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Keywords:

  • Blnk;
  • Interferon;
  • NF-kB;
  • PDC

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

Plasmacytoid dendritic cells (PDC) are the main type I interferon (IFN-I) producers and play a central role in innate and adaptive immunity. CD303 (BDCA-2) is a type II c-type lectin specifically expressed by human PDC. CD303 signaling induces tyrosine phosphorylation and Src kinase dependent calcium influx. Cross-linking CD303 results in the inhibition of IFN-I production in stimulated PDC. Here, we demonstrate that PDC express a signalosome similar to the BCR signalosome, consisting of Lyn, Syk, Btk, Slp65 (Blnk) and PLCγ2. CD303 associates with the signaling adapter FcR γ-chain. Triggering CD303 leads to tyrosine phosphorylation of Syk, Slp65, PLCγ2 and cytoskeletal proteins. Analogous to BCR signaling, CD303 signaling is likely linked with its internalization by clathrin-mediated endocytosis. Furthermore, CD303 signaling leads to reduced levels of transcripts for IFN-I genes and IFN-I-responsive genes, indicating that the inhibition of IFN-I production by stimulated PDC is at least partially regulated at the transcriptional level. These results support a possible therapeutic value of an anti-CD303 mAb strategy, since the production of IFN-I by PDC is considered to be a major pathophysiological factor in systemic lupus erythematosus patients.

See accompanying commentary at http://dx.doi.org/10.1002/eji200737944

Abbreviations:
Btk:

Bruton tyrosine kinase

CHC:

clathrin heavy chain

CME:

clathrin-mediated endocytosis

DCIR:

human dendritic cell immunoreceptor

dectin-2:

dendritic cell-associated c-type lectin 2

PDC:

plasmacytoid dendritic cell

PMF:

peptide mass fingerprint

SLE:

systemic lupus erythematosus

Slp65:

Src homology 2 domain-containing leukocyte protein of 65 kDa

Syk:

spleen tyrosine kinase

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

Plasmacytoid dendritic cells (PDC) constitute a subset of immature dendritic cells that are identified in humans by the expression of antigens such as CD4, CD45RA, ILT-3, CD123 and HLA-DR. They lack expression of markers for other hematopoietic lineages (CD3, CD14, CD16, CD19, CD20, CD56) as well as antigens associated with myeloid dendritic cells, for example CD11c or ILT-1 13.

PDC play a central role in immunity 4. Although a rare population of circulating blood dendritic cells with a median frequency of 0.4% of peripheral blood mononuclear cells 5, PDC are major producers of type I interferon (IFN-I) enabling a fast and reliable reaction of the innate immune system upon viral and microbial infection 6, 7. Beside this role as natural IFN-producing cells, they can also act as antigen-presenting cells able to induce adaptive immune responses 8, 9.

CD303, also known as BDCA-2, is a type II c-type lectin that is specifically expressed by human PDC 5. CD303 has a putative mannose specificity due to an EPN motif in its C-terminal, extracellular domain; its natural ligand, however, remains unknown. Ligand binding to CD303 can be mimicked by mAb ligation leading to rapid internalization of the lectin-antibody complex 9. CD303 is targeted to the late endosome (unpublished data), which is consistent with the expression of an acidic triad (EEE) within its cytoplasmic tail 10. Thereby, it is possible to target antigen to the MHC class II compartment for presentation to antigen-specific CD4 T cells, as we have shown previously 11.

Intriguingly, CD303-mediated signaling leads to inhibition of the IFN-I production of PDC, irrespective of the origin of the IFN-I-inducing stimulus 11. IFN-I production can be induced in PDC by pathogen-associated molecular patterns such as single-stranded RNA or unmethylated CpG motif-containing DNA (CpG), in line with their Toll-like-receptor (TLR)7 and TLR9 expression 7, 12, influenza virus, complexes consisting of DNA and anti-double-stranded DNA antibodies, a hallmark of systemic lupus erythematosus (SLE) 13, or SLE serum. Although lacking an intrinsic signaling motif, the CD303 signaling capacity comprises Src family kinase-dependent calcium influx and protein tyrosine phosphorylation, suggesting recruitment of a signaling adapter molecule such as FcR γ-chain or DAP12 11, 14.

PDC are presumably involved in the pathophysiology of several immunological diseases, including granulomatous lymphadenitis 15, Kikuchi's lymphadenitis 16, dermal lesions and non-inflammatory skin of SLE patients 17, 18, and skin lesions from psoriasis vulgaris patients 19. Many SLE patients exhibit increased IFN-I serum concentrations 20, decreased frequencies of circulating PDC in accordance with large numbers of activated PDC infiltrating the skin lesions 18, 21, coinciding with PDC-triggered B cell differentiation into plasma cells 22.

Since IFN-I production is central to PDC biology, this study was performed to elucidate CD303 signaling properties that can lead to the inhibition of IFN-I production in PDC. We demonstrate that CD303 signaling is mediated by a BCR-like signalosome, comprising Lyn, spleen tyrosine kinase (Syk), Bruton tyrosine kinase (Btk), Src homology 2 domain-containing leukocyte protein of 65 kDa (Slp65) and phospholipase C-gamma (PLCγ) 2. By association with the FcR γ-chain, CD303-initiated protein tyrosine kinase activity is presumably linked with the internalization of the c-type lectin, thereby targeting it into the late endosomal compartment by clathrin-mediated endocytosis (CME). The CD303 regulatory capacity on IFN-I production is likely dependent on the early signaling and CD303 alteration of gene expression mainly confined to IFN-I genes and IFN-I-responsive genes.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

CD303 triggering leads to regulation of IFN-I subtypes and responsive genes

To elucidate downstream effects of CD303 signaling in PDC, microarray analysis was performed using the Affymetrix HG-U133A platform and peripheral blood PDC pooled from ten donors. Four experimental conditions were compared: PDC stimulated with CpG oligonucleotides and treated with anti-CD303 antibody or an isotype control antibody, and unstimulated PDC treated with anti-CD303 antibody or an isotype control antibody. Cells were harvested after 15 h of culture. The U133A array comprises 22 283 probe sets. Considering all four samples, approximately 50% of these probe sets had received a present call by the MAS 5.0 algorithm. Analysis was accomplished using high-performance chip data analysis (unpublished data) with a relational database, filtering all genes with significantly differential regulation. It was confined to genes that exhibited at least a twofold change in expression. Expression of 721 genes was affected when comparing non-stimulated PDC and CpG-stimulated PDC, 97 genes were regulated when comparing non-stimulated and anti-CD303 antibody-treated PDC, and 179 genes were regulated when comparing CpG-stimulated and CpG-stimulated + anti-CD303 antibody-treated PDC (Supporting Information Table 1–3).

Treatment of PDC with CpG in the presence of isotype control mAb resulted in the induction of all IFN-I subtypes, including IFN-ω and IFN-β. Triggering CD303 in CpG-stimulated PDC inhibited the induction of all of these transcripts (Fig. 1A). Similarly, the expression of IFN-I-responsive genes, including 2′,5′-oligoadenylate synthetase and synthetase-like proteins, MX2 and IFIT1-3, was induced by CpG stimulation 23. The same inducing effect by CpG was observed for a cluster of chemokines including CCL3, CCL4, CCL5, CXCL10 and CXCL11. Triggering CD303 in non-stimulated PDC led to down-regulation of these transcripts. In CpG-stimulated PDC, CD303 triggering either inhibited the induction of these genes or even decreased their expression to a level below the one of non-stimulated cells (Fig. 1B). Thus, CD303 triggering resulted for IFN-I-responsive and inducible transcripts in a converse gene regulation compared to the CpG stimulus 24. The gene regulation data were verified by quantitative RT-PCR for CCL5, CXCL11 and CLEC4A (also named human DC immunoreceptor, DCIR) with consistent results (Supporting Information Table 4), and the regulation of CCL5 and CXCL11 was also confirmed on the protein level by ELISA. The chemokine concentrations detected in supernatants of cultured PDC showed the expected inhibitory effect of CD303 treatment on chemokine secretion of CpG-stimulated cells (Fig. 1C). The expression pattern of PDC triggered with anti-CD303 antibody remains remarkably similar to unchallenged cells beside the lack of IFN-I.

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Figure 1. Alteration of gene expression in PDC upon CD303 triggering. The effect of CD303 triggering in CpG-stimulated and non-stimulated PDC was assessed by Affymetrix HG-U133A microarray analysis and ELISA after 15 h of culture. (A) Expression profile of 15 IFN-I genes in PDC after CpG stimulation with (grey bars) or without (black bars) CD303 triggering. The y axis is in arbitrary units for relative fluorescence intensity (RFI). (B) Comparison of regulated genes with a minimum of a twofold change upon either CpG stimulation or CD303 triggering showed that the gene regulation prominently influenced IFN-I-responsive genes and a group of chemokines. Shown is the regulation by CpG stimulus (black), by triggering CD303 in non-stimulated (white) and CpG-stimulated (grey) PDC. Gene regulation depicted as log 2 ratios. (C) Protein levels for CCL5, CXCL11 and IFN-α in supernatants of cultured PDC were quantified by ELISA. Data are presented as means ± SD of triplicate quantifications. Representative protein levels for PDC treated with isotype, CD303, CpG and CpG + CD303 are shown for one donor of five tested.

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CD303-induced tyrosine phosphorylation signature

To elucidate the signaling capacity of CD303, we focused on our previous finding that CD303 triggering induces an overall protein tyrosine phosphorylation signature compared to untriggered PDC within 5 min 11. For this, PDC were treated with isotype control or anti-CD303 antibody for 5 min. PDC lysates were then separated by SDS-PAGE, in some cases with prior isoelectric focusing (2D), and phospho-tyrosine-specific immunoblots were generated to serve as blueprints for protein extraction from Coomassie-stained duplicate gels. Peptide mass fingerprint (PMF) analysis results of significant (p <0.05) tyrosine phosphorylation targets are shown in Table 1. Proteins involved in cytoskeletal rearrangements such as cytoplasmic (β-/γ-) actin, alpha actinin, tubulin and profilin were primarily found, but also proteasome activator subunit 1 and clathrin heavy chain (CHC) were identified.

Table 1. Tyrosine phosphorylation targets upon CD303 triggering according to PMF analysis a)
Tyrosine phosphorylation target PMF resultProbability-based Mowse score
  1. a) To characterize early CD303-induced tyrosine-phosphorylation events, PDC treated for 5 min with anti-CD303 and isotype control antibody were compared. PDC lysates were separated by SDS-PAGE, in some cases with prior isoelectric focusing for discrete protein preparation and gels treated in parallel subjected to either phospho-tyrosine-specific PY20 immunoblot or Coomassie staining. The PY20 immunoblots served as the blueprints to extract proteins for subsequent PMF analysis. Shown are the probability-based Mowse scores (p <0.05 if score >64).

α-actinin 190
β-actin178
γ-actin221
α-tubulin69
β-tubulin72
profilin138
proteasome activator subunit 1119
clathrin heavy chain 1118

CD303 signaling leads to CHC tyrosine phosphorylation

Signaling of immunoreceptors like B cell receptors (BCR) and T cell receptors (TCR) is linked to CHC phosphorylation, which plays a role in the regulation of CME 25, 26. To elucidate the function of CHC in CD303 internalization, CHC from PDC lysates treated for 5 or 10 min with anti-CD303 antibody was immunoprecipitated, and levels of phosphorylation were measured by immunoblotting with a phospho-tyrosine-specific antibody (Fig. 2A). In untriggered PDC, no CHC phosphorylation was observed whereas 5 min after CD303 triggering CHC phosphorylation was detected that further increased after 10 min. TCR treatment with CD3 MacsiBeads on Jurkat cells used as positive control showed a similar CHC tyrosine phosphorylation.

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Figure 2. Early CD303 signaling is required for IFN-I inhibition. (A) Tyrosine phosphorylation of CHC upon immunoreceptor ligation was confirmed by immunoprecipitation of CHC from Jurkat T cells treated with CD3 MacsiBeads and PDC treated with anti-CD303 antibody. Cell lysates were subjected to anti-CHC (clone X22) immunoprecipitation. Shown are anti-CHC antibody (clone TD.1) and phospho-tyrosine (PY20)-specific immunoblots of CHC immunoprecipitates (one representative shown of three experiments performed). (B) Transcript levels (depicted as relative fluorescence intensity (RFI)) of immunoreceptor signalosome components of unstimulated, isotype control-treated PDC are shown. None of the listed genes was regulated by a minimum of twofold change by CD303 triggering. For comparison, CD19 was listed which had received an absent call by the MAS 5.0 algorithm. (C) Comparison of the CD303 (CLEC4C) amino acid sequence with those of related lectins. Putative transmembrane (TM) regions of CD303, CLEC4D (CLECSF8) and CLEC4A (DCIR) are underlined. (D) Anti-HA immunoblot of CD303 immunoprecipitates. Lysates derived from HEK 293T FcR γ-chain CD303, CD303-CLEC4D-TM and CD303-CLEC4A-TM transfectants, respectively, were analyzed. TM sequences of the CD303 chimeras correspond to amino acid sequences as shown in (C). Aliquots of the lysates before immunoprecipitation were used as controls. (E) Phospho-tyrosine (PY20)-specific immunoblots of HEK CD303 FcR γ-chain lysates separated by 2-D gel electrophoresis are shown, comparing the effect after 5 min of isotype and anti-CD303 mAb treatment on the induction of tyrosine phosphorylation. PMF analysis identified the corresponding protein to the tyrosine phosphorylation event marked as “1” as proteasome activator subunit 1.

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CD303 associates with the FcR γ-chain

The short cytoplasmic tail of CD303 lacks any known signaling motif. Tyrosine phosphorylation of CHC occurs after cross-linking of the TCR and BCR as part of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling 25. We therefore analyzed the gene expression profiles of PDC for the expression of ITAM-containing signaling adapters and identified DAP12 and the FcR γ-chain (Fig. 2B). Dendritic cell-associated c-type lectin 2 (dectin-2), the closest homologue of CD303, associates with the FcR γ-chain 27. Therefore, we focused on a potential interaction of CD303 with the FcR γ-chain. The co-immunoprecipitation of CD303 with the FcR γ-chain from primary PDC lysates proved ineffective, presumably because the endogenous protein levels of CD303 and the FcR γ-chain were too low to allow detection of the co-immunoprecipitated FcR γ-chain. Therefore, the possible interaction of CD303 with the FcR γ-chain was studied in transfectants. For this, HEK 293T cells expressing an HA-tagged FcR γ-chain were transfected with CD303 wild type or CD303 chimeras with either the transmembrane domain of CLEC4D or CLEC4A (Fig. 2C) as negative control, thereby altering the CD303 wild-type transmembrane amino acid sequence by 6 and 16 amino acids, respectively.

Performing anti-HA immunoblot analysis of CD303 immunoprecipitates (Fig. 2D) from CD303 transmembrane chimeras showed co-immunoprecipitation of the FcR γ-chain only in the case of CD303 wild-type transmembrane conformation. Furthermore, triggering CD303 in HEK 293T CD303 HA-tagged FcR γ-chain transfectants resulted in the induction of tyrosine phosphorylation, exemplarily shown by PY20 immunoblots of isoelectrically focused lysates (Fig. 2E). We thereby identified proteasome activator subunit 1 to be tyrosine phosphorylated upon triggering CD303 in HEK 293T transfectants as well as PDC (Table 1).

CD303 utilizes a BCR-like signaling network

The presence of PLCγ2, Btk, Lyn, Syk and Slp65 in the absence of PLCγ1, Itk, Fyn, ZAP70 and Slp76 in the PDC expression profile resembles the BCR signalosome (Fig. 2B). The expression of Syk, Slp65 and PLCγ2 by PDC was confirmed by immunoblotting (Fig. 3A). Using phospho-specific antibodies, we identified Syk to be subjected to phosphorylation upon CD303 triggering at tyrosine residues 525 and 352 (Fig. 3A). CD303 triggering resulted also in the phosphorylation of tyrosine 96 of Slp65 and an increase of the phosphorylation levels of tyrosine 759 of PLCγ2 (Fig. 3A).

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Figure 3. CD303 signaling resembles BCR signaling and interferes with the induction of the canonical NF-κB pathway in PDC. (A) Immunoblot analysis of PDC lysates with or without CD303 stimulation using control anti-Syk, anti-phospho-Syk-Y352, anti-phospho-Syk-Y525/526 (upper panel) and anti-Slp65 and anti-phospho-Slp65-Y96 (middle panel) antibodies. Results are representative of five donors tested. Lysates from PDC enriched from leukapheresis products of two donors were subjected to PLCγ2 and phospho-PLCγ2-Y759 immunoblot analysis (lower panel). (B) The effect of PLCγ activity on the IFN-I production in stimulated PDC was assessed by treating PDC with ionomycin and PMA. IFN-α production in PDC was quantified after 15 h by ELISA. Viable cell numbers at that time were determined by FACS (diamonds). Results are representative of five experiments performed. Data are presented as means ± SD of triplicate quantifications. (C) PDC express conventional PKCα/β and novel PKCδ as shown by immunoblot of PDC lysates using pan-PKC-, PKCα/β-, PKCθ- and PKCδ-specific antibodies. (D) Immunoblot analysis of IκBα (loading control) and phospho-IκBα levels in PDC were performed. Time-dependent induction of phosphorylation of IκBα Ser32 was assessed for PDC treated either with anti-CD303 antibody (left), CpG (middle) or TNF-α. Lysates derived from PDC after 5, 10, 20 and 30 min exposure to anti-CD303 mAb, isotype control and TNF-α (left panel). Increase of phospho-IκBα-Ser32 levels after CpG treatment was detectable within 16 min (middle). The effect of CpG treatment compared to TNF-α is shown in the middle panel. TNF-α- and CpG-induced IκBα phosphorylation is inhibited upon pretreatment for 30 min with anti-CD303 antibody (right). Representative immunoblots of a minimum of three donors tested are shown.

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PLCγ activity has been reported to be involved in the formation of signalosomes in TCR and BCR, resulting in Vav/Nck-dependent cytoskeleton rearrangements upon receptor triggering 28. PLCγ contains two SH2 domains capable of binding phosphorylated tyrosine motifs and, once activated, initiates secondary messenger release. Diacylglycerol (DAG) and inositol 1,4,5-trisphosphate are released, leading to protein kinase C (PKC) activity and calcium flux 29. Therefore, the effect of PLCγ activity on the IFN-I production in CpG-stimulated PDC was assessed by treatment with PMA and ionomycin, because these substances mimic DAG and calcium release, respectively 30. While ionomycin did not affect the production of IFN-I (unpublished data), treatment of PDC with PMA led to a dose-dependent reduction of IFN-I secretion. The application of a dose higher than 50 pg/mL PMA led to the inhibition of IFN-I, mimicking CD303 treatment, and this inhibitory effect was independent of calcium mobilization. DMSO, in which ionomycin had been dissolved, had no effect on cell viability or IFN-I production, while the combined treatment of PDC with ionomycin and higher concentrations of PMA resulted in reduced cell viabilities (Fig. 3B). On the transcriptional level, PDC express PKCB1, PKCD and PKCI (Fig. 2B). By subjecting PDC lysates to immunoblot analysis for PKC subtypes, the presence of conventional PKCα/β and novel PKC was confirmed (Fig. 3C).

CD303 signaling decreases IκBα S32 phosphorylation levels

Activation of PKC in B and T cells can lead to the activation of the inhibitor of kappa B (IκB) kinase complex and subsequently the NF-κB pathway 31, 32. Indicative of the activation of the canonical NF-κB pathway are phosphorylation levels of serine 32 of IκBα. Pro-inflammatory stimuli such as tumor necrosis factor (TNF)-α and CpG lead to the activation of the canonical NF-κB pathway in PDC 33. To elucidate whether CD303 signaling interferes with the signaling pathway responsible for the induction of IFN-I production in PDC, the effect of triggering CD303 on the NF-κB pathway was assessed by immunoblot analysis (Fig. 3D). While TNF-α and CpG induced the phosphorylation of IκBα in PDC, CD303 signaling did not change the phosphorylation status of IκBα S32. Interestingly, pretreamtent of PDC with anti-CD303 antibody for 30 min before stimulation with TNF-α or CpG decreased the phosphorylation level of IκBα at S32 compared to isotype control-treated PDC.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

PDC are the major IFN-I producers and play a central role in innate and adaptive immunity. CD303 is a type II c-type lectin exclusively expressed by human PDC. Triggering CD303 by mAb ligation inhibits the IFN-I production of stimulated PDC. CD303 signaling leads to a Src family kinase-dependent calcium mobilization and induces protein tyrosine phosphorylation 5, 9, 11. The focus of this study was the characterization of the CD303 signaling mechanism in human PDC associated with the inhibition of IFN-I production.

Here, we show that the signaling after CD303 cross-linking in PDC in many aspects resembles the signaling of the BCR after cross-linking of membrane IgM in B cells 34. CD303 associates with an ITAM-containing signaling adapter, and signals via Syk, Slp65 and PLCγ2 within a BCR-like signalosome. Similar to the cytoskeletal rearrangement and the CME induced after BCR cross-linking, CD303 triggering leads to tyrosine phosphorylation of cytoskeletal proteins and CHC. However, in contrast to BCR signaling, CD303 signaling appears to extenuate the activation of the NF-κB pathway in PDC, and CD303 triggering-induced gene regulation appears to be confined to IFN-I transcripts and IFN-I-responsive genes.

PDC express the ITAM-containing signaling adapters FcR γ-chain and DAP12. We show that CD303 associates with the FcR γ-chain. Although this interaction was shown after overexpression of both molecules in HEK transfectants, the fact that the controls with an exchange of the transmembrane region of CD303 did not result in immunoprecipitation of the FcR γ-chain supports the specificity of the interaction. After CD303 cross-linking, the proteasome activator subunit 1 is tyrosine phosphorylated both in PDC and in HEK 293T cells expressing the CD303 FcR γ-chain. Similar to CD303, cross-linking of immunoglobulin-like transcript 7 (ILT7), which also associates with the FcR γ-chain, results in the inhibition of IFN-I production of stimulated PDC 35. Furthermore, considering that the expression of a receptor is stabilized by the expression of its signaling adapter 36, the sustained surface expression of CD303 in DAP12-deficient PDC argues against a possible interaction with DAP12 37. For the initiation of signal transduction after BCR cross-linking, the ITAM-containing signaling adapters CD79a and CD79b are crucial 38. The interaction of CD303 with the FcR γ-chain might be of similar importance for the initiation of CD303 signaling.

PDC express Lyn, Syk, Slp65, Btk and PLCγ2, indicative of a BCR-like signalosome, whereas we observe a lack of expression of central components of TCR signaling (Itk, Lck, Fyn, ZAP70, Slp76 and PLCγ1). After BCR cross-linking, the initial activation of Lyn and Syk, key BCR signaling molecules, is connected to downstream effectors by recruitment of Slp65, also known as Blnk (B cell linker). SLP65 is recruited to serve as docking adapter for Btk, PLCγ2 and Vav 39, 40. We show here the tyrosine phosphorylation of Syk, Slp65 and PLCγ2 in PDC after cross-linking of CD303. Syk tyrosine phosphorylation of Tyr352 has been correlated with its association with PLCγ 41 and phosphorylation of Tyr525/526 is essential for its function 42. The phosphorylation of tyrosine 96 of Slp65 is indicative of Btk association 43. The phosphorylation of Tyr759 of PLCγ2 and its activity are Btk dependent 44. For BCR signaling, PLCγ2 activity is crucial for the generation of the second messengers inositol 1,4,5-trisphosphate and DAG leading to calcium flux and PKC activity 45. Therefore, PLCγ2 activation might explain the reported calcium mobilization after CD303 cross-linking 11. The mimicry of CD303-mediated inhibition of IFN-I production in PDC by phorbolester treatment indicates that the involvement of downstream events of PLCγ2 activity in CD303 signal transduction needs further elucidation with focus on the protein kinase C family members as promising targets.

CD303 is likely internalized via CME. CD303 triggering results in the induction of tyrosine phosphorylation of CHC in PDC with an increase of signal intensities from 5 to 10 min, again similar to downstream events after BCR cross-linking. Membrane IgM cross-linking results in the tyrosine phosphorylation of CHC 26. By detection of CHC tyrosine phosphorylation, the initiation of CME in the case of antigen receptor can be monitored 25. Indeed, upon ligation, the BCR can be found in clathrin-coated pits and vesicles, indicative of a clathrin-mediated internalization process 46.

CD303 trafficking is likely dependent on functional actin and tubulin filaments for both internalization by CME and vesicular transport to late endosomes. We show the tyrosine phosphorylation of α-actinin, cytoplasmic actin, tubulin and profilin upon CD303 ligation. Profilin, α-actinin and cytoplasmic actin are the components of a growing actin filament 47. Upon BCR cross-linking, the tyrosine phosphorylation of tubulin is Syk dependent 48 and tyrosine phosphorylation of actin is associated with the polymerization of actin filaments linked with lipid raft recruitment of the BCR 49. Profilin and cytoplasmic actin were shown to be substrates for tyrosine phosphorylation of constitutive active tyrosine kinases in tumor cell lines undergoing cytoskeletal rearrangement 50. Furthermore, trafficking of the BCR to late endosomes depends on the actin cytoskeleton 51. With CD303 containing an EEE (late endosomal sorting motif) in its cytoplasmic tail, reported to facilitate trafficking of CD205 into the late endosome 10, the tyrosine phosphorylation of cytoskeleton proteins after CD303 triggering can be interpreted to be indicative for the targeting of CD303 to the late endosomal compartment.

Despite the similarity with BCR signaling mentioned so far, CD303 signaling, on the contrary, does not lead to the activation of the canonical NF-κB pathway that can be monitored by the IκBα S32 phosphorylation status 52. In fact, we find that prior CD303 triggering markedly reduces the level of phosphorylation of IκBα S32 by TNF-α. The decrease of canonical NF-κB pathway activity induced by CD303 triggering correlates with the decreased CCL5 expression as a NF-κB marker gene presented in this study 53, suggesting that the negative regulation of CD303 is linked to early signaling events. BCR cross-linking can result in the activation of NF-κB 54, and this process appears to be linked in B celIs to PKCβ by recruiting IκB kinase into lipid rafts 55. Activation of NF-κB in B cells is also linked to PLCγ2 activity 39. However, activation of the canonical NF-κB in anergic T and B cells is still a matter of debate and the activation of the NF-κB pathway by the BCR signalosome is tightly regulated, depending on the activation and developmental state of the cell 56.

Focusing on downstream events of CD303 signaling, we show here that the alteration of gene expression in PDC by CD303 mAb ligation appears mainly to be confined to the inhibition of all IFN-I genes and IFN-I-responsive genes. Triggering CD303 altered the expression more than twofold of only 97 and 179 genes of all detectable genes in non-stimulated and CpG-stimulated PDC, respectively. Most of these genes were down-regulated by CD303 triggering. Ito et al.57 showed the induction of all IFN-I genes by CpG in PDC. By triggering CD303, this induction of the IFN-I genes is inhibited. The expression of IFIT1–3, GBP1, IFI27 and IFI35, MX2, OAS1, OASL as well as the expression of the chemokine transcripts CCL3–5, CXCL10 and CXCL11 is inhibited in non-stimulated and CpG-stimulated PDC by CD303 triggering. In addition to the IFN-I genes themselves whose expression is inhibited by CD303 triggering, many of the other genes are known IFN-I-inducible genes or at least known to be expressed in the presence of IFN-I 23, 24. Therefore, we conclude that the inhibition of IFN-I production in CpG-stimulated PDC is at least partially regulated at the transcriptional level.

In summary, we propose a model (Fig. 4) whereby CD303, associated with the FcR γ-chain, signals within a BCR-like signalosome. Triggering CD303 results in the activation of Syk, the recruitment of SLP65 and PLCγ2 activity. Within PDC, the PLCγ2-PKC axis is possibly exerting the negative regulation of the NF-kB pathway and inhibition of IFN-I genes after CD303 triggering.

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Figure 4. Model of CD303 signaling. CD303 associates with the ITAM-containing signaling adapter FcR γ-chain. CD303 signals via Syk, Slp65 and PLCγ2 within a BCR-like signalosome, leading to cytoskeletal rearrangements likely mediated by Vav and Nck activity. The release of inositol 1,4,5-trisphosphate (IP3) and DAG after PLCγ2 activation likely induces calcium influx and possibly functions as negative regulator in PDC, possibly mediating the IFN-I inhibition and decreased activation of NF-κB by CD303 triggering.

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There is growing evidence that PDC as the major IFN-I producers might play a role in the pathogenesis of SLE 58. SLE is one of the most common autoimmune disorders, characterized by high levels of autoantibodies against nuclear proteins and autoantibodies against DNA 59. Patients suffering from SLE show decreased frequencies of PDC in the blood 21, likely caused by PDC infiltration of cutaneous lupus erythematosus lesions and migration to lymphoid tissue 17, 18. Elevated IFN-I serum levels have been reported in SLE patients and correlate with the disease severity and progression 60. The results of this study emphasize the possible therapeutic value of a CD303-mediated inhibition of IFN-I production and reduction of pro-inflammatory cytokine levels in stimulated PDC.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

Reagents used in this study

For immunoblot analysis and immunoprecipitation experiments, phosphorylation state-specific antibody PY20 horseradish peroxidase (HRP) conjugate (Becton Dickinson, Heidelberg, Germany), rabbit anti-human PLCγ2, anti-phospho-Y759 PLCγ2, anti-Syk, anti-phospho-Y525/526 Syk, anti-phospho-Y352 Syk, anti-phospho-Y323 Syk, anti-Blnk, anti-phospho-Y96 Blnk, anti-phospho-PKC antibody sampler kit, anti-IκB-α antibody, anti-phospho-IκBα (Ser32), anti-rabbit HRP [all from Cell Signaling Technology (New England Biolabs, Frankfurt, Germany)], mouse anti-CHC clone TD.1 (Sigma-Aldrich, Munich, Germany), mouse anti-CHC clone X22 (Calbiochem, Darmstadt, Germany), and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Newmarket, UK) were used. For IFN-I induction, CpG (ODN 2216: 5′-ggGGGACGATCGTCgggggG-3′ 9) was obtained from Metabion (Martinsried, Germany). Phorbol 12-myristate 13-acetate (PMA), ionomycin and dimethyl sulfoxide (DMSO) used for elucidating PLCγ activity on IFN-I production in stimulated PDC was purchased from Sigma-Aldrich. I-TAC and RANTES ELISA kits were obtained from R&D Systems (Wiesbaden, Germany) and IFN-α ELISA from BenderMed Systems (Vienna, Austria).

Cell preparations

Buffy coats from normal healthy volunteers were obtained from the Institute for Transfusion Medicine (Dortmund, Germany). Peripheral blood mononuclear cells (PBMC) were prepared from buffy coats by standard Ficoll-Paque (Amersham Pharmacia Biotech, Freiburg, Germany) density gradient centrifugation. PDC were isolated from PBMC by direct magnetic labeling with CD304 (BDCA-4) mAb (clone AD5-17F6)-conjugated microbeads (CD304 MicroBead kit; Miltenyi Biotec, Bergisch-Gladbach, Germany) and enrichment of labeled cells using a high-gradient magnetic cell sorting device (MiniMACS; Miltenyi Biotec). PDC used for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), IκBα phosphorylation, CHC immunoprecipitation and PKC isotyping experiments were isolated from leukapheresis products by CliniMACS CD304 (BDCA-4) MicroBeads labeling followed by enrichment of labeled cells using a clinically approved immunomagnetic selection device (CliniMACS; Miltenyi Biotec). Leukapheresis products from non-mobilized volunteers were obtained from the DRK-Blutspendezentrale Ulm (Ulm, Germany). As determined by staining with PE-conjugated anti-CD303 (BDCA-2) mAb (AC144; Miltenyi Biotec) and flow cytometric analysis using a FACSCalibur (Becton Dickinson) and CellQuest software (Becton Dickinson), PDC purities of 92–98% were routinely obtained.

In vitro assays

PDC were cultured at a cell density of 106 cells/mL in medium [RPMI 1640 supplemented with 2 mM L-glutamine, 10% FCS (PAA, Linz, Austria), 100 µM sodium pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin] at 37°C in a humidified 5% CO2-containing atmosphere in the presence of 10 ng/mL IL-3 (Biosource, Solingen, Germany) for 15 h. PDC were challenged with either isotype control anti-cytokeratin mAb (clone CK3–6H5, mIgG1; Miltenyi Biotec) or CD303 mAb (clone AC144, mIgG1; Miltenyi Biotec) at 10 µg/mL concentrations. For IFN-I production, 2 µg/mL ODN2216 (CpG) was added.

For short incubations, PDC were warmed to 37°C in RPMI 1640 (Miltenyi Biotec) without supplements for 5 min and incubated with mAb as specified.

Constructs and transfectants

Comparison of the transmembrane domains of type II c-type lectins shows that two groups can be distinguished: those with a high homology to CD303 like CLEC4D (CLECSF8), displaying a short cytoplasmic tail without intrinsic signaling motifs; or those with longer cytoplasmic regions, carrying an ITIM as signaling unit as is the case for CLEC4A. FcR γ-chain amplified from PDC cDNA (Superscript II; Invitrogen, Karlsruhe, Germany) was cloned into the Bgl II-Not I sites of pDisplay (Invitrogen) using the primers 5′-CTAGAGATCTGAGCCTCAGCTCTGCTATAT-3′ (forward), 5′-TAATGCGGCCGCATCCGTAAACAGCATCTGAGC-3′ (reverse) (Metabion) taken from Merck et al.36. Full-length CD303 derived from Dzionek et al.11 and the CD303 CLEC4A and CLEC4D transmembrane chimeras were cloned into the Bam HI-Not I sites of the pEF/Myc-HIS vector (Invitrogen). For CD303-CLEC4A and CD303-CLEC4D transmembrane chimeras, nucleotide sequences were synthesized according to GenBank accession numbers NM_016184 and NM_080387, respectively, at Entelechon (Entelechon GmbH, Regensburg, Germany). For confirmation, DNA sequencing was performed by Agowa (Berlin, Germany) for all constructs generated.

HEK 293T cells were transfected with 2 µg HA-FcR γ-chain-pDisplay using FuGene (Roche, Mannheim, Germany). Stable transfectants were obtained by labeling cells with biotin-conjugated anti-HA mAb followed by anti-biotin microbead enrichment using a high-gradient magnetic cell sorting device (MiniMACS; Miltenyi Biotec). HEK 293T FcR γ-chain transfectants were transfected with 5 µg CD303, CD303-CLEC4D-TM and CD303-CLEC4A-TM constructs (Fugene; Roche) according to the manufacturer's protocol. Selection was performed by administration of G418 and blasticidin (Invitrogen).

Microarray analysis

Unstimulated or ODN2216-stimulated PDC from ten donors treated with either anti-CD303 or isotype control mAb were cultured for 15 h. For cytokine quantification by ELISA, supernatants of PDC cultures were retained. PDC were pooled and lysed with the use of RLT lysis buffer (Qiagen, Hilden, Germany) containing β-mercaptoethanol. Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and was treated with an RNase-free DNase Set (Qiagen) to remove genomic DNA. Double-stranded cDNA was synthesized using SuperScript II. Reverse Transcriptase and the SuperScript II Kit (Invitrogen) and was converted to biotin-labeled cRNA viain vitro transcription, by use of the BioArray HighYield Transcription Labeling Kit (Enzo, Farmingdale, NY). The labeled cRNA was then fragmented and hybridized to Affymetrix HG-U133A chips. All preparation procedures and quality controls were performed in accordance with the Affymetrix standard protocol. Data evaluation was performed using the Affymetrix GeneChip software Microarray Suite 5.0 according to the manufacturer's instructions.

2-D PAGE

PDC were solubilized in 500 µL lysis buffer (8 M urea, 4% CHAPS, 2% IPG buffer pH 4–7) with the sample grinding kit (Amersham Pharmacia Biotech). Protein concentrations were quantified with the 2D Quant kit (Amersham Pharmacia Biotech). Aliquots of 100 µg lysate, equaling approximately 2 × 107 PDC, were isoelectrically focused with 8 kV h using a Multiphore II unit. The focused Immobiline strip was loaded onto the second-dimension gel, after two-step equilibration first with 45 mM dithiothreitol followed by 90 mM iodoacetamide treatment in 50 mM Tris-HCl pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, 0.002% w/v bromophenol blue (all Amersham Pharmacia Biotech). Proteins were transferred onto Hybond-P membranes for subsequent phospho-tyrosine-specific immunoblotting or visualized by Phast Gel Blue Coomassie staining for protein extraction.

Peptide mass fingerprint analysis

PMF analysis was performed at the Zentrum fuer Molekulare Medizin (Zentrale Bioanalytik, Cologne, Germany).

Immunoblotting

For analysis of Syk, Slp65, PLCγ2, PKC and IκBα, PDC were lysed in Laemmli protein sample buffer (0.06 M Tris HCl pH 6.8, 2% SDS, 10% glycerine, 0.005% bromphenol blue), sonicated, boiled, resolved on NuPage gradient gels, transferred onto Hybond-P membranes followed by BSA block and immunoblotted according to the manufacturer's protocol.

CD303 and CHC immunoprecipitation

Cells (5 × 106) were lysed in 1 mL buffer containing 2% Triton X-100, 500 mM Tris-HCl pH 7.4, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors (complete protease inhibitor cocktail tablets; Roche) on ice for 30 min. For immunoprecipitation with anti-CD303 antibody, protein G-Sepharose (Amersham Pharmacia Biotech) -precleared PDC lysates were incubated with AC144 mAb at 5 µg/mL for 1 h at 4°C and then precipitated with protein G for 1 h at 4°C. For CHC immunoprecipitation, 4 µg/mL X22 mAb was used. Precipitated proteins were resolved on NuPage gradient gels, transferred onto Hybond-P membranes and blocked in 50 mM Tris-HCl pH 7.8 containing 0.5% Tween-20 and 5% BSA overnight at 4°C. For detection of CD303 co-immunoprecipitated HA-tagged FcR γ-chain, HRP-conjugated anti-HA antibody (clone GG8–1F3, Miltenyi Biotec) was used. For detection of immunoprecipitated CHC and its tyrosine phosphorylation status, phospho-tyrosine-specific HRP-conjugated PY20 and anti-CHC (clone TD.1) antibodies, followed by incubation with HRP-conjugated goat anti-mouse IgG, were used. Blots were visualized with enhanced chemiluminescence (ECL Plus, Amersham Pharmacia Biotech).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

We thank Dr. J. C. Campbell for critical reading and discussion of the manuscript, and Dr. K. Hofmann for the extensive bioanalytical comparison of the members of the CLECS-family.

  • 1

    WILEY-VCH

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    WILEY-VCH

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    WILEY-VCH

  • 4

    WILEY-VCH

Appendix

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

Conflict of interest: J.R., E.S., J.S., and G.W. are employed by Miltenyi Biotec GmbH, Germany. The other authors declare no competing financial interests.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Appendix
  9. Supporting Information

Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2040/2007/37711_s.pdf or from the author.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.