BDCA2/Fc?RIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells

Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter FcɛRIγ. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with FcɛRIγ, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.     

BDCA2/FcɛRIγ Complex Signals through a Novel BCR-Like Pathway in Human Plasmacytoid Dendritic Cells

Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter FcRIγ. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with FcRIγ, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.     

BDCA2/Fc epsilon RI gamma complex signals through a novel BCR-like pathway in human plasmacytoid dendritic cells

Dendritic cells are equipped with lectin receptors to sense the extracellular environment and modulate cellular responses. Human plasmacytoid dendritic cells (pDCs) uniquely express blood dendritic cell antigen 2 (BDCA2) protein, a C-type lectin lacking an identifiable signaling motif. We demonstrate here that BDCA2 forms a complex with the transmembrane adapter Fc epsilon RI gamma. Through pathway analysis, we identified a comprehensive signaling machinery in human pDCs, similar to that which operates downstream of the B cell receptor (BCR), which is distinct from the system involved in T cell receptor (TCR) signaling. BDCA2 crosslinking resulted in the activation of the BCR-like cascade, which potently suppressed the ability of pDCs to produce type I interferon and other cytokines in response to Toll-like receptor ligands. Therefore, by associating with Fc epsilon RI gamma, BDCA2 activates a novel BCR-like signaling pathway to regulate the immune functions of pDCs.     

CD2AP/SHIP1 complex positively regulates plasmacytoid dendritic cell receptor signaling by inhibiting the E3 ubiquitin ligase Cbl

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.     

Dendritic cell immunoactivating receptor,a novel C-type lectin immunoreceptor,acts as an activating receptor through association with Fc receptor gamma chain

An increasing number of C-type lectin receptors are being discovered on dendritic cells, but their signaling abilities and underlying mechanisms require further definition. Among these, dendritic cell immunoreceptor (DCIR) induces negative signals through an inhibitory immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. Here we identify a novel C-type lectin receptor, dendritic cell immunoactivating receptor (DCAR), whose extracellular lectin domain is highly homologous to that of DCIR. DCAR is expressed similarly in tissues to DCIR, but its short cytoplasmic portion lacks signaling motifs like ITIM. However, a positively charged arginine residue is present in the transmembrane region of the DCAR, which may explain its association with Fc receptor gamma chain and its stable expression on the cell surface. Furthermore, cross-linking of DCAR in the presence of gamma chain activates calcium mobilization and tyrosine phosphorylation of cellular proteins. These signals are mediated by the immunoreceptor tyrosine-based activating motif (ITAM) of the gamma chain. Thus, DCAR is closely related to DCIR, but it introduces activating signals into antigen-presenting cells through its physical and functional association with ITAM-bearing gamma chain. The identification of this activating immunoreceptor provides an example of signaling via a dendritic cell-expressed C-type lectin receptor.     

The assembly of diverse immune receptors is focused on a polar membrane-embedded interaction site

The majority of receptors responsible for activation of distinct cell types within the immune system assemble with dimeric signaling modules through interaction of a basic transmembrane residue with a pair of acidic residues of the signaling dimer. Because assembly of other membrane proteins requires specific interactions along extended stretches of transmembrane helices, we examined how transmembrane sequences flanking the polar interaction site contribute to assembly for three receptors that associate with different signaling modules—the natural killer cell receptors KIR and NKG2D and the Fc receptor for IgA, FcαRI. The KIR and NKG2D receptors assembled with the DAP12 and DAP10 dimers, respectively, even when the entire KIR or NKG2D transmembrane domains were replaced by polyleucine sequences with a properly positioned basic residue. In contrast, a high degree of specificity for the basic side chain could be observed because the KIR–DAP12 and FcαRI–Fcγ interactions favored lysine or arginine, respectively. Steric hindrance among incompatible extra-membranous domains and competition for signaling modules also contributed to specificity of assembly. These results demonstrate that these interactions are focused on the polar site created by three ionizable transmembrane residues, and explain how the DAP12 and Fcγ signaling modules can assemble with large, non-overlapping sets of receptors that have highly divergent transmembrane sequences.     

Evaluation of the BDCA2-DTR Transgenic Mouse Model in Chronic and Acute Inflammation

Background and Aims

Plasmacytoid dendritic cells (pDCs) are a small subset of dendritic cells and the main producers of type I interferons. Besides their contribution to tolerance, they are known to be involved in autoimmune diseases and have recently been implicated in atherosclerosis. However, their precise involvement, particularly in advanced lesion development, remains elusive. Hence, we investigated the role of pDCs in atherogenesis vs atheroprogression by specifically depleting this cell population using the BDCA2-DTR mouse model bred to Apolipoprotein E (Apoe ^-/-) deficient mice.

Methods and Results

Our results revealed that continuous diphtheria toxin-induced pDC depletion in Apoe ^-/- BDCA2-DTR mice receiving a high-fat diet (HFD) for 4 weeks did not alter lesion size or composition. Instead, these mice displayed increased B cell numbers and altered levels of inflammatory cytokines. Analysis of depletion efficiency showed that complete pDC depletion could only be sustained for one week and reoccurring pDCs sorted after 4 weeks did not express DTR anymore. Consequently, we analyzed lesion development in a model of partial carotid ligation, inducing established lesions after 5 weeks of HFD feeding, and only depleted pDCs during the last week of 5 weeks HFD feeding. Despite short-term, but efficient pDC depletion, we observed no differences in atherosclerotic lesion development, but changes in inflammatory cytokine titers. To assure the functionality of the BDCA2-DTR model in acute settings, we additionally examined the effect of pDC depletion in an indirect acute lung injury (iALI) model. This time, efficient pDC depletion resulted in a significantly reduced macrophage and neutrophil accumulation in the lung 12 hours after LPS challenge, underlining a pro-inflammatory role of pDCs in the innate immune response in iALI.

Conclusion

Taken together, the BDCA2-DTR mouse model only allows efficient pDC depletion for one week, which subsequently restricts its usability to more acute but not chronic inflammatory disease models.     

免疫融合蛋白sFcεRIα/mIg(IgG2)的研制

哮喘、过敏性鼻炎、特应性皮炎及食物过敏等疾病为一类常见疾病,但存在治疗效果不佳,患者病程长等特点。IgE 分子是导致过敏性疾病的关键分子。抑制IgE分子与效应细胞膜表面FcεRI受体的结合可抑制过敏反应的发生。通过克隆FcεRI受体α亚基的cDNA与IgG2的稳定区铰链区、CH2和CH3的cDNA连接,以二聚化融合蛋白sFcεRIα/mIg(IgG2)的形式在CHO细胞中表达,达到提高sFcεRIα生物半衰期的目的。表达载体构建和初步的功能性试验等一系列研究证实,所表达的融合蛋白的分子量为170kDa,并与人IgE和鼠IgE有较好的结合活性。这些研究为该融合蛋白最终实现产业化打下良好基础。     

Rat Macrophage C-Type Lectin Is an Activating Receptor Expressed by Phagocytic Cells

Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.     

Crystal structures of carbohydrate recognition domain of blood dendritic cell antigen‐2 (BDCA2) reveal a common domain‐swapped dimer

We report on crystal structures of a carbohydrate recognition domain (CRD) of human C‐type lectin receptor blood dendritic cell antigen‐2 (BDCA2). Three different crystal forms were obtained at 1.8–2.3 Å resolution. In all three, the CRD has a basic C‐type lectin fold, but a long loop extends away from the core domain to form a domain‐swapped dimer. The structures of the dimers from the three different crystal forms superimpose well, indicating that domain swapping and dimer formation are energetically stable. The structure of the dimer is compared with other domain‐swapped proteins, and a possible regulation mechanism of BDCA2 is discussed. Proteins 2014; 82:1512–1518. © 2013 Wiley Periodicals, Inc.     

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains ¹. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout ^{2, 3}. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml^-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses ^{4, 5}. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease ^{6, 2, 3}.     

Regulation of TLR7/9 signaling in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), also known as type I interferon (IFN)-producing cells, are specialized immune cells characterized by their extraordinary capabilities of mounting rapid and massive type I IFN response to nucleic acids derived from virus, bacteria or dead cells. PDCs selectively express endosomal Toll-like receptor (TLR) 7 and TLR9, which sense viral RNA and DNA respectively. Following type I IFN and cytokine responses, pDCs differentiate into antigen presenting cells and acquire the ability to regulate T cell-mediated adaptive immunity. The functions of pDCs have been implicated not only in antiviral innate immunity but also in immune tolerance, inflammation and tumor microenvironments. In this review, we will focus on TLR7/9 signaling and their regulation by pDC-specific receptors.     

Specific post-transcriptional inhibition of mRNA for ligand binding chain of IgE high affinity receptor

IgE high affinity receptor (FcεRI) plays an important role in triggering type I allergic reactions. In this study, we have investigated the ability of four synthetic and sequence-specific RNA interfering antisense oligodeoxynucleotides (AS-ODNs) to reduce the expression of FcεRIα gene in granulocytes of allergy sufferers in vitro. Only AS1 out of four AS-ODNs specifically inhibited the FcεRIα gene expression and the dose response assay revealed that AS1 was capable of specific inhibition of target mRNA expression over a linear concentration range without affecting the expression of house keeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Together, these results indicate that sequence-specific RNA interfering ODNs can be effectively used to silence the expression of key genes like IgE high affinity receptor that are involved in chronic inflammatory diseases.     

Plasmacytoid dendritic cells in antiviral immunity and autoimmunity

Plasmacytoid dendritic cells (pDCs) represent a unique and crucial immune cell population capable of producing large amounts of type I interferons (IFNs) in response to viral infection. The function of pDCs as the professional type I IFN-producing cells is linked to their selective expression of Toll-like receptor 7 (TLR7) and TLR9, which sense viral nucleic acids within the endosomal compartments. Type I IFNs produced by pDCs not only directly inhibit viral replication but also play an essential role in linking the innate and adaptive immune system. The aberrant activation of pDCs by self nucleic acids through TLR signaling and the ongoing production of type I IFNs do occur in some autoimmune diseases. Therefore, pDC may serve as an attractive target for therapeutic manipulations of the immune system to treat viral infectious diseases and autoimmune diseases.     

The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

Background

IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium.

Methodology/Principal Findings

FcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains.

Conclusions/Significance

Our data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested.     

Regulation of Fc?RI Signaling in Mast Cells by G Protein-coupled Receptor Kinase 2 and Its RH Domain

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on FcϵRI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2^flox/flox/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ∼50% decrease in antigen-induced Ca²⁺ mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the FcϵRI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of FcϵRIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates FcϵRI signaling in mast cells via at least two mechanisms. One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt.