRNA of human immature moDC. The following DCIR primers were used in PCR reactions (forward primer: 5⬘-ATG ACT TCG

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DCIR/clathrin colocalization studies Cells were allowed to adhere to fibronectin-coated coverslips, fixed in 1% paraformaldehyde in PBS, and permeabilized with 0.1% Triton X-100. To block unspecific antibody binding, cells were incubated in PBS, supplemented with 1% human serum, 3% BSA, and 10 mM glycine for 30 min at 4°C. Cells were then incubated with murine anti-DCIR and anticlathrin mAb for 30 min at 4°C. Unbound antibodies were washed away. Cells were stained with goat anti-mouse IgG1-Alexa488 and anti-mouse IgG2b-Alexa568. Isotype control mouse IgG1 and IgG2b did not show any staining. Cells were mounted in Mowiol and analyzed by CLSM.

Cytokine production and analysis of CD80 and CD86 up-regulation Immature moDC (5⫻104) were stimulated with zymosan (100 ␮g/mL), Pam3Cys (5 ␮g/ml), CpG (5 ␮g/ml), R848 (4 ␮g/ml), or LPS (200 ng/ml) in RPMI-1640/10% FCS in 96-well plates coated with mouse anti-DCIR mAb or mouse IgG1 isotype control (5 ␮g/ml). Supernatants were collected after 24 h, and DCs were harvested. TNF-␣ and IL-12 production was measured with capture and biotinylated anti-mouse antibodies (Endogen, Woburn, MA, USA)

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using standard ELISA procedures. IL-6 and IL-10 were measured using the Luminex technology, a beads-based cytokine detection system, according to the manufacturer’s instructions (Bio-Rad/Luminex). DCs were labeled with murine anti-CD80-PE and anti-CD86-PE antibodies. Matched PE-labeled isotype controls were included. Cells were analyzed by FACS.

Statistical analyses Statistical differences were determined using unpaired Student’s t-test. Significance was accepted at the P ⬍ 0.05 level.

RESULTS Human APCs express DCIR We first assessed DCIR expression on human APCs, such as monocytes, macrophages, and immature and mature DCs. Expression on B cells and freshly isolated myeloid and plasmacytoid DCs was also analyzed, and the ITAM-containing CLR dectin-1 was included as a control (Fig. 1A). DCIR expression was readily found on monocytes, macrophages, B cells, and immature DCs, thereby confirming previous studies [13]. High dectin-1 expression was also detected on these cells, except for B cells that exhibited low or undetectable dectin-1 expression. Freshly isolated myeloid DCs expressed DCIR and dectin-1. Interestingly, plasmacytoid DCs also expressed DCIR but no dectin-1. Following moDC maturation with IL-6, TNF-␣, PGE2, and IL-1␤, expression of DCIR as well as dectin-1 was decreased significantly. DCIR and dectin-1 are thus coexpressed by all APC subsets analyzed with the exception of B cells and plasmacytoid DCs, which show low or absent dectin-1 expression.

DCIR triggering induces receptor internalization C-type lectins are known to mediate endocytosis of bound ligands [2]. DCIR ligands have not yet been identified. To gain insight into DCIR function in human immature moDC, we first confirmed the presence of DCIR as a 37-kD protein in monocytes and immature DCs by Western blot analysis (Fig. 1B). DCIR internalization was then studied in immature DCs after incubation with anti-DCIR antibodies. Anti-DC-SIGN and anti-␤2-integrin antibodies were included as positive and negative controls, respectively. DCIR internalization was already detected after 1 h incubation at 37°C and increased further in time (Fig. 2A). Internalization of DCIR was comparable with that of the known endocytic receptor DC-SIGN (positive control). Incubation of the cells at 4°C did not result in receptor uptake. The ␤2-integrin antibody was not internalized (negative control; Fig. 2A). Similarly, we observed that DCIR internalization by antibody triggering also occurs in a CHO cell line stably transfected with DCIR-EGFP (Supplemental Fig. 1). As receptor shedding after antibody binding cannot be formally excluded as an explanation for the reduced DCIR levels, DCIR internalization into immature moDC was studied further by CLSM (Fig. 2B). Immature DCs were incubated at 4°C with anti-DCIR, followed by fluorochrome-conjugated secondary antibodies. Cell surface was labeled with anti-MHC class I antibodies. CLSM showed that elevation of the incubation temperature to 37°C induced clear receptor internalization. We note that a significant amount of DCIR colocalized with MHC

Fig. 1. Human professional APCs coexpress DCIR and dectin-1, (A) whose expression was analyzed by flow cytometry on freshly isolated monocytes, monocyte-derived macrophages, mature and immature DCs, B cells, and plasmacytoid and myeloid DCs (pDCs and mDCs, respectively). All professional APCs express DCIR (gray-shaded areas), and dectin-1 is not expressed on plasmacytoid DCs. Dectin-1 expression on B cells and mature DCs is low to undetectable. Matched isotype control antibodies (dashed lines) did not show any binding. Data represent three independent experiments. (B) DCIR expression by monocytes and moDC was demonstrated by immunoprecipitation (IP) and Western blot (WB) analysis with anti-DCIR antibodies. A single band of 37 kD corresponding to the predicted molecular weight of DCIR was detected specifically after immunoprecipitation with anti-DCIR and not with matched isotype control antibodies. Data shown are representative of four independent experiments. mIgG1, Mouse IgG1.

Meyer-Wentrup et al. DCIR, endocytic receptor, and inhibitor of TLR8 signaling

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Fig. 2. DCIR triggering on DCs induces receptor internalization. (A) Immature moDC were incubated with anti-DCIR, anti-DC-SIGN, and anti-␤2-integrin antibodies. Elevation of the incubation temperature to 37°C induced DCIR and DC-SIGN internalization over time (thin lines: 1 h incubation; thick lines: 2 h) compared with incubation at 4°C (shaded areas). Labeling of ␤2-integrin, which is not endocytosed after cross-linking, was included as a negative control. (B) Immature DCs internalize DCIR after receptor triggering. DCs were labeled with anti-DCIR antibody followed by a fluorochromeconjugated secondary antibody. The cell membrane was stained with anti-HLA class I antibodies. Cells were analyzed by CLSM. Incubation for 1 h at 37°C induced receptor internalization (lower panel). Control cells incubated at 4°C did not internalize DCIR (upper panel). Matched isotype control antibodies did not show any binding. Data represent three independent experiments.

class I, suggesting that not all DCIR was internalized after 1 h. No DCIR was internalized into cells that were kept at 4°C (Fig. 2B). The reduced DCIR expression detected after DCIR triggering is thus at least partly a result of receptor endocytosis and cannot be explained simply by receptor shedding.

DCIR internalization into human DCs is clathrindependent Receptor-mediated endocytosis often occurs via clathrincoated pits [18]. To study whether DCIR internalization is clathrin-dependent, we first performed colocalization studies on immature moDC. DCs were allowed to adhere and stretch on fibronectin-coated coverslips and were incubated with antiDCIR and anticlathrin antibodies at different conditions. CLSM revealed that DCIR partially colocalizes with clathrin in distinct clusters at the cell surface (Fig. 3A). Clathrin-mediated endocytosis is known to be inhibited by incubation of cells in hypertonic media. Incubation in media containing 450 mM sucrose results in disruption of clathrincoated pits and induces formation of nonfunctional clathrin microcages. This process is fully reversible once cells are returned into isotonic media [19, 20]. To further confirm clathrin-mediated endocytosis as the mechanism underlying DCIR 4


internalization, uptake experiments were therefore performed in the presence of 450 mM sucrose. Immature moDC were incubated with anti-DCIR and fluorochrome-conjugated secondary antibodies at 37°C. The internalized DCIR pool was then detected by stripping off noninternalized anti-DCIR/secondary antibody complexes with a pH shift (Fig. 3B). Receptor internalization thus resulted in an increasing fluorescence signal (left panel). This internalization was inhibited completely once DCs were incubated with antibodies in hypertonic medium containing 450 mM sucrose (middle panel). The inhibitory effect of hypertonic medium was fully reversible, so that DCIR was again internalized after wash-out of hypertonic medium (right panel). Figure 3C depicts the increase of DCIR internalization at 37°C over time. Incubation at 4°C or in the presence of hypertonic medium results in complete inhibition of receptor internalization. The association with clathrin and the effects of disruption of clathrin-coated pits on DCIR internalization were also clearly visible in a CHO cell line stably transfected with DCIR-EGFP (Supplemental Figs. 2 and 3). DCIR colocalized with clathrin on the surface of CHO-DCIR cells (Supplemental Fig. 2) and was internalized under isotonic conditions after antibody triggering (Supplemental Figs. 1 and 3). Hypertonic shock resulted in total inhibition of DCIR internalization. Importantly, CLSM showed that hypertonic conditions did not induce visible changes in cellular morphology (Supplemental Fig. 3, and data not shown). Taken together, these findings show that receptor triggering induces clathrindependent DCIR internalization.

DCIR translocates into the endosomal/lysosomal compartment, including LAMP-1⫹ lysosomes On APCs, C-type lectins are known to internalize ligands for subsequent antigen processing and presentation. Therefore, we analyzed in which cellular compartment DCIR localizes after receptor endocytosis. To this end, immature moDC were incubated with anti-DCIR and fluorochrome-conjugated secondary antibodies and counterstained with an antibody detecting the lysosomal protein LAMP-1. Incubation at 37°C for 20 min induced receptor clustering with subsequent receptor internalization after 60 min. Part of the DCIR molecules colocalized with the LAMP-1-positive lysosomal compartment (Fig. 4, A and B). DCIR molecules not colocalizing with the lysosome were found in large clusters in close proximity to the lysosomal compartment. Whether these DCIR molecules are entering or leaving a LAMP-1⫹ compartment or are present in another compartment is currently unclear. When the internalization experiments were performed with moDC stretched out on glass coverslips, similar results were obtained with DCIR partially colocalizing with the lysosome. Interestingly, in both experiments, a significant percentage of DCIR molecules remained at the cell surface even after 60 min incubation at 37°C (Fig. 4B). As expected, no colocalization with lysosomes was detectable after incubation at 4°C for 60 min, as DCIR was still present in small patches in the cell membrane. At 37°C, DCIR triggering therefore induces receptor internalization, followed by receptor translocation into intracellular compartments, including lysosomes. http://www.jleukbio.org

Fig. 3. DCIR internalization is clathrin-dependent. (A) DCIR colocalizes with clathrin on the surface of human DCs, and human immature DCs were plated on fibronectin-coated coverslips. Cells were incubated with anti-DCIR and fluorochrome-conjugated secondary antibodies for 10 min at 37°C, fixed, permeabilized, and counterstained with anticlathrin antibodies, followed by fluorochrome-conjugated secondary antibodies. DCIR (green) is detected in distinct clusters on the DC surface and colocalizes in part with clathrin (red). Data shown are representative of two independent experiments. (B) Incubation in hypertonic media inhibits DCIR internalization. Immature DCs were incubated with anti-DCIR and fluorochrome-conjugated secondary antibodies in isotonic medium for 10 (dotted lines), 30 (dashed lines), and 60 (thick lines) min at 37°C to induce receptor internalization. Noninternalized anti-DCIR/ secondary antibody complexes remaining at the cell surface were then stripped off by a pH shift, as described in Materials and Methods, allowing detection of the internalized DCIR pool. Receptor internalization thus results in increasing fluorescence signal (left). This internalization is inhibited completely once the DCs are incubated with antibodies in hypertonic medium containing 450 mM sucrose (middle). The inhibitory effect of hypertonic medium is fully reversible, so that DCIR is again internalized after “wash-out” of hypertonic medium (right). The shaded histograms depict total surface DCIR expression. A matched isotype control did not bind to the cells (thin lines). One representative experiment out of three is shown. (C) Immature DCs were treated as described above. Receptor internalization was monitored over time. At 37°C, DCIR is readily internalized, and there is not internalization at 4°C. DCIR internalization is fully inhibited by 450 mM sucrose, and sucrose wash-out followed by incubation in isotonic medium restores DCIR uptake. The differences between cells incubated in hyper- versus isotonic medium and after sucrose wash-out were statistically significant (*, P⬍0.05; **, P⬍0.01). The data shown represent the mean ⫾ SEM of three independent experiments.

DCIR triggering inhibits TLR8-dependent inflammatory cytokine production DCIR contains an intracellular ITIM motif with a putative immune inhibitory function. Cross-talk between CLR and TLR signaling pathways has been shown previously to modulate APC function. We therefore decided to first study the effects of DCIR triggering on DC maturation induced by the TLR4 ligand LPS and the TLR8 ligand R848. As a result of the lack of a specific DCIR ligand, we used mAb-mediated DCIR crosslinking to trigger DCIR. TLR engagement in the presence of an isotype control was performed as negative control. Both TLR ligands induced robust up-regulation of the B7 family members CD80 and CD86 (Fig. 5A). Triggering of DCIR neither influenced CD80 nor CD86 expression. Similar data were obtained for CD83 (data not shown). Effects of endogenous DCIR triggering on cytokine signaling have not yet been reported. A possible way of immune inhibition mediated by DCIR could be suppression of cytokine production induced by immune stimulatory agents such as TLR ligands. To test this hypothesis, immature moDC were stimulated with the TLR ligands LPS (TLR4), zymosan (TLR2 and TLR4), polyIC (TLR3), and R848 (TLR8) on plates coated with anti-DCIR or isotype control antibody. Supernatants were taken after 24 h, and TNF-␣, IL-6, IL-10, and IL-12 production was measured (Fig. 5B). DCIR triggering inhibited TLR8mediated TNF-␣ and IL-12 production by 45% and 36%, respectively. It did, however, not affect TLR4-induced TNF-␣ and IL-12 production. IL-10 secretion was only detected in

response to zymosan and was not altered by DCIR crosslinking (data not shown). DCIR triggering also lowered TLR8mediated IL-6 production, but this difference was not statistically significant (Fig. 5B). Furthermore, DCIR triggering alone did not induce any cytokine secretion. Collectively, the data show that DCIR triggering does not affect TLR-induced upregulation of CD80 and CD86 but that it inhibits TLR8mediated IL-12 and TNF-␣ production selectively in immature DCs and leaves Il-10 secretion unaffected.

DISCUSSION The present study demonstrates for the first time that human APCs express the ITAM- and ITIM-containing CLRs dectin-1 and DCIR. We have detected DCIR, not only on moDC but also on freshly isolated myeloid and plasmacytoid DCs. DCIR triggering with specific antibodies induced clathrin-dependent receptor internalization followed by trafficking to endosomal/ lysosomal compartments. Furthermore, DCIR cross-linking selectively inhibited TLR8-mediated IL-12 and TNF-␣ production in moDC. Detection of DCIR on moDC and decreased expression after DC maturation are in line with earlier findings reported in the literature [13]. In addition, we found DCIR expression on all professional APCs, including myeloid and plasmacytoid DCs. DCIR was not only detected by flow cytometry but could also be immunoprecipitated from monocytes and immature DCs and labeled by Western blot analysis as a 37-kD protein band.


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Fig. 4. DCIR is internalized into the lysosome. (A) Human moDC were incubated with anti-DCIR and fluorochrome-conjugated secondary antibodies (green) for different time-points. Cells were fixed and counterstained with anti-LAMP-1 antibodies (blue) to label the lysosomal compartment. Incubation at 37°C induced receptor clustering followed by receptor internalization and translocation to the lysosome. After 60 min incubation at 37°C, DCIR partially colocalizes with LAMP-1, and the remaining molecules are localized mostly in large clusters in close proximity to the lysosome. Incubation at 4°C does not lead to receptor internalization. DCIR is instead detected in small patches localized in the cell membrane. Samples were analyzed by CLSM. Matched isotype controls did not show any binding. Similar data were obtained in three independent experiments. (B) The same experiment was performed with moDC stretched out on coverslips. Again, DCIR (green) partially colocalizes in a large cluster with LAMP-1 (red). Some DCIR molecules remain at the cell surface.

DCIR is, next to DCAL-2, the only known ITIM-containing C-type lectin expressed by human APCs. Until now, nothing was known about the function of endogenous DCIR in human APCs. DCIR ligands have not yet been identified, making functional studies rather difficult. So far, functional analysis of DCIR has therefore relied on chimeric molecules containing its intracellular domain and immunoprecipitation experiments with peptides comprising the cytoplasmic ITIM domain. An immune inhibitory function for DCIR was first implied by the finding that BCR coligation with chimeric Fc␥RIIB molecules containing the intracellular domain of murine DCIR inhibits BCR-mediated Ca2⫹ mobilization and protein tyrosine phos6


phorylation [15]. Recently, a DCIR peptide containing the phosphorylated ITIM domain has been shown to bind to the protein tyrosine phosphatases SHP-1 and SHP-2, thus further substantiating an immune-inhibitory role for DCIR [16]. Our data now show that DCIR is internalized into moDC after receptor triggering. The level of internalization is comparable with that of the CLR DC-SIGN, for which a role in antigen uptake has been defined and has been used to target antigens to DCs [4, 21]. When we studied putative mechanisms involved in DCIR internalization, we found that DCIR colocalized with clathrin in distinct clusters on the APC surface. An involvement of clathrin in DCIR internalization was substantiated further by the finding that inhibition of clathrin-mediated endocytosis by incubation in hypertonic media totally abrogated DCIR internalization into DCs. This block was fully reversible after returning DCs into isotonic medium. The same was found in DCIR-transfected CHO cells. DCIR internalization could thus be inhibited by disruption of clathrin-coated pits in hyperosmolar medium [19, 20] in moDC and CHO cells. These findings are supported further by our recent observation that DCIR internalization into human plasmacytoid DCs can also be inhibited significantly by disrupting clathrin-mediated endocytosis. Additionally, we have found that cotransfection of dominant-negative dynamin into the DCIR-expressing CHO cell line results in inhibition of DCIR internalization [22]. Detailed analysis of effects of DCIR triggering showed that it also inhibits TLR-induced cytokine production and leaves TLR-induced CD80 and CD86 expression unaffected. IL-12 and TNF-␣ production induced by R848, which triggers TLR8 on moDC [23], was inhibited by 36% and 45%, respectively, after simultaneous DCIR cross-linking. The inhibitory effects of DCIR were selective, as cytokine production mediated by TLR4, TLR3, or zymosan was not affected by DCIR triggering. Inhibition was clearly observed for IL-12 and TNF-␣, to a lesser extent for IL-6 (not statistically significant), and was absent for IL-10 production. An important question still to be answered is at which step in the TLR8 signal transduction cascade DCIR exerts its inhibitory function. TLR8 is triggered by viral RNA and is known to signal via I␬B␣ phosphorylation and IL-1R-associated kinase-dependent NF-␬B and JNK activation [24]. Uptake of RNA viruses containing DCIR ligands could inhibit TLR8 signaling by recruitment of phosphatases such as SHP-1 and SHP-2 via the phosphorylated ITIM domain of DCIR. These phosphatases could in turn dephosphorylate members of the TLR8 signaling cascade such as I␬B, resulting in immune inhibition. DCIR ligands would therefore control and counterbalance the antiviral immune response to protect the host. DCIR-mediated immune inhibition could, however, be exploited by viruses to escape immune responses. Intriguingly, we have shown recently that DCIR triggering also inhibits TLR9-mediated IFN-␣ and TNF-␣ production by human plasmacytoid DCs by 40% and 28%, respectively [22]. Taken together, these results point at an important role for DCIR in limiting and modifying DC-mediated immune activation. Alternatively, DCIR signaling could be regulated via clathrinmediated endocytosis, as it has been demonstrated for TGFR␤II, where interfering with clathrin-dependent trafficking blocks TGF-␤-induced signaling [25, 26]. After internalizahttp://www.jleukbio.org

Fig. 5. DCIR triggering inhibits TLR8-dependent cytokine production by human DCs, and it does not affect TLR-mediated CD80 and CD86 expression. (A) DCs were cultured on anti-DCIR or match isotype control-coated plates. The TLR4 ligand LPS and the TLR8 ligand R848 were added, and cells were incubated for 24 h. CD80 and CD86 expression was analyzed by FACS. Data are depicted as ⌬-mean fluorescence intensity (MFI; MFI of specific signal–MFI of isotype control). The data shown represent the mean ⫾ SEM of three independent experiments. CD80 and CD86 up-regulation in response to both TLR stimuli was not affected by DCIR triggering. (B) DCs were cultured on anti-DCIR or matches isotype control-coated plates. Various TLR ligands were added for 24 h, and secretion of TNF-␣, IL-6, IL-10, and IL-12 was analyzed. DCIR cross-linking inhibited R848-mediated TNF-␣ and IL-12 production significantly by 45 ⫾ 8 (P⬍0.01) and 36 ⫾ 18% (P⬍0.05), respectively. TNF-␣ and IL-12 secretion, mediated by the other TLRs, was not changed after DCIR triggering. R848-induced IL-6 production was also inhibited to some degree by DCIR triggering. This inhibition was, however, not statistically significant. IL-10 secretion was only detectable after adding zymosan and was not influenced by DCIR cross-linking (data not shown). ND, Not detectable. PolyIC, Polyinosinic:polycytidylic acid. The data shown represent the mean ⫾ SEM of three independent experiments.

tion, DCIR was detected partially in or in close proximity to the endosomal/lysosomal compartment. Interestingly, some DCIR remained at the cell surface even after 60 min at 37°C. In analogy to our findings about dectin-1 [11], this could possibly be explained by association of DCIR with large molecular complexes in the cell membrane such as the tetraspanin web, allowing only free DCIR to be internalized. As DCIR is expressed by all professional APCs, it may be possible to deliver antigens or immune-modulatory compounds to DCs via antiDCIR mAb, opening the exciting prospective to target/modulate multiple APCs simultaneously. We have shown recently that antigen targeting to human plasmacytoid DCs via antiDCIR antibodies results in specific presentation [22] and have performed similar experiments with human moDC. We could demonstrate proliferation of PBLs in response to moDC loaded with anti-DCIR-keyhole limpet hemocyanin (KLH) targeting constructs (Supplemental Fig. 4). Preincubation with antiDCIR antibodies or soluble DCIR (sDCIR) inhibited T cell proliferation. However, incubation with an isotype control resulted in similar inhibition, suggesting that at least part of the observed effect is mediated by FcRs. It is known that moDC express a different repertoire of FcRs compared with plasmacytoid DCs, which can explain our findings. Identification of specific DCIR carbohydrate ligands and their conjugation to tumor antigens could be a way to target DCIR specifically without interfering with FcR-mediated endocytosis. Finally, the results presented may help to identify putative DCIR ligands. These could be diverse, ranging from viral

proteins to ligands, expressed by regulatory T cells. Expression of DCIR ligands may serve as a viral immune escape mechanism, but it may also play an important role in controlling and regulating DC-mediated immunity. Ligand identification may provide us with tools to manipulate DCIR function to prevent and treat immune-mediated diseases.

ACKNOWLEDGMENTS This work was supported in part by the Deutsche Forschungsgemeinschaft (Grant ME 2051/2-1 to F. M-W.) and by The Netherlands Organization for Scientific Research (NWO; ZonMw 912.02.034 to G. J. A., NWO-TOP Grant 912.06.030 to C. G. F., and VENI Grant 916.66.028 to A. C.). The authors thank Paul Tacken, Ph.D. (NCMLS, UMC Nijmegen, The Netherlands), for critical reading and discussion of this manuscript.

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