The generation of pathogen-specific immune responses is dependent on the signaling capabilities of pathogen-recognition receptors. DC-SIGN is a C-type lectin that mediates capture and internalization of viral, bacterial, and fungal pathogens by myeloid dendritic cells. DC-SIGN–interacting pathogens are thought to modulate dendritic cell maturation by interfering with intracellular signaling from Toll-like receptor molecules. We report that engagement of DC-SIGN by specific antibodies does not promote dendritic cell maturation but induces ERK1/2 and Akt phosphorylation without concomitant p38MAPK activation. DC-SIGN ligation also triggers PLCγ phosphorylation and transient increases in intracellular calcium in dendritic cells. In agreement with its signaling capabilities, a fraction of DC-SIGN molecules partitions within lipid raft–enriched membrane fractions both in DC-SIGN–transfected and dendritic cells. Moreover, DC-SIGN in dendritic cells coprecipitates with the tyrosine kinases Lyn and Syk. The relevance of the DC-SIGN–initiated signals was demonstrated in monocyte-derived dendritic cells, as DC-SIGN cross-linking synergizes with TNF-α for IL-10 release and enhances the production of LPS-induced IL-10. These results demonstrate that DC-SIGN–triggered intracellular signals modulate dendritic cell maturation. Since pathogens stimulate Th2 responses via preferential activation of ERK1/2, these results provide a molecular explanation for the ability of DC-SIGN–interacting pathogens to preferentially evoke Th2-type immune responses.

IntroductionThe identification of the lectin gene cluster at chromosome 19p13.2 1 has led to the realization that some C-type lectins are capable of mediating intercellular adhesion, pathogen-binding, and antigen internalization for induction of T cell responses. 2 The paradigmatic example of this type of lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), which efficiently internalizes antigens, 3 mediates dendritic cell intercellular adhesions, 4 and recognizes a wide range of microorganisms through binding to mannose-and Lewis-containing glycans. 5 C-type lectins on dendritic cells enhance their ability for pathogen recognition 6 and contribute to modulation of toll-like receptor (TLR)-initiated signals. 7 Consequently, the definition of the range of dendritic cell lectins and their binding specificities might provide adequate targets for immune intervention and prevention of pathogen entrance and spreading.The lectin gene cluster at chromosome 19p13.2 includes the genes encoding for the type II C-type lectins DC-SIGN, liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN), CD23, and liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin). 1,4,8,9 DC-SIGN is expressed on myeloid dendritic cells, 4,10 and alternatively activated in vitro on macrophages. 11 In vivo it is found on interstitial dendritic cells, 12 a subset of CD14ϩ peripheral blood DC, 13 human microvascular endothelial cells, 8 and on synovial, placenta, lymph node, and alveolar macrophages. 14-16 By contrast, L-SIGN is exclusively expressed on endothelial cells of the liver, lymph nodes, and placenta, 17,18 but not on myeloid cells.The LSECtin (CLEC4G) gene is located between the CD23 and DC-SIGN genes with the three genes arranged in the same orientation. 9 LSECtin encodes a protein with a lectin domain followed by a 110-residue stalk region, a transmembrane domain, and a 31-residue cytoplasmic domain. 9 LSECtin has been previously detected on liver and lymph node sinusoidal endothelial cells at the protein and RNA level. 9 LSECtin functions as an attachment factor for Ebola virus and SARS, but it does not bind HIV or hepatitis C virus. 19 We now describe the expression of LSECtin isoforms in ex vivo isolated human peripheral blood and thymic dendritic cells as well as in dendritic cells and macrophages generated in vitro. LSECtin exhibits ligand-induced internalization, and its sugar recognition specificity differs from that of DC-SIGN. The presence of LSECtin on myeloid cells should therefore contribute to expanding their antigen-capture and pathogen-recognition capabilities. Materials and methodsThe study described was approved by the Centro de Investigaciones Biologicas (CSIC) Institutional Review Board. The study did not involve any direct contact with human subjects. Cell cultureHuman peripheral blood mononuclear cells were isolated from buffy coats from normal donors over a Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient according to sta...

The expression of the new Ly108 isoform H1 weakens lupus-like disease of C57BL/6.Sle1b mice.

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other cell surface structures, including the CD38 co-receptor molecule. Here, we show that in TCR ؉ T cells that express a CD3-lacking the cytoplasmic domain, cross-linking with CD38-or CD3-specific monoclonal antibodies induces tyrosine phosphorylation of CD3-⑀, -associated protein-70, linker for activation of T cells, and Shc. Moreover, in these cells, anti-CD38 or anti-CD3 stimulation leads to protein kinase B/Akt and Erk activation, suggesting that the CD3--immunoreceptor tyrosinebased activation motifs are not required for CD38 signaling in T cells. Interestingly, in unstimulated T cells, lipid rafts are highly enriched in CD38, including the T cells lacking the cytoplasmic tail of CD3-. Moreover, CD38 clustering by extensive cross-linking with an anti-CD38 monoclonal antibody and a secondary antibody leads to an increased resistance of CD38 to detergent solubilization, suggesting that CD38 is constitutively associated with membrane rafts. Consistent with this, cholesterol depletion with methyl-␤-cyclodextrin substantially reduces CD38-mediated Akt activation while enhancing CD38-mediated Erk activation. CD38/raft association may improve the signaling capabilities of CD38 via formation of protein/lipid domains to which signaling-competent molecules, such as immunoreceptor tyrosine-based activation motif-bearing CD3 molecules and protein-tyrosine kinases, are recruited.

IntroductionHuman CD38 is the prototype of a family of proteins that share structural similarities and ectoenzymatic activities involved in the production of calcium-mobilizing compounds. [1][2][3] Aside from its ectoenzymatic activities and, apparently with independent modalities, CD38 may perform as a receptor, ruling adhesion and signaling in T 4 and B lymphocytes, 5 monocytes, 6 and natural killer (NK) cells. 7,8 The receptor functions of CD38 are regulated through interaction with a counterreceptor, identified as CD31. 9 The signaling events initiated by interactions between CD38 and CD31 (and fully mimicked by agonistic anti-CD38 monoclonal antibodies [mAbs]) were initially studied in the dynamic context of circulating CD38 ϩ T lymphocytes adhering to CD31 ϩ endothelial cells. 10 Use of this model allowed definition of some of the events that take place after the interaction and that include calcium (Ca ϩϩ ) mobilization from cytosolic stores, tyrosine phosphorylation of selected substrates, activation of nuclear factors, and secretion of cytokines. 11 It is generally agreed that CD38 controls a specific signaling pathway in T cells, B cells, NK cells, and monocytes. In spite of this evidence, the modalities through which the signal is initiated remain elusive. The molecule has neither the canonical structure of a receptor nor the hallmark domains. Indeed, the cytoplasmic tail is short and lacks docking sites and it is not tyrosine phosphorylated on activation. 12,13 Such negative characteristics are even more evident in CD157, the other member of the protein family, whose signaling features are known, notwithstanding a glycophosphatidylinositol linkage to the cell membrane. 14,15 Some clues can be extrapolated from cocapping experiments, which show that CD38 associates on the cell membrane with professional signaling receptors such as the T-cell receptor (TCR)-CD3 complex in T cells, the B-cell receptor (BCR) in B cells, and CD16 in NK cells. 16 A hypothesis to explain the signaling properties of CD38 is that the molecule exploits the signaling machinery of professional receptors to deliver its own independent signals. This idea was first supported by experiments using CD38 ϩ T-cell lines deficient in components of the signaling apparatus of the TCR-CD3 complex. 17,18 The inability of CD38 to signal in these cells was overcome by reconstituting a complete TCR-CD3 complex, thereby indicating that CD38 signaling depends on the presence of a functional TCR. These observations were recently expanded by studies using T lymphocytes purified from the intestinal The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From lamina propria as a model in which the TCR complex is physiologically impaired. 19 A comparative analysis of circulating versus residential T lymphocytes from the s...