The ability of fungal-derived β-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-α, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-α in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-α in response to fungi, a critical step required for the successful control of these pathogens.

The phagocytosis of pathogens is a critical event in host defense, not only for clearance of the invading microorganism, but also for the subsequent immune response. We have examined Dectin-1, a proinflammatory nonopsonic receptor for ␤-glucans, and show that it mediates the internalization of ␤-glucan-bearing ligands, including yeast particles. Although requiring tyrosine phosphorylation and the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-like motif, uptake mediated by Dectin-1 was different from any previously reported phagocytic receptor and was not dependent on Syk-kinase in macrophages. IntroductionPhagocytosis plays a critical role in innate immunity, both by facilitating the removal and killing of pathogens and by priming the adaptive immune response. The phagocytic process is initiated by the cross-linking of an array of dedicated surface receptors, some capable of direct recognition, the so-called pattern recognition receptors (PRRs), and others that recognize opsonins coating the pathogens. Of these receptors, the opsonic Fc␥ (Fc␥Rs) and complement receptors (CRs) are the best described and exhibit different phagocytic mechanisms and subsequent cellular responses that reflect important differences in their signaling pathways. 1,2 Nonopsonic PRRs, such as the macrophage mannose receptor, scavenger receptors, and recently CEACAM3, [3][4][5] have also been suggested to possess phagocytic capacity, but the mechanisms underlying these activities are less clear.We have identified Dectin-1 as the major macrophage PRR for ␤-glucans, carbohydrate polymers that possess anti-infective and antitumorigenic properties in vivo. [6][7][8] Dectin-1, which is also expressed on the surface of other innate immune cells, including neutrophils and dendritic cells, 9 is a C-type lectinlike transmembrane receptor containing an immunoreceptor tyrosine-based activation motif (ITAM)-like motif in its cytoplasmic tail, which becomes tyrosine phosphorylated on ligand binding. 10,11 We and others have shown this motif was required for proinflammatory cytokine production, in collaboration with the Toll-like receptors (TLRs), and for induction of the respiratory burst in response to ␤-glucan ligands, including fungal pathogens. 11,12 The proinflammatory properties of Dectin-1 are similar to those of the ITAM-containing Fc␥Rs, which also mediate the internalization of immune complexes. 13 Phagocytosis by Fc␥R, after ligand binding, is thought to be initiated by src kinase-mediated tyrosine phosphorylation of the receptor ITAM domains leading to the recruitment of p72Syk, a protein tyrosine kinase that is required for subsequent cellular activation and ligand internalization. 1,14 Although the exact downstream pathways leading from Fc␥ and other receptors to actin polymerization and phagocytosis is currently unclear, other molecules, including phosphatidylinositol (PI)-3 kinase, protein kinase C (PKC), and the Rho guanosine triphosphatases (GTPases), are known to be involved. 1 We determined if Dectin-1 could als...

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal b-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the b-glucan receptor (bGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional bGR isoforms -bGR-A and bGR-B -were expressed by these cell populations in peripheral blood; however, only bGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other b-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major b-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory Ctype lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the ␤-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the ␤-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.The functional balance of the immune system is regulated, in part, by inhibitory and activatory receptors found on all leukocytes as well as on many non-immune cells (1). Within these activatory and inhibitory receptor families, cytoplasmic consensus motifs have been identified and shown to associate with particular signaling mechanisms. These motifs include the immunoreceptor tyrosine-based activation motifs (ITAMs), 1 which become phosphorylated upon stimulation, sending their activatory signals through a variety of intracellular enzyme cascades and resulting in a wide range of cellular outcomes, and the immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which suppress such activatory signals by recruiting phosphatase enzymes that dephosphorylate ITAMs as well as other components of the activatory pathways.The array of inhibitory and activatory receptors expressed by natural killer cells has recently been the focus of much investigation (2). In humans, these receptors can be divided into two families: the Ig superfamily, including the killer cell Ig-like receptors (3) that bind classical major histocompatibility complex class I or HLA G (non-classical), and the C-type lectin-like family (4), including the NKG2 receptors, which bind the nonclassical HLA E or other distantly related molecules that are up-regulated on stressed, transformed, or infected cells (5, 6).The C-type lectin and lectin-like proteins are classified into 14 groups based on the arrangement of their C-type lectin-like domains (CTLDs).2 In particular, group V receptors include the lectin-like NKG2 family mentioned above, but also a distinct subgroup of these receptors that are expressed predominantly on myeloid and endothelial cells (7). The members of this subgroup appear to bind diverse ligands, as exemplified by LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) (8) and the ␤-glucan receptor (BGR), which is an activatory phagocytic receptor for ␤-glucans (9).3 Although they share structuralhomologywithclassic...

The murine molecule dectin-1 (known as the b-glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin-1 extracellular domain is a C-type lectin-like domain, and functional studies have established that it binds fungal b-glucans. We report several dectin-1 crystal structures, including a high-resolution structure and a 2.8 Å resolution structure in which a short soaked natural b-glucan is trapped in the crystal lattice. In vitro characterization of dectin-1 in the presence of its natural ligand indicates higher-order complex formation between dectin-1 and b-glucans. These combined structural and biophysical data considerably extend the current knowledge of dectin-1 structure and function, and suggest potential mechanisms of defense against fungal pathogens.Keywords: immune recognition; fungal pathogen; b-glucan; protein crystallography; C-type lectin-like domain Dectin-1 is a cell-surface immune receptor for b-glucans, which are major structural cell wall components that are conserved in fungi (Brown and Gordon 2001). Recognition of b-glucans by dectin-1 can trigger phagocytosis of fungal pathogens and protective inflammatory responses. Originally identified as a dendritic cell receptor in the mouse (Ariizumi et al. 2000), dectin-1 is now known to be widely expressed in both mouse and human cells, particularly on monocytes/macrophages and neutrophils (Brown 2006). Primary sequence analysis indicates that dectin-1 is a 28-kDa type II membrane protein. An extracellular Ctype lectin-like domain (CTLD) is connected by a stalk to a transmembrane region, followed by a cytoplasmic tail containing an immunoreceptor tyrosine-based activation (ITAM)-like motif (Ariizumi et al. 2000). First recognized as a calcium-dependent carbohydrate-binding domain, the CTLD fold is also seen in non-calciumdependent protein recognition interactions (Drickamer 1999). Few of the residues required for calcium coordination in classical CTLDs are conserved in dectin-1. The dectin-1 CTLD has two potential N-linked glycosylation sites, whereas its human homolog, the b-glucan receptor (bGR), has none. In addition to its role in b-glucan binding, it has been suggested that dectin-1 recognizes an endogenous T-cell ligand in a carbohydrate-independent manner (Ariizumi et al. 2000), but there is no evidence as to the nature of this potential ligand. Abbreviations: AUC, analytical ultracentrifugation; bGR, human b-glucan receptor; BGC, b-glucan; CTLD, C-type lectin-like domain; DLS, dynamic light scattering; TTX, Triton X-100.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Inhibitory receptors are required for the control of cellular activation and they play essential roles in regulating homeostasis and immunity. We previously identified a human inhibitory C-type lectin-like receptor, MICL (CLEC12A), a heavily glycosylated monomer predominantly expressed on myeloid cells. Here we characterise the murine homolog of MICL (mMICL), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (hMICL), although there are some notable differences. mMICL is expressed as a dimer and is not heavily glycosylated; however, like hMICL, the receptor can recruit inhibitory phosphatases upon activation, and is down-regulated on leukocytes following stimulation with selected TLR agonists. Using novel monoclonal antibodies, we demonstrate that, like the human receptor, mMICL is predominantly expressed by myeloid cells. However, mMICL is also expressed by B cells and CD8 + T cells in peripheral blood, and NK cells in the bone marrow. Finally, we show that mMICL recognises an endogenous ligand in a variety of murine tissues, suggesting that the receptor plays a role in homeostasis.

Host defence against pathogens requires the recognition of conserved microbial molecules, or 'pathogen-associated molecular patterns' (PAMPs), by their receptors termed 'pattern recognition receptors' (PRRs), represented most notably by toll-like receptors (TLRs) and C-type lectins. The 'non-classical' C-type lectins (these that lack the residues involved in calcium binding, required for carbohydrate binding) are traditionally thought of as being restricted to natural killer (NK) or T cells, playing important roles in immune surveillance. In recent years, however, a growing number of these receptors have been identified on myeloid cells, both of human and mouse origin. In contrast to their NK counterparts that primarily control cellular activation through recognition of major histocompatibility antigen (MHC) class I and related molecules, the myeloid-expressed receptors appear to have a far more diverse range of functions and ligands, including those of exogenous origin. Some of C-type lectin-like molecules possess activating/inhibitory signalling motifs that trigger downstream signalling events, suggesting the role for these receptors as positive/negative regulators of granulocyte and monocyte functions. With the exception of a few myeloid NK-like lectins, the natural ligands for most of these receptors remain unidentified, making it difficult to define their functions in normal physiological, inflammatory or pathological conditions. Importantly, in some cases, these novel C-type lectin-like lectins, encoded by genes from the same gene cluster, can act as receptor/ligand pairs, additionally contributing to the regulation of myeloid cell functions or their interaction with other (like NK) cell types. However, the relevance and importance of such interactions still needs to be assessed. Although few of the myeloid-expressed C-type lectins have been characterized in detail, we review here each of these receptors and highlight their prospective roles in innate and adaptive immunity.