Syk Kinase-Coupled C-type Lectin Receptors Engage Protein Kinase C-δ to Elicit Card9 Adaptor-Mediated Innate Immunity

1The innate immune system uses germline-encoded pattern recognition receptors (PRRs) to sense microbial pathogens or other forms of danger, leading to the induction of inflammatory responses and host defense. Bona fide PRRs with signaling capacities that reprogram gene expression include Toll-like receptors (TLRs), nucleotide-oligomerization domain (Nod)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like helicases (RLRs), and the recently emerging family of spleen tyrosine kinase (Syk)-coupled C-type lectin receptors (CLRs). Central to the ability of these PRRs to elicit inflammation and innate immunity is their capacity to activate the canonical NF-κB signaling cascade.

2Dectin-1, the first Syk-coupled CLR found to be crucial for mammalian host protection, still serves as a paradigm for CLR signaling. This receptor recognizes β-glucan carbohydrates in the cell walls of fungi. Genetic studies in humans and mice have revealed essential functions for Dectin-1 in antifungal host protection. To induce signaling, Dectin-1 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like motif in its intracellular tail, which is responsible for Syk engagement. Additional Syk-coupled CLRs with critical roles in antifungal immunity are Dectin-2 and Mincle, which associate with the ITAM-containing adaptor molecule Fc receptor γ chain (FcRγ). Notably, ITAM-Syk-coupled CLRs are not only involved in antifungal defense but are also implicated in innate immunity to mycobacteria or helminths, as well as in inflammatory responses triggered by viruses or self-ligands.

3CLR signaling is initiated by receptor proximal Src family kinases that phosphorylate ITAMs or ITAM-like structures, resulting in Syk recruitment and activation. Syk is then responsible for tyrosine phosphorylation of signaling intermediates that trigger downstream pathways, including mitogen-activated protein kinase (MAPK) and NF-κB signaling. A general mediator of CLR function is the adaptor protein Card9, which controls canonical IκB-kinase (IKK) and NF-κB activation and which is required for inflammatory responses induced by Dectin-1, Dectin-2, and Mincle. Consistent with the essential roles of the Card9 pathway in host defense, mice or humans with a genetic loss of Card9 function are immunodeficient and highly susceptible to fungal infections. Still, even though the Syk-Card9 pathway is nonredundant in innate immunity, the molecular mechanisms that couple CLR proximal events to Card9 activation are undefined.

4Here we used a proteomic approach to search for intracellular mediators of CLR-Syk function. With Dectin-1 signaling as a model, we report that PKCδ (encoded by Prkcd) is activated upon CLR stimulation and specifically required to induce Card9-Bcl10 complex assembly for TAK1 activation and canonical NF-κB control. We found that Prkcd−/− dendritic cells are defective in innate immune responses to CLR stimulation and that Prkcd−/− mice are highly susceptible to fungal infection. These results identify a fundamental mechanism for inflammatory cell activation, with essential roles in CLR-mediated innate immunity.

5To gain insights into the mechanisms of Dectin-1-Syk signaling, we stimulated primary bone marrow-derived dendritic cells (BMDCs) from mice with zymosan. Zymosan is a fungal cell wall preparation composed primarily of β-glucans and functions as a strong Dectin-1 agonist. In addition, it contains ligands for Dectin-2 and TLR2. As expected, zymosan stimulation induced tyrosine phosphorylation of multiple target proteins (Figure 1A). To reveal the identity of these molecules in an unbiased manner, we affinity purified the tyrosine-phosphorylated proteins and analyzed these by mass spectrometry (MS) (Figure 1B). The 20 specifically tyrosine-phosphorylated proteins with the highest Mascot scores included four kinases: the receptor proximal Src family members Lyn and Fgr, together with Syk and PKCδ (data not shown). Because we were particularly interested in putative Syk effector molecules, we focused our attention on PKCδ.

6To validate zymosan-induced PKCδ phosphorylation, we immunoprecipitated this specific kinase from stimulated BMDCs and subsequently performed immunoblots with phospho-tyrosine antibodies. Consistent with our MS data, zymosan treatment induced strong PKCδ tyrosine phosphorylation (Figure 1C). Then, we performed analyses with a phosphospecific antibody raised against PKCδ tyrosine (Tyr) 311, a residue involved in PKCδ activation. Robust, activation-specific PKCδ Tyr311 phosphorylation was rapidly detected in response to zymosan treatment (Figure 1D). To test whether this PKCδ phosphorylation was mediated via Src and Syk signaling, we pharmacologically blocked these receptor proximal tyrosine kinases with the inhibitors PP2 or R406, respectively, before stimulation of the cells. Both inhibitors impeded zymosan-induced PKCδ Tyr311 phosphorylation (Figure 1E). Thus, we conclude from this first set of experiments that innate recognition of zymosan activates PKCδ through a Src and Syk tyrosine kinase-dependent mechanism.

7Next, we were interested in the functional roles of PKCδ in Dectin-1-mediated innate immune responses. We therefore pretreated BMDCs with the selective PKC inhibitor panPKC LMWI prior to stimulation. panPKC LMWI inhibited TNF, IL-10, and IL-2 production in response to zymosan stimulation or upon specific Dectin-1 triggering with curdlan, a pure β-(1,3)-glucan polymer and selective agonist for Dectin-1 (Figure 2A; ). In contrast, treatment of the cells with panPKC LMWI did not inhibit cytokine secretion induced by TLR4 stimulation with LPS or TLR9 stimulation with CpG-DNA (Figure 2A).

8Because panPKC LMWI inhibits not only PKCδ but also other PKC isoforms, we moved on to a genetic approach to dissect the functions of individual PKCs in CLR signaling. To this end, we generated BMDCs from mouse strains that are deficient in either PKCα (encoded by Prkca), PKCβ (encoded by Prkcb), PKCβ and PKCθ (encoded by Prkcq), or PKCδ. Interestingly, BMDC differentiation was not impaired by the lack of any of these PKC isoforms (data not shown). Moreover, BMDCs deficient in PKCα, PKCβ, or PKCβ and PKCθ produced regular amounts of TNF and IL-10 in response to zymosan treatment or upon specific Dectin-1 stimulation with curdlan (Figure 2B and data not shown). In sharp contrast, Prkcd−/− BMDCs were severely impaired in zymosan- or curdlan-induced cytokine production, although responses to TLR1-TLR2 stimulation with Pam3CSK4 or TLR3 triggering with long poly(I:C) were not reduced (Figure 2B). These findings indicate an essential and specific role for PKCδ in the Dectin-1 pathway. To characterize this function in more detail, we performed dose-response experiments with different PRR agonists. Again, we observed a critical requirement for PKCδ in zymosan- or curdlan-mediated TNF and IL-10 production, but cytokine production in response to TLR4 stimulation with LPS was not affected by the deletion of PKCδ (Figure 3A).

9Because Dectin-1 signaling triggers not only the synthesis of cytokines but also the phagocytosis of fungal components, as well as the production of reactive oxygen species (ROS), we studied the role of PKCδ in these pathways. In contrast to cytokine production, the phagocytosis of zymosan particles was independent of PKCδ, as assessed by either fluorescence microscopy or flow cytometric quantification (Figures 3B and 3C). Moreover, the generation of ROS upon zymosan treatment was not significantly impaired in Prkcd−/− BMDCs (Figure 3D).

10Next we investigated whether PKCδ functions in Dectin-1-independent CLR responses by stimulating Prkcd−/− cells with agonistic antibodies against Dectin-2 or with trehalose-6,6-dibehenate (TDB), a synthetic adjuvant analog of the mycobacterial cord factor that functions as a selective and specific agonist for Mincle. Similar to the Dectin-1 responses, both Dectin-2- and Mincle-induced IL-10 and TNF production were severely impaired in the absence of PKCδ (Figure 3E and data not shown). These findings indicate that PKCδ plays a general role in Syk-coupled CLR response pathways, where it is specifically required for the control of cytokine synthesis.

11To define the molecular function of PKCδ in CLR signaling, we focused again on zymosan stimulation and selective Dectin-1 triggering (Figure 4). The overall tyrosine phosphorylation pattern, the activation of Syk, as well as the phosphorylation of the Dectin-1 signal transducer PLCγ2 did not differ substantially between zymosan- or curdlan-stimulated WT and Prkcd−/− cells (Figure 4A and data not shown). Moreover, PKCδ was to a large extent dispensable for Erk1 and Erk2 MAPK activation, as shown by the fact that Erk1 and Erk2 phosphorylations were only slightly reduced in Prkcd−/− cells (Figure 4B). In sharp contrast, signaling to the canonical NF-κB pathway, a key driver of cytokine production, was almost completely blocked in zymosan-treated or Dectin-1-stimulated Prkcd−/− BMDCs, as indicated by the lack of phosphorylation in the activation loops of IKKα and IKKβ (Figure 4C).

12As mentioned above, Dectin-1-Syk signaling engages the Card9 adaptor protein for canonical IKK-dependent NF-κB activation. To study the potential involvement of PKCδ in the Card9 signaling cascade, we first stimulated Card9−/− BMDCs with zymosan and investigated PKCδ tyrosine phosphorylation. PKCδ was normally activated in the absence of Card9 (Figure 5A). Then, we tested the possibility that PKCδ might directly phosphorylate Card9. Indeed, in vitro kinase assays with recombinant proteins demonstrated that PKCδ can phosphorylate Card9 but not bovine serum albumin (Figure 5B). To search for putative PKCδ phosphorylation sites within Card9, we performed a bioinformatic analysis applying both the NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) and Scansite (http://scansite.mit.edu/) target site prediction algorithms. Three conserved PKC phosphorylation sites were predicted in both murine and human Card9. These were Thr95, Thr231, and Ser303 (Figure 5C). Because Thr95 and Thr231 are within the only two conserved TxK or TxR motifs in Card9, we next performed immunoblot analysis with proteins from the PKCδ-Card9 kinase reaction and a phosphoThreonine-x-Arginine (pTxR) antibody that recognizes phosphorylated Thr followed by Arg or Lys at the +2 position. Consistent with the bioinformatic prediction, PKCδ-phosphorylated Card9 reacted with the pTxR antibody, indicating phosphorylation at TxK motifs (Figure 5D). To further map PKCδ phosphorylation sites within Card9, we used site-directed mutagenesis and transiently transfected expression plasmids encoding tagged forms of WT-Card9, Card9(T95A), Card9(T231A), or Card9(S303A) in HEK293 cells. We were technically unable to efficiently express the Card9(T95A) version in mammalian cells, possibly because of effects of this mutation on protein stability. Nevertheless, WT-Card9, Card9(T231A), and Card9(S303A) were equally expressed in HEK293 cells (data not shown) and were used upon purifications as substrates for in vitro PKCδ kinase assays (Figure 5E). The quantification of radio-phospho incorporation revealed that WT-Card9 and Card9(S303A) were equally phosphorylated by PKCδ in vitro. In contrast, the phosphorylation of the Card9(T231A) mutant was reduced by 50% compared to the WT, demonstrating that PKCδ phosphorylates Card9 at position Thr231 and presumably on additional sites.

13To study the functional relevance of the Card9 Thr231 phosphorylation in CLR signaling, we next generated retroviral vectors that express WT-Card9, Card9(T231A), or Card9(S303A) to reconstitute Card9 gene-deficient BMDCs. Subsequently, we stimulated the reconstituted cells with the Dectin-1 agonist Curdlan and measured the production of IL-10 as a readout for productive signaling (Figure 5F). Although the Card9(S303A) mutant restored the signaling defect of Card9-deficient cells to the same extent as WT-Card9, Card9(T231A) was unable to rescue Dectin-1 signaling for cytokine production. Together with the biochemical results above, these data indicate that PKCδ mediates Thr231 phosphorylation of Card9 and that this phosphorylation is essential for Card9 function.

14Upon cellular stimulation, Card9 forms a signaling complex with its effector protein Bcl10 leading to canonical IKK-dependent NF-κB signaling. To investigate the requirement for PKCδ in these events, we analyzed Card9-Bcl10 complex assembly in Prkcd−/− cells. To this end, we immunoprecipitated Bcl10 from zymosan-stimulated WT and Prkcd−/− cells and studied its association with Card9 by protein immunoblot. Card9-Bcl10 complexes assembled only in zymosan-stimulated WT BMDCs but not in cells lacking PKCδ (Figure 6A), indicating an essential function for PKCδ activity in Card9-Bcl10 complex formation.

15How the Card9-Bcl10 signalosome activates IKKs has not been well defined. However, given that Bcl10 can utilize the kinase TAK1 for T cell receptor-mediated NF-κB activation, we speculated that TAK1 could also be activated through the Card9-Bcl10 complex during innate immune response. To test this hypothesis, we stimulated BMDCs of the different genotypes with zymosan and investigated TAK1 phosphorylation. Indeed, zymosan-induced TAK1 phosphorylation was strictly dependent on Card9 (Figure 6B). Moreover and in line with our finding that Prkcd−/− BMDCs were defective in Card9-Bcl10 activation, zymosan-induced TAK1 activation was abrogated in PKCδ-deficient cells (Figure 6C). To directly investigate the function of TAK1 in zymosan-induced NF-κB signaling, we pretreated BMDCs with the selective chemical TAK1 inhibitor (5Z)-7-Oxozeaenol before cell stimulation. Although TAK1 inhibition did not block zymosan-induced PKCδ activation, it prevented the activation of IKKs (Figure 6D). Together, these findings indicate that PKCδ plays an essential role upstream of the Card9-Bcl10 module, which is critical for the subsequent activation of TAK1 and the engagement of the canonical NF-κB pathway.

16After uncovering the molecular functions of PKCδ in CLR signaling, we were interested in its role in a pathophysiologically relevant setting. As a clinically important model pathogen we chose C. albicans because its PAMPs are recognized by Dectin-1, Dectin-2, and Mincle and they drive Syk- and Card9-mediated innate immunity and host defense. Although C. albicans infection of WT BMDCs induced a robust and dose-dependent production of cytokines, TNF, IL-10, and IL-1β, production of these cytokines was almost completely abolished in the absence of PKCδ (Figure 7A). Also on the biochemical level we observed that, in spite of normal Syk phosphorylation, Prkcd−/− BMDCs had severe deficiencies in activating NF-κB signaling after stimulation with C. albicans hyphae, a potent agonist of Dectin-2. In line with our results above, Prkcd−/− cells furthermore showed defects in IKK activation, as well as in IκBα phosphorylation and degradation (Figure 7B) after C. albicans recognition.

17Finally, to study the relevance of these findings for host protection in vivo, we infected Prkcd−/− mice with C. albicans. Compared to the wild-type, PKCδ-deficient mice exhibited significantly greater weight loss upon infection (Figure 7C) and had much lower survival rates (Figure 7D). In an independent set of experiments, we sacrificed the animals 8 days after infection and assessed intravital fungal growth. Consistent with an essential role for PKCδ in innate resistance in vivo, we observed massive fungal infiltration in the kidneys of Prkcd−/− mice by histopathology (Figure 7E) and detected significantly higher titers of C. albicans in the kidneys, livers, small intestines, and spleens (Figure 7F).

18Our results defined an essential role for PKCδ in the activation of CLR-mediated, Card9-dependent innate immune responses. Our findings are in line with the established functions of CLRs and Card9 in antifungal defense, and we showed an essential activity of PKCδ in host resistance in vivo.

19The detection of fungal particles by Dectin-1 and the subsequent activation of Syk triggers various intracellular signaling pathways. We observed that the recognition of zymosan elicits a Src- and Syk-dependent phosphorylation of PKCδ at Tyr311, indicating that PKCδ activation occurs downstream of Syk. Moreover, Prkcd−/− BMDCs showed impaired Card9-Bcl10 complex assembly and NF-κB control in spite of normal Syk activation, demonstrating that PKCδ acts upstream of Card9. Together, these findings indicate that PKCδ operates as a missing link between Syk signaling and Card9 complex formation for the activation of innate immunity. As shown by the fact that only Prkcd−/− BMDCs but not cells lacking PKCα, PKCβ, or PKCθ were defective in Dectin-1-induced cytokine production, PKCδ is the specific PKC isoform for signaling in the Dectin-1 pathway.

20Treatment of the cells with a small molecule PKC kinase inhibitor blocked zymosan- or curdlan-induced cytokine production. This indicates that the enzymatic serine-threonine kinase activity and not merely a scaffold function of PKCδ is responsible for its activity in Dectin-1 signaling. In vitro kinase assays revealed that Card9 is a direct PKCδ substrate and that PKCδ phosphorylates Card9 at position Thr231. PKCδ is likely to phosphorylate other Card9 residues because the Card9(T231A) mutant was still substantially phosphorylated by PKCδ in vitro. Another PKCδ target site could be Thr95 because global proteomic approaches have demonstrated Card9 phosphorylation at Thr95 in vivo. Because we were unable to express the Card9(T95A) mutant in mammalian cells, we were not able to determine the physiological function of Thr95 phosphorylation but speculate that it might be important for protein stability. Nevertheless, our genetic experiments with Card9-deficient cells that were reconstituted with the phosphorylation-defective Card9(T231A) mutant revealed that the Thr231 residue phosphorylated by PKCδ is absolutely required for downstream signaling and cytokine production. In addition, we observed that PKCδ signaling is essential for Card9-Bcl10 complex assembly and for Card9-dependent TAK1 activation. Thus, we postulate a molecular model in which Syk-induced PKCδ activity mediates direct Card9 phosphorylation, finally resulting in Card9-Bcl10 complex assembly for TAK1 activation. TAK1 then most probably mediates Dectin-1-induced IKK activation in a fashion similar to its mode of triggering NF-κB activity in response to stimuli from other immune receptors, such as TLRs. This would be consistent with the fact that pharmacological TAK1 blockage inhibited zymosan-induced IKK activation.

21Intriguingly, although the Card9 signaling pathway was severely defective in Prkcd−/− BMDCs, the phagocytosis of zymosan and the production of ROS were largely intact and the activation of Erk MAPK signaling was only slightly reduced. These findings indicate that PKCδ controls only specific subsets of the Dectin-1 responses. Because Dectin-1 ligation can activate the serine-threonine kinase Raf-1 through alternative mechanisms, it is possible that Raf-1 might be responsible for Dectin-1-triggered and PKCδ-independent Erk activation, consistent with the role of Raf-1 in activating MAPK pathways in numerous settings. Interestingly, Prkcd−/− mice were similar to Card9−/− mice in being highly susceptible to fungal infections. The specific PKCδ-Card9 effector response downstream of CLRs is therefore absolutely critical for host defense. The aforementioned pivotal functions of Dectin-1 and Card9 in human antifungal immunity were recently demonstrated in genetic studies. In the future it will thus be important to investigate whether genetic defects in PKCδ might also cause human immunodeficiency syndromes.

22Many of our experiments aimed at identifying the principle mechanisms of CLR signaling utilized zymosan stimulation or selective Dectin-1 triggering. Yet in the absence of PKCδ, activation of the NF-κB pathway as well as cytokine production were also severely impaired in response to whole C. albicans cells. Recent studies have indicated that C. albicans cells, and particularly C. albicans hyphae, are potent activators of Dectin-2 signaling. Presumably the hyphae in addition also activate Mincle. Thus, we propose that PKCδ also couples signals from those CLRs to the Card9-controlled NF-κB pathway. This hypothesis is in line with our observation that Prkcd−/− BMDCs were defective in cytokine responses to selective agonists for Dectin-2 and Mincle, which formally establishes PKCδ as a general integrator of CLR function.

23We believe that our findings have implications beyond antifungal immunity. Mincle and Dectin-1 detect ligands on mycobacteria and Card9−/− mice are impaired in the Mincle-induced inflammatory response to the mycobacterial cord factor. These animals also succumb rapidly to aerosol lung infection with Mycobacterium tuberculosis. In addition, Schistosoma mansoni activates Dectin-2 and Card9 and viruses such as the Dengue virus induce inflammatory responses through the ITAM-coupled CLR Clec5a. Moreover, Dectin-1 also binds to unknown endogenous structures on T cells, the Syk-coupled CLR Clec9a recognizes ligands that are exposed upon cellular necrosis, and Mincle triggers Card9-dependent inflammatory responses upon binding to SAP130 from necrotic cells under sterile conditions. Together with our results, these data imply that PKCδ might also mediate innate responses to bacteria, parasites, or viruses or could be involved in immune responses to conditions of noninfectious cell injury. These hypotheses need to be investigated.

24Finally, CLR-triggered Card9 signaling not only regulates the immediate innate antimicrobial responses but also couples innate recognition to the activation of adaptive immunity. Triggering of the Card9 pathway by Dectin-1, Dectin-2, or Mincle ligands on antigen-presenting cells controls the synthesis of distinct cytokines. This cytokine milieu instructs the development of antigen-specific Th17 cell responses and additionally induces Th1 cell immunity. Th17 cell responses are often associated with autoimmunity and polymorphisms in Card9 are recurrently detected in human inflammatory diseases. It will therefore be important to test whether PKCδ signaling in innate cells couples to Th17 cell responses and whether aberrant activity in CLR-induced PKCδ-Card9 signaling contributes to human inflammatory disease. In this context, PKCδ is a drugable target and PKC inhibitors are in clinical trials. Thus, our findings raise the possibility of therapeutically manipulating CLR-mediated Card9 signaling by targeting PKCδ function.

25Prkca−/−, Prkcb−/−, Prkcq−/−, Prkcd−/−, and Card9−/− mice have been described. Mice were infected with 1 × 104–5 × 104 colony-forming units (c.f.u.) of C. albicans (strain SC5314) as described and monitored daily for health and survival, according to institutional guidelines. For fungal burden determination, the organ homogenates were plated in dilutions on Sabouraud agar. For histological analyses, kidney sections were stained with hematoxylin and eosin or periodic acid-Schiff according to standard protocols.

26BMDCs were derived from bone marrow as described and stimulated with zymosan (Sigma), curdlan (Wako), LPS, CpG, Pam3CSK4, poly(I:C) (all from Invivogen), Dectin-2 monoclonal antibody (AbD Serotec), TDB (Avanti Polar Lipids), or C. albicans strain SC5314, as indicated. Where indicated, cells were pretreated for 20 min with the Syk inhibitor R406 (Rigel), the Src kinase inhibitor PP2 or its inactive analog PP3 (both Calbiochem), the PKC inhibitor panPKC LMWI (ALTANA Pharma, Konstanz, Germany), or the TAK1 inhibitor (5Z)-7-Oxozeaenol (Calbiochem Merck).

272 × 108 BMDCs were stimulated for 10 min with zymosan (300 μg ml−1) and lysed in RIPA buffer. The lysates were then subjected to immunopurification with 20 μg of either an antibody against phospho-tyrosine (P-Tyr-100) or mouse IgG1 as an isotype control coupled to Protein A Sepharose. Immunopurified proteins were eluted with Phenylphosphate (100 mM) in PBS, concentrated by ultrafiltration, and separated by 1-D PAGE. Whole gel lanes were cut into 23 slices. After protein digestion, MS-MS spectra of individual gel slices were obtained with a liquid chromatography coupled LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Electron). Peak lists were searched against NCBInr database with Mascot v.2.2.06 as a search engine and MudPIT for protein scoring.

28Recombinant PKCδ was purchased from Proqinase (Heidelberg) and in vitro kinase assays were performed according to manufacturers' instructions. Recombinant Card9 as a substrate was purified from E. coli by standard protocols. Alternatively, the indicated Card9 mutants containing a Strep-tag were transiently transfected into HEK293 cells and were purified with Streptactin-sepharose (IBA BioTAGnology). In brief, 70 ng PKCδ were incubated with 2 μg BSA, 2 μg Card9 (E. coli), or 1 μg HEK293 expressed Card9-versions in 70 mM HEPES-NaOH (pH 7.5); 3 mM MgCl2; 3 mM MnCl2; 3 μM Na3VO4; 1.2 mM DTT; 50 μg ml−1 PEG20.000; 100 nM ATP; 5 μCi γ-[32P]ATP; and 1% (v/v) DMSO in 20 μl for the indicated times. Samples were boiled in Laemmli buffer, separated by SDS-PAGE, and analyzed by autoradiography. For quantification, densitometric phospho-band signals were normalized to Coomassie Brilliant Blue stain intensities.

29Retroviral particles were generated by transfection of Phoenix-E cells with pMIG retroviral vectors expressing the indicated mutants of human Card9, as previously described. Bone marrow cells of Card9−/− mice were retrovirally transduced on day 1 and 2 of BMDC differentiation. Successful transduction was verified by FACS analysis of GFP expression from the bicistronic pMIG retroviral vectors and protein immunoblot analysis of Card9 expression, as previously described.

30Concentrations of TNF, IL-10, IL-2, and IL-1β secreted into cell culture supernatants of untreated and stimulated cells were analyzed with plate-bound ELISA-kits (ELISA Ready-SET-Go!, eBioscience or BD OptEIA, BD Biosciences PharMingen) according to manufacturers' recommendations.

31BMDCs were left untreated or incubated for 30, 120, or 180 min with FITC-labeled zymosan particles (100 μg ml−1, Molecular Probes). After washing, cells were analyzed either by microscopy for FITC fluorescence or by flow cytometry for CD11c expression and FITC fluorescence in the CD11c-positive population with a FACSCanto II (BD Biosciences) and FlowJo Software (Tree Star, Inc.) according to standard protocols. Fluorescently labeled antibodies were from eBioscience.

322 × 105 BMDCs were plated in 100 μl culture medium without GM-CSF into a 96-well luminometer plate (Nunc). For stimulation, 100 μl HBSS containing 100 μM Luminol and zymosan was added to each well. The luminensence was measured for 1 hr in 10 min intervals at a Mithras LB940 according to manufacturers' instructions. As a measure for ROS generation, relative light units (RLU) were integrated over time.

33Protein immunoblots were performed as described with primary antibodies against Syk, phospho-Syk (Tyr525/526), PLCγ2, phospho-PLCγ2 (Tyr759), phospho-Tyrosine (P-Tyr-100), PKCδ, phospho-PKCδ (Tyr311), phospho-IKKα/β (Ser176/180), p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), IκBα, phospho-IκBα (Ser32/36), phospho-TxR, TAK1, phospho-TAK1 (Thr184/187), Bcl10 (all Cell Signaling Technology), IKKβ, IKKα (Upstate Millipore), Card9 (Santa Cruz Biotechnology), or β-actin (Sigma). For coimmunoprecipitation experiments, BMDCs were stimulated with zymosan as indicated and lysed in NP-40-containing buffer. Lysates were cleared by incubation with Protein G Sepharose (GE Healthcare 4 Fast Flow) and then incubated with a Bcl10 antibody. Immune complexes were precipitated with Protein G Sepharose and subjected to protein immunoblotting as indicated.