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The Journal of Immunology, 2007, 178: 154-162.
Copyright © 2007 by The American Association of Immunologists, Inc.

Dectin-1 Interaction with Tetraspanin CD37 Inhibits IL-6 Production1

Friederike Meyer-Wentrup*, Carl G. Figdor*, Marleen Ansems*, Peter Brossart{dagger}, Mark D. Wright{ddagger}, Gosse J. Adema2,* and Annemiek B. van Spriel*

* Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, University Medical Centre, Nijmegen, The Netherlands; {dagger} Department of Hematology, Oncology and Immunology, University of Tübingen, Tübingen, Germany; and {ddagger} Leucocyte Membrane Protein Laboratory, Austin Research Institute, Heidelberg, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
C-type lectins are pattern-recognition receptors important for pathogen binding and uptake by APCs. Evidence is accumulating that integration of incoming cellular signals in APCs is regulated by grouping of receptors and signaling molecules into organized membrane complexes, such as lipid rafts and tetraspanin microdomains. In this study, we demonstrate that C-type lectin dectin-1 functionally interacts with leukocyte-specific tetraspanin CD37. Dectin-1 and CD37 colocalize on the surface of human APCs. Importantly, macrophages of CD37-deficient (CD37–/–) mice express decreased dectin-1 membrane levels, due to increased dectin-1 internalization. Furthermore, transfection of CD37 into a macrophage cell line elevated endogenous dectin-1 surface expression. Although CD37 deficiency does not affect dectin-1-mediated phagocytosis, we observed a striking 10-fold increase of dectin-1-induced IL-6 production in CD37–/– macrophages compared with wild-type cells, despite reduced dectin-1 cell surface expression. Importantly, the observed increase in IL-6 production was specific for dectin-1, because signaling via other pattern-recognition receptors was unaffected in CD37–/– macrophages and because the dectin-1 ligand curdlan was used. Taken together, these findings show that tetraspanin CD37 is important for dectin-1 stabilization in APC membranes and controls dectin-1-mediated IL-6 production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antigen-presenting cells, including B cells, macrophages, and dendritic cells (DCs)3 are crucial for initiating immune responses leading to pathogen clearance and immunity. APCs express various pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). The most prominent PRRs are TLRs and C-type lectins. TLRs recognize a broad array of ligands (1), while C-type lectins bind exclusively to specific carbohydrate moieties through their carbohydrate-recognition domain (2, 3). The host-pathogen interaction is a highly complex and dynamic process. Immune cells express multiple PRRs that can interact with various PAMPs expressed on a single pathogen at a given time point. They must therefore integrate those incoming signals to generate an appropriate immune response. An important mechanism to regulate ligand binding and subsequent signal transduction events is to cluster membrane receptors and signaling molecules into organized complexes (4, 5), such as lipid rafts or tetraspanin microdomains.

We have recently shown that clustering of the C-type lectin DC-SIGN on immature DCs improves ligand binding (6). These findings prompted us to take a closer look at the membrane organization of dectin-1 on APCs. Dectin-1 is the only C-type lectin that contains a cytoplasmic ITAM motif. It is the major beta-glucan receptor and plays a prominent role in the immune response against fungi such as Candida albicans (7). Dectin-1 is expressed by various APCs, including immature DCs, macrophages, and B cells (8). Human and murine dectin-1 are alternatively spliced resulting in two major functional isoforms (A and B) that differ by a stalk region between the carbohydrate recognition domain and the transmembrane region that is present only in the A isoform. Minor functional differences between A and B isoforms have been described recently (9). Additionally, six nonfunctional isoforms have been identified (10). Cooperative signaling of dectin-1 with TLR2 in response to live C. albicans and the yeast cell wall component zymosan has been demonstrated in dectin-1-transfected cells and bone marrow-derived macrophages (11, 12). Furthermore, dectin-1 is the first C-type lectin for which direct cellular signaling through an interaction with Syk kinase has been reported (13, 14). Dectin-1 triggering induces production of proinflammatory cytokines by murine macrophages and DCs (8, 13, 15) including the key cytokine IL-6, known to be important in fungal infections (16, 17, 18). However, little is known about dectin-1 organization in APC membranes in relation to its function.

Tetraspanins are members of the transmembrane four superfamily that influence a wide variety of fundamental biological processes including adhesion, proliferation, Ag presentation, endocytosis, and exocytosis (19, 20). Tetraspanins modulate signal transduction by means of their capacity to laterally interact with important (immuno-) receptors, signaling molecules, and each other. They create "tetraspanin microdomains" in the plasma membrane of cells, also referred to as the "tetraspanin web" (21, 22, 23). Interestingly, dectin-1 was recently found to interact with the ubiquitously expressed tetraspanin CD63 (24). It has been suggested that tetraspanin expression is modulated during infections (25, 26), thus supporting an important role for tetraspanins in the immune response to pathogens. In contrast, tetraspanins may be exploited by certain pathogens such as hepatitis C (27) and HIV (28) and parasites such as Plasmodium falciparum (29) to infect host cells. Tetraspanin CD37 is, in contrast to CD63 and most other tetraspanins, expressed exclusively in the immune system (30). CD37-deficient mice show impaired T cell-dependent B cell responses and T cell hyperproliferation (31, 32). However, CD37 function on APCs has not been unraveled.

In this report, we demonstrate that CD37 on APCs interacts with dectin-1 and stabilizes it in the cell surface. Moreover, CD37 inhibits dectin-1-mediated IL-6 production by APCs. To our knowledge, this is the first functional interaction between a tetraspanin and a C-type lectin identified so far.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and reagents

All reagents were purchased from Sigma-Aldrich 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) and 300 U/ml IL-4 (Strathmann) in complete RPMI 1640 (Invitrogen Life Technologies)/10% FCS for 6 days to generate immature DCs. DC maturation was induced by addition of 200 ng/ml LPS for 24 h. Human macrophages were generated by culturing monocytes in complete RPMI 1640/10% human serum for 4–6 days. Raji B cells were cultured in complete RPMI 1640/10% FCS. HEK293 cells were cultured in complete DMEM (Invitrogen Life Technologies)/10% FCS.

Zymosan (Sigma-Aldrich), prepared from the Saccharomyces cerevisiae cell wall, consists of protein-carbohydrate complexes and contains high amounts of beta-glucans. Curdlan (WAKO) is a dectin-1 ligand consisting of linear beta-1,3-glucan polymers derived from Alcaligenes faecalis (33, 34). RAW 264.7 cells were transfected with murine CD37-pEGFP or pEGFP using FuGene 6 (Roche). Enhanced GFP (EGFP)-expressing cells were sorted (Elite; Coulter) and cultured under 0.5 mg/ml G418 (Calbiochem) in RPMI 1640/10% FCS.

Isolation of mouse macrophages

CD37–/– mice were generated by homologous recombination (31) and backcrossed 10 times to the C57BL/6J background. Mice were bred at the Central Animal Laboratory (Radboud University, Nijmegen, The Netherlands) and used at 6–10 wk of age. Age- and sex-matched C57BL/6J wild-type (WT) mice were obtained from Charles River Laboratories. Animal studies were approved by the Nijmegen Animal Experiments Committee. Resting macrophages were isolated from the peritoneum by abdominal lavage with 4 ml of ice-cold RPMI 1640. Adherent cells were stimulated with 20 ng/ml murine GM-CSF (Tebu-Bio) for 16 h. In addition, mice were injected with 0.75 ml of thioglycolate (Difco), killed after 4 days, and 4 ml of ice-cold RPMI 1640 were injected into the abdominal cavity to harvest thioglycolate-induced macrophages. Cells were cultured in 24-wells plates for 2 h after which nonadherent lymphocytes were removed. Flow cytometry analysis revealed adherent cells to consist of 95% (±3%) macrophages (F4/80-positive cells).

PCR and DNA constructs

cDNA was generated from mRNA isolated from Raji B cells, monocytes, mature and immature DCs, and PBLs by reverse transcription. Dectin-1 primers detecting the two major isoforms were used in the PCR (forward primer: 5'-ATG GAA TAT CAT CCT GAT TTA G-3', reverse primer: 5'-TTA CAT TGA AAA CTT CTT CTC AC-3'). Isoform B of human dectin-1 was isolated from the predominant 650-bp band of a PCR product of an RT-PCR performed with cDNA of immature DCs and dectin-1-specific primers. The coding sequence was cloned into a pECFP-C1 vector (BD Biosciences/BD Clontech) from which the enhanced cyan fluorescent protein sequenced had been removed and replaced by a C-terminal hemagglutinin (HA) tag. Human and murine CD37 were cloned into pEGFP-N1 vector placing the EGFP moiety on the C terminus of the protein (BD Biosciences/BD Clontech). Murine CXCL16 was cloned into an expression vector containing the coding sequence for an HA tag. Human DC-SIGN was cloned into pRC-CMV. All constructs were checked for correct reading frame and mutations at the Department of Antropogenetics (Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands).

Antibodies

Polyclonal goat anti-human dectin-1 (R&D Systems), monoclonal murine anti-human CD37 (clone HH1, DakoCytomation; clone WR17, Serotec), and monoclonal anti-{alpha}Lbeta2 Ab (clone Ts2/4) (35) were used in FACS analysis and copatching experiments. FACS analysis was performed with a FACSCalibur using CellQuest software (BD Biosciences). To detect murine dectin-1 monoclonal rat anti-mouse-dectin-1 Ab (clone 2A11; Serotec) was applied. MHC class II molecules were stained with rat mAb (clone m5/114) (36). Rat anti-mouse {alpha}Lbeta2 integrin (clone M17/4), rat anti-mouse {alpha}5 integrin (clone 5H10-27; BD Biosciences), rat anti-mouse Fc{gamma}R (anti-CD16/32, clone 2.4G2; BD Biosciences), allophycocyanin-conjugated hamster anti-mouse B220 (BD Biosciences), and allophycocyanin-conjugated hamster anti-mouse CD11c (BD Biosciences) Abs were used to analyze murine leukocytes. Secondary Abs used in FACS analysis were PE or FITC labeled (BD Biosciences). For confocal imaging, donkey anti-goat-Alexa 568- and rabbit anti-mouse-Alexa 647-labeled secondary Abs were used (Molecular Probes). Donkey anti-goat-Alexa 647-conjugated Ab (Molecular Probes) was used to detect anti-human dectin-1 Ab in FACS studies. For Western blot analysis, rat monoclonal anti-HA (clone 3F10; Sigma-Aldrich), rabbit polyclonal anti-GFP (a gift from F. van Kuppeveld, Department of Medical Microbiology, University Medical Centre, Nijmegen, The Netherlands; Ref. 37), rabbit polyclonal against dectin-1 (38) and rabbit polyclonal anti-DC-SIGN Ab were used. The latter was obtained after immunization of rabbits with the following peptide from DC-SIGN coupled to keyhole limpet hemocyanin: CSRDEEQFLSPAPATPNPPPA. Immunoprecipitations (IPs) were performed with murine anti-HA (clone 12Ca5; Roche), murine anti-GFP (clones 7.1 and 13.1; Roche), murine anti-dectin-1 (clone 259931; R&D Systems), goat anti-dectin-1 (R&D Systems), murine anti-CD37 (clone WR17, Serotec; clone HH1, DakoCytomation), and murine anti-DC-SIGN (clone AZND1; Beckman Coulter) Abs.

Coimmunoprecipitation (co-IP) and Western blot analysis

Dectin-1-HA, CXCL16-HA, DC-SIGN, and human CD37-EGFP or empty pEGFP were transiently cotransfected into HEK293 cells using lipofectamine (Invitrogen Life Technologies). Cells were lysed in lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1% Triton X-100, or 1% CHAPS, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 mM PMSF 16–19 h after transfection. Raji B cell lysates were precleared with bare protein G Sepharose beads (Amersham Biosciences) and isotype control Abs bound to protein G Sepharose beads. Lysates were then incubated with either anti-HA, anti-GFP, anti-CD37, or anti-dectin-1 Abs 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% 2-ME was added. Samples were boiled, separated by PAGE, and blotted onto nitrocellulose membranes. Membranes were blocked in PBS, 3% BSA, 1% skim milk powder at 4°C overnight, and stained with specific Abs. Ab signals were detected with HRP coupled secondary Abs using an enhanced luminescence detection kit (Pierce).

Mouse dectin-1 expression

Murine macrophages and RAW cells were stained with rat anti-mouse dectin-1 2A11 (10 µg/ml) or rat IgG2b isotype control (BD Biosciences), followed by allophycocyanin-labeled goat anti-rat IgG (BD Biosciences), and analyzed by FACS. For microscopy studies, macrophages were adhered to poly-L-lysine-coated glass cover slips for 1 h at 37°C, fixed with 2% paraformaldehyde, and blocked with 3% BSA, 2% goat serum, 10 mM glycine in PBS. Membranes were opened with 0.5% saponin, 3% BSA in PBS, and cells were stained with rat anti-mouse dectin-1 or rat IgG2b isotype control, followed by goat anti-rat-Alexa 488-conjugated secondary Ab (Molecular Probes). Cells were counterstained with propidium iodine to visualize nuclei. Samples were mounted in mowiol and analyzed by confocal laser scanning microscopy (Bio-Rad MRC1024).

Dectin-1 internalization assay

Freshly isolated thioglycolate-elicited peritoneal macrophages were incubated with anti-dectin-1 mAb 2A11 (10 µg/ml) or anti-MHC class II Ab m5/114 (10 µg/ml) in RPMI 1640, supplemented with 2% goat serum, on ice for 30 min. Free Ab was washed away and cells were incubated at 37°C for 0–120 min. The amount of dectin-1-mAb or MHC class II-mAb complexes remaining on the cell surface was then determined by FACS analysis after staining cells with anti-rat IgG-allophycocyanin. Isotype control rat IgG2b did not show any staining. The percentage of dectin-1 or MHC class II was calculated from mean fluorescence intensity values of each sample relative to the values at the 0-min time point.

Phagocytosis assay

Phagocytosis of zymosan was performed by incubating WT and CD37–/– macrophages (1 x 105) with zymosan (1 or 10 particles/cell) for 30 min at 37°C. Samples were analyzed by light microscopy. FITC-labeled zymosan (0.1 mg/ml FITC (Sigma-Aldrich) in 0.1 M sodium phosphate buffer) was used to quantify uptake using flow cytometry.

Copatching experiments

Raji B cells (2 x 106) were washed with ice-cold PBS and blocked in PBS, 1% human serum, 3% BSA, 10 mM glycine for 30 min on ice. Cells were incubated with primary Abs or matched isotype controls (10 µg/ml) for 30 min on ice. After washing with ice-cold PBS, cells were incubated with rabbit anti-mouse-Alexa 647- and donkey anti-goat-Alexa 568-coupled secondary Abs for 30 min on ice. The temperature was elevated to 12°C and cells were incubated for another hour. After washing in cold PBS cells were fixed in 2% paraformaldehyde and mounted on poly-L-lysine-coated cover slips. Samples were analyzed by confocal laser scanning microscopy.

IL-6 production

CD37–/– and WT macrophages (5 x 105) were stimulated with zymosan (10 particles/cell), curdlan (100 µg/ml), or LPS (1 µg/ml) in RPMI 1640/10% FCS in 24-well plates. In blocking experiments, anti-mouse-dectin-1 Ab 2A11 or isotype control (10 µg/ml) were included during stimulations. In alternative experiments, macrophages were stimulated with 2 µg/ml TLR ligands Pam3Cys, MALP-2 (both from EMC Microcollections) or R848 (PharmaTech). Supernatant was collected after 16 h and IL-6 production was measured with capture and biotinylated anti-mouse IL-6 Abs (BD Biosciences) using standard ELISA procedures.

TNF-{alpha} production

CD37–/– and WT macrophages (5 x 105) were stimulated with zymosan (10 particles/cell) or LPS (1 µg/ml) in RPMI 1640/10% FCS in 24-well plates. Dectin-1-blocking experiments were performed as described above. Supernatant was collected after 16 h and TNF-{alpha} production was measured using Luminex technology, a bead-based cytokine detection system, according to the manufacturer’s instructions (Bio-Rad/Luminex).

Statistical analyses

Statistical differences were determined using the unpaired Student t test. Significance was accepted at the p < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Dectin-1 interacts with tetraspanin CD37

To explore a putative interaction between dectin-1 and CD37, co-IP experiments in HEK293 cells transfected with CD37-EGFP and dectin-1-HA fusion proteins were performed. Because strength and nature of tetraspanin-protein interactions are commonly classified according to their degree of detergent resistance (39), co-IP experiments were done under mild (CHAPS) and rigid (Triton X-100) lysis conditions. Interactions that are stable in 1% Triton X-100 buffers are usually considered to be strong and direct, while stability only in mild detergents indicates indirect interactions. Under both conditions large amounts of CD37-EGFP could be coimmunoprecipitated with dectin-1-HA (Fig. 1A, data shown for Triton X-100). To demonstrate specificity of the CD37-dectin-1 interaction, we performed co-IPs of CD37-EGFP with the C-type lectin DC-SIGN and the chemokine receptor CXCL16-HA as negative controls. In both cases, no co-IP of CD37-EGFP was observed (Fig. 1A). Furthermore, dectin-1 did not coimmunoprecipitate GFP. Importantly, dectin-1-HA could also be coimmunoprecipitated with CD37-EGFP, showing that CD37-dectin-1 co-IPs worked in both directions (Fig. 1B). Thus, these data demonstrate a strong interaction between dectin-1 and tetraspanin CD37.


Figure 1
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  		FIGURE 1. Dectin-1 coimmunoprecipitates CD37 and vice versa. A, HEK293 cells were transiently transfected with the indicated constructs and lysed in buffer containing 1% Triton X-100. Dectin-1-HA (23 kDa) and CXCL16-HA (50 kDa) were immunoprecipitated with anti-HA and DC-SIGN (48 kDa) was immunoprecipitated with anti-DC-SIGN Abs. Total lysates (TL) and immunoprecipitates (IP) were run on 10% polyacrylamide gels under denaturing conditions and Western blots were probed with the indicated Abs. Dectin-1 strongly coimmunoprecipitates CD37 (black arrowhead) in contrast to CXCL16 and DC-SIGN. Due to heavy glycosylation, CD37 can be detected in three individual bands or a single broad band (47 ). The 19-kDa bands running below immunoprecipitated dectin-1 most likely correspond to partially degraded protein. Experiments were repeated four times providing similar data. B, HEK293 cells were transiently transfected with the indicated constructs and lysed in buffer containing 1% Triton X-100. CD37-EGFP and free EGFP were immunoprecipitated with anti-GFP Abs. Gels were run and Western blots were probed with anti-GFP and anti-HA Abs. In the absence of CD37, there is no precipitation of dectin-1. CD37 strongly coimmunoprecipitates dectin-1-HA (black arrowhead). Thus, dectin-1 can be coimmunoprecipitated with CD37 and vice versa.

 
Dectin-1 and CD37 are coexpressed on APCs

To assess whether CD37 could play a role in dectin-1 function on APCs, dectin-1 and CD37 surface expression were first analyzed on primary human APCs (Fig. 2). Human macrophages and monocytes clearly expressed dectin-1. Expression on immature DCs was lower and decreased further after maturation with LPS. We observed that dectin-1-positive macrophages and monocytes coexpressed CD37. CD37 expression on immature DCs was low and absent on mature DCs, consistent with dectin-1 expression. In addition, high levels of dectin-1 and CD37 were detected on the cell surface of Raji B cells. Primary B cells also coexpressed both molecules (data not shown). The Ab used to detect dectin-1 is directed against its carbohydrate recognition domain, which is shared by both dectin-1 isoforms (A and B). To assess which isoform is predominantly expressed in APCs, dectin-1 mRNA expression in immature and mature DCs, Raji B cells, PBMC, and PBLs was analyzed using RT-PCR. DCs (immature and mature) and PBLs predominantly expressed the B isoform. In PBMC and Raji B cells, A and B isoforms could be detected at similar levels (data not shown).


Figure 2
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  		FIGURE 2. Professional APCs coexpress CD37 and dectin-1. Human macrophages and DCs were cultured from monocytes as described in Materials and Methods. Immature DCs were matured by adding LPS (200 ng/ml) for 24 h. Cells were stained with anti-CD37 and anti-dectin-1 Abs (thick lines) or the respective isotype controls (thin lines) and analyzed by flow cytometry.

 
Endogenous dectin-1 and CD37 colocalize on human APC membranes

Next, we evaluated the membrane localization of CD37 and dectin-1 in human APCs. Because Raji B cells express both endogenous dectin-1 and CD37 at high levels (Fig. 2), copatching studies were performed with live Raji B cells. Confocal laser scanning microscopy revealed distinct colocalization of dectin-1 and CD37 in membrane patches indicating an interaction between the two molecules (Fig. 3A, upper panel). As negative control, the same experiment was done with CD37 and the {alpha}Lbeta2 integrin, which did not show copatching (Fig. 3A, lower panel). Furthermore, we performed IPs of endogenous CD37 and dectin-1 from Raji B cells (Fig. 3B). Endogenous CD37 was immunoprecipitated with two different Abs that both coimmunoprecipitated dectin-1 under 1% CHAPS conditions. Control IPs performed with a mix of goat and mouse IgG showed no co-IP of dectin-1. To analyze the nature of the upper bands (~27 kDa, *), the Western blot was stained with anti-mouse secondary Abs (data not shown). Murine Ig H and L chains were detected in approximately equal amounts in all lanes except for the IP with goat anti-dectin-1 Ab (lane 1), indicating that the upper band corresponds to Ig L chains cross-reacting with the secondary Ab. Due to unavailability of anti-CD37 Abs suitable for Western blot analysis, reciprocal co-IPs could not be performed. Taken together, endogenous dectin-1 and CD37 interact and colocalize on the surface of human APCs.


Figure 3
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  		FIGURE 3. Endogenous dectin-1 and CD37 colocalize in distinct clusters on the surface of human Raji B cells and can be coimmunoprecipitated. A, Live Raji B cells were incubated on ice with anti-dectin-1 and anti-CD37 Abs, washed, incubated with fluorochrome-labeled secondary Abs, and copatching was induced at 12°C. Cells were then fixed, mounted on poly-L-lysine-coated cover slips, and analyzed by confocal laser scanning microscopy. CD37 is labeled in green and dectin-1 in red. The merge (yellow) shows colocalization (white arrowheads) (upper panel). To exclude nonspecific colocalization, the same experiment was performed with anti-CD37 and anti-{alpha}Lbeta2-integrin Abs as negative control (lower panel). Similar results were obtained in three independent experiments. B, Raji B cells were lysed in buffer containing 1% CHAPS. Endogenous dectin-1 and CD37 were immunoprecipitated with goat polyclonal and mouse monoclonal anti-dectin-1, and mouse anti-CD37 mAbs HH1 and WR17, respectively. IPs were separated on 10% polyacrylamide gels under denaturing conditions and the Western blot was probed with rabbit polyclonal anti-dectin-1 Ab. Dectin-1 is coimmunoprecipitated in both CD37-IPs at 23 kDa (black arrowhead). The negative control IP was performed with a mix of goat polyclonal IgG and mouse IgG (right lane). The upper bands (~27 kDa, *) correspond to Ig L chains cross-reacting with the secondary Ab. Reciprocal IPs could not be performed in the endogenous setting due to unavailability of anti-CD37 Abs suitable for Western blot analysis.

 
Dectin-1 surface expression is stabilized by CD37

To study the significance of the dectin-1 interaction with CD37 on APCs, dectin-1 surface expression was examined in CD37 knockout (CD37–/–) mice. We first analyzed on which murine APC subsets dectin-1 was expressed. In line with literature (40), clear dectin-1 expression was detected on murine thioglycolate-elicited WT macrophages, whereas dectin-1 expression on B cells and splenic immature DCs was low/absent (Fig. 4A). Strikingly, dectin-1 membrane expression was significantly reduced on CD37–/– macrophages (70%), while expression of other surface receptors tested ({alpha}4beta1 integrin, {alpha}Lbeta2 integrin, and Fc{gamma}R) was similar between WT and CD37–/– macrophages (Fig. 4A). Reduced dectin-1 membrane expression on CD37–/– macrophages was confirmed by confocal microscopy studies (Fig. 4B). In addition, CD37 deficiency resulted in decreased intracellular expression of dectin-1 in macrophages. To substantiate that the observed differences in dectin-1 expression were indeed due to the interaction with CD37 and not to general cellular activation caused by CD37 deficiency in vivo, we analyzed resting peritoneal macrophages. Dectin-1 expression on resting macrophages was low, which is in line with literature (41) (Fig. 4C). In vitro stimulation with GM-CSF resulted in higher dectin-1 surface expression on WT macrophages compared with CD37–/– cells, similar to thioglycolate-induced macrophages. Thus, these experiments demonstrate that expression of CD37 on APCs increases the amount of cell surface dectin-1.


Figure 4
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  		FIGURE 4. Cell surface dectin-1 expression is reduced on CD37–/– macrophages. A, Dectin-1 expression on WT and CD37–/– B cells and splenic immature DCs (upper panel) and on thioglycolate-elicited macrophages (middle panel). Mean fluorescence intensity of dectin-1 surface levels on CD37–/– macrophages is reduced by 70% compared with WT cells (overlay). CD37–/– and WT thioglycolate-elicited macrophages were labeled with anti-{alpha}4beta1-integrin, anti-{alpha}Lbeta2, and anti-Fc{gamma}R Abs (thick line) or matching isotype controls (thin line) and analyzed by flow cytometry (lower panel). FACS data are representative of four independent experiments. B, Dectin-1 expression is reduced in CD37–/– cells. WT and CD37–/– macrophages were fixed, permeabilized, labeled with anti-dectin-1 Ab and propidium iodine (PI), and analyzed by confocal laser scanning microscopy. A matched isotype control did not bind to the cells. Original magnification: x400 and x1000 (insets). C, Resting WT and CD37–/– peritoneal macrophages express low dectin-1 levels (upper). Dectin-1 expression is higher in WT compared with CD37–/– macrophages stimulated in vitro with GM-CSF (lower).

 
To explore the underlying mechanisms, dectin-1 internalization studies were performed. Macrophages from WT and CD37–/– mice were incubated with dectin-1 Ab and dectin-1 surface expression was analyzed by flow cytometry after incubating cells at 37°C. WT macrophages still expressed all (96%) of surface dectin-1 after 30 min. Interestingly, on CD37–/– macrophages this number decreased significantly to 75%. After 60 min, 76% dectin-1 expression on WT and only 56% expression on CD37–/– cells could still be detected (Fig. 5A). To exclude differences in nonspecific protein turnover in membranes of CD37–/– compared with WT macrophages, endocytosis of MHC class II molecules was analyzed as control. Similar MHC class II internalization was observed between WT and CD37–/– macrophages (Fig. 5B). Thus, CD37 increases dectin-1 surface expression by stabilizing dectin-1 at the APC membrane leading to reduced protein internalization.


Figure 5
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  		FIGURE 5. CD37 stabilizes dectin-1 in the cell membrane. A, Macrophages from WT and CD37–/– mice were incubated with anti-dectin-1 Ab 2A11 on ice for 30 min. Free Ab was washed away and cells were incubated at 37°C for the indicated time points. Dectin-1 surface expression was measured by FACS analysis after adding anti-rat-allophycocyanin-labeled secondary Ab. Isotype control did not show any staining. To take into account the different dectin-1 expression between WT and CD37–/– cells, results are shown as percentages of dectin-1 surface expression relative to time point 0 min. The percentage of dectin-1 on the membrane was calculated from mean fluorescence intensity values of each sample. Differences observed between WT and CD37–/– cells were statistically significant (p values of 0.028, 0.0009, and 0.006 at 10, 30, and 60 min, respectively). Results are depicted as average of three independent experiments ± SEM. B, CD37 does not affect MHC class II internalization. Internalization studies with macrophages from WT and CD37–/– mice were performed as described above using anti-MHC class II Ab. Percentage of MHC class II surface expression (±SEM) at the indicated time points relative to time point 0 is shown.

 
Finally, we transfected murine Raw macrophages, which are known to express low levels of endogenous dectin-1 (11), with murine CD37-EGFP and measured dectin-1 surface expression by flow cytometry. Overexpression of CD37 clearly increased endogenous dectin-1 surface expression compared with untransfected cells or cells transfected with EGFP alone (Fig. 6). These experiments show that CD37 is a direct regulator of dectin-1 protein expression. In addition, we performed dectin-1 quantitative PCR on RAW cells transfected with CD37-EGFP or EGFP. Dectin-1 mRNA levels were not increased by CD37 (data not shown) in contrast to dectin protein levels (Fig. 6). Thus, CD37 is not involved in protein synthesis of dectin-1, but CD37 controls dectin-1 protein stabilization.


Figure 6
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  		FIGURE 6. CD37 transfection increases endogenous dectin-1 cell surface expression. Murine raw macrophages were transfected with EGFP or CD37-EGFP. Cells were labeled with anti-dectin-1 Abs or matching isotype controls and analyzed by flow cytometry. Untransfected macrophages express dectin-1 at low levels comparable to EGFP-transfected cells, while transfection of CD37-EGFP results in substantially increased dectin-1 surface levels.

 
CD37 inhibits dectin-1-mediated IL-6 production

To investigate whether dectin-1 interaction with CD37 has functional consequences, we first studied phagocytic capacity of murine macrophages. Uptake of the fungal cell wall preparation zymosan by WT and CD37–/– thioglycolate-elicited peritoneal macrophages was similar (Fig. 7). Dectin-1 triggering with beta-glucan containing fungi induces production of proinflammatory cytokines by murine macrophages and DCs (8, 15, 42) including the key cytokines IL-6 and TNF-{alpha}. IL-6 is known to be important in immune defense against fungal infections (16, 17, 18). Therefore, WT and CD37–/– macrophages were stimulated with zymosan that contains highly concentrated beta-glucans and IL-6 secretion was measured. Surprisingly, we observed a 10-fold increase in IL-6 response of CD37–/– cells compared with WT macrophages (2900 vs 210 pg/ml), despite the decreased dectin-1 expression on CD37–/– cells (Fig. 4). As control, WT and CD37–/– cells were stimulated with LPS at concentrations ranging from 0.001 to 1 µg/ml, which revealed no differences in IL-6 production (Fig. 8A, data shown for 1 µg/ml LPS). Similarly, CD37–/– macrophages produced more TNF-{alpha} in response to zymosan than WT cells. There was no difference in the response to LPS (Fig. 8B). Also, stimulating CD37–/– and WT macrophages with other TLR ligands (Malp-2, R848, Pam-3-Cys) resulted in comparable IL-6 production (data not shown). Zymosan-induced IL-6 could be blocked only partially with the dectin-1 Ab 2A11 (40% reduction, data not shown). This may be explained by the presence of ligands for other C-type lectins in zymosan cell wall preparations. To further characterize dectin-1-mediated IL-6 production in the absence and presence of CD37, we therefore used the more specific dectin-1 ligand curdlan. Curdlan has recently been shown to bind dectin-1 (34) and to trigger dectin-1 signaling in murine APCs independently of TLR2 (33). Curdlan stimulation significantly enhanced IL-6 production by CD37–/– macrophages compared with WT macrophages (495 vs 115 pg/ml). Importantly, curdlan-induced IL-6 production by both CD37–/– and WT macrophages was almost completely blocked with the anti-dectin-1 Ab 2A11 (Fig. 8C). Similar data were obtained for TNF-{alpha} (data not shown). IL-6 release induced by LPS was not inhibited by the anti-dectin-1 Ab (data not shown). Thus, these data demonstrate that curdlan-induced IL-6 production is dectin-1 dependent. In summary, we conclude that CD37 controls dectin-1-mediated signaling leading to decreased IL-6 secretion. In the absence of CD37, dectin-1-induced IL-6 release by APC is strongly enhanced.


Figure 7
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  		FIGURE 7. CD37-deficiency does not change phagocytic capacity of macrophages. A, Phagocytosis of zymosan particles by CD37–/– macrophages (left) and WT cells (right) after 30 min of coincubation. Arrowheads indicate ingested zymosan particles. B, The graph shows percentage of zymosan-positive WT and CD37–/– macrophages using different ratios of macrophage:zymosan (1:1 and 1:10) and measured by flow cytometry (n = 3). Error bars indicate SEM.

 

Figure 8
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  		FIGURE 8. CD37 inhibits dectin-1-mediated IL-6 production. A, WT and CD37–/– thioglycolate-elicited macrophages were stimulated with zymosan (10 particles/cell) or LPS (1 µg/ml) for 16 h. CD37–/– macrophages produced significantly more IL-6 in response to zymosan (p = 0.022) than WT cells, while LPS-induced IL-6 production was similar. B, CD37–/– macrophages also produce more TNF-{alpha} than WT cells in response to zymosan (10 particles/cell). C, Curdlan (100 µg/ml)-induced IL-6 production is mediated through dectin-1. Preincubating WT and CD37–/– macrophages with anti-dectin-1 Ab 2A11 (10 µg/ml) blocks curdlan-induced IL-6 production in WT (p = 0.0014) and CD37–/– macrophages (p = 0.00007), while incubation with a matched isotype control (ratIgG2b) does not. One representative experiment of three is shown. Error bars indicate SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Little is known about dectin-1 cell surface distribution and putative interactions with other proteins in APC membranes. In this study, we demonstrate not only that dectin-1 interacts with the leukocyte-specific tetraspanin CD37, but also that the dectin-1-CD37 association affects dectin-1-mediated IL-6 secretion. The interaction with dectin-1 is the first functional interaction demonstrated for CD37. Our data support a new model in which C-type lectin function is regulated by tetraspanins (Fig. 9).


Figure 9
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  		FIGURE 9. Model for CD37-dectin-1 interaction at the cell surface and inhibition of dectin-1-mediated IL-6 secretion by CD37. Under normal conditions, CD37 binds to dectin-1, stabilizes it in the cell membrane, and inhibits dectin-1-mediated IL-6 production, possibly through recruiting yet unknown tyrosine phosphatases. In the presence of CD37, moderate amounts of IL-6 are released in response to dectin-1 triggering (left panel). In the absence of CD37, dectin-1 internalization increases, leading to reduced dectin-1 surface expression. In parallel, dectin-1-mediated IL-6 secretion is deblocked and increases dramatically (right panel).

 
The interaction between CD37 and dectin-1 in transfected HEK293 cells is stable under Triton X-100 conditions, which is indicative for a primary (or direct) interaction. In Raji B cells it could so far only be detected under mild conditions (CHAPS). A possible explanation for this discrepancy could be that on the APC surface the interaction is more dynamic depending on cellular activation status. Furthermore, APCs may express other (immuno)-proteins not expressed by HEK293 cells, which compete for interaction with CD37 or dectin-1. Additionally, IP of endogenous CD37 from Raji cells was hampered by low efficiency of CD37 Abs in IP (data not shown). The stalk region present in the larger A isoform of dectin-1 is apparently not required for the interaction because co-IPs were successfully performed with the B isoform. In addition, the B isoform was coimmunoprecipitated with CD37 from Raji B cells. The exact domains of CD37 and dectin-1 involved in the interaction and the stoichiometry have yet to be defined.

The interaction between CD37 and dectin-1 is specific, because other leukocyte receptors (C-type lectin DC-SIGN and the chemokine receptor CXCL16) used as controls did not coimmunoprecipitate CD37. We observed that human APC subsets coexpress dectin-1 and CD37 on their surface, and that both receptors colocalized in distinct clusters on the cell membrane, confirming the interaction of endogenous CD37 and dectin-1 in APCs. Furthermore, endogenous dectin-1 and CD37 could be coimmunoprecipitated from Raji B cells. Dectin-1 has been found to associate with the ubiquitously expressed tetraspanin CD63 on human immature DCs. This interaction was demonstrated only under mild lysis conditions, and its functional significance has not yet been elucidated (24). CD63 is mainly present in intracellular vesicles and may play a role in protein routing to MHC class II compartments. CD37, in contrast, is expressed on the cell surface and is, like dectin-1, leukocyte specific.

In this study, we report that CD37 stabilizes dectin-1 in the cell membrane and in intracellular compartments of APCs. CD37 deficiency results in low dectin-1 surface and intracellular expression in murine APCs and increases dectin-1 internalization. Although we cannot exclude that loss of dectin-1 from the cell membrane is due to cleavage of dectin-1 extracellular domain, there is to our knowledge no experimental evidence for this. Moreover, our findings are confirmed by studies with RAW cells, in which CD37 overexpression resulted in higher cell surface levels of endogenous dectin-1. Our data are consistent with studies reporting tetraspanins to influence cellular distribution as well as protein turnover and signaling capacity (43).

Recently, it was shown that dectin-1 triggering by the fungus Aspergillus fumigatus induces IL-6 production (15). Moreover, IL-6-deficient mice show impaired immune responses to C. albicans (16, 18, 44), substantiating a crucial role for IL-6 in host defense against fungi. To determine the functional significance of decreased dectin-1 expression, we first compared IL-6 production by WT and CD37-deficient macrophages in response to the yeast cell wall preparation zymosan. Zymosan is rich in the dectin-1 ligand beta-glucan. Interestingly, despite the reduced expression of dectin-1 on CD37–/– macrophages, a massive increase in IL-6 production was observed, which specific dectin-1 Abs could block only partially. Because zymosan contains ligands for other PRRs, and CD37 is likely to affect other proteins, macrophages were stimulated with the more specific dectin-1 ligand curdlan. Curdlan has recently been shown to bind dectin-1 and to induce release of proinflammatory cytokines (33, 34). Stimulation of CD37–/– macrophages with curdlan induced significantly higher IL-6 production compared with WT macrophages, which could be blocked by specific dectin-1 Abs. In summary, these data demonstrate that CD37 1) stabilizes dectin-1 in the cell membrane by reducing protein internalization and 2) inhibits signaling pathways leading to dectin-1-mediated IL-6 production. Our data provide a new concept in which the function of dectin-1 is negatively regulated by its interaction with CD37 (Fig. 9). In the presence of CD37, moderate amounts of IL-6 are released in response to dectin-1 triggering. However, IL-6 production is increased due to unleashed dectin-1 signaling in CD37-deficient APCs. Because we did not observe differences in zymosan uptake in CD37–/– compared with WT macrophages, altered ligand phagocytosis is not responsible for the observed increase in dectin-1-mediated IL-6 production. Our findings correspond to literature reporting independent signaling pathways for dectin-1-mediated phagocytosis and cytokine production (45). The underlying mechanism may involve recruitment of tyrosine phosphatases by CD37 that dephosphorylate molecules involved in dectin-1-mediated signaling cascades. Tetraspanin association with tyrosine phosphatases has been documented before (46). Dectin-1 has been shown to signal directly via its interaction with the protein kinase Syk (13, 14) and to induce cytokine secretion. In addition, dectin-1 has been described to augment TLR2-mediated TNF-{alpha} and IL-12 production by an as yet unknown mechanism. Interestingly, we detected that CD37-deficient APCs produce more TNF-{alpha} in response to zymosan, but not to LPS. So far, a direct physical interaction between dectin-1 and TLR2 was not detected (12). How the CD37-dectin-1 interaction relates to dectin-1 synergy with the TLR2-signaling pathway remains to be determined. It is tempting to speculate that CD37 directly links dectin-1 and TLR2 signal transduction pathways.

The association of dectin-1 with CD37 (and possibly other tetraspanins) is likely to be dynamic, allowing cells to cluster surface PRRs to achieve specificity in the immune response to multivalent pathogens displaying various PAMPs. The next challenge is to define the constituents of the tetraspanin web at different stages of APC activation under immune stimulatory vs immune inhibitory conditions. We hypothesize that recruitment of C-type lectin receptors into tetraspanin domains determines their signaling potential.


   Acknowledgments
 
We thank Mihai Netea and Gerben Ferwerda (Department of Internal Medicine, University Medical Centre, Nijmegen, The Netherlands) for helpful and inspiring discussions during the preparation of this manuscript. Stefan Nierkens and Roger Sutmuller helped us with the experiments performed with murine APCs. We are grateful to Dr. Z. Orinska (Department of Immunology and Cell Biology, Research Centre, Borstel, Germany) for providing CD37-deficient mice. Furthermore, we greatly appreciate the technical help from the Central Animal Facility of the University Medical Centre.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the Dutch Cancer Association (Koningin Wilhelmina Fonds Fellowship to A.B.v.S.), by the Deutsche Forschungsgemeinschaft (Grant ME 2051/2-1 to F.M.-W.), and by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (ZonMw 912-02-034 to G.J.A.). Back

2 Address correspondence and reprint requests to Prof. Gosse J. Adema, Department of Tumor Immunology, Radboud University Medical Centre, Nijmegen, Nijmegen Centre for Molecular Life Sciences/278 TIL, Post Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail address: g.adema{at}ncmls.ru.nl Back

3 Abbreviations used in this paper: DC, dendritic cell; PRR, pattern-recognition receptor; PAMP, pathogen-associated molecular pattern; IP, immunoprecipitation; co-IP, coimmunoprecipitation; EGFP, enhanced GFP; WT, wild type; HA, hemagglutinin. Back

Received for publication June 23, 2006. Accepted for publication October 10, 2006.


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 Introduction
 Materials and Methods
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 Disclosures
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