Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1α and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

SummaryThe b-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized that dectin-1 has potent synergistic effects with both TLR2 and TLR4 in human PBMCs and macrophages. Human PBMCs and monocyte-derived macrophages were stimulated with curdlan, a linear b-1,3-glucan-polymer derived from Alcaligenes faecalis with specific ligand affinity for dectin-1, in combination with the synthetic TLR2 ligand Pam3Cys and the ultrapure TLR4 ligand LPS. TNF-a and IL-10 production was measured in the supernatants with ELISA. Curdlan is a specific dectin-1 ligand without TLR2-or TLR4-stimulating properties. Human primary monocytes and macrophages express dectin-1 on the cell membrane. Stimulation of human PBMCs with curdlan in combination with Pam3Cys or LPS leads to synergistic increase in TNF-a production that was inhibited by GE2, a neutralizing dectin-1 antibody. Dectin-1-dependent synergy between curdlan and TLR agonists was also apparent in human monocyte-derived macrophages. Conclusively, dectin-1 synergizes with both TLR2 and TLR4 pathways for the production of TNF-a in human primary PBMCs and in monocyte-derived macrophages.

C-type lectin receptors (CLRs) fulfill multiple functions within the immune system by recognition of carbohydrate moieties on foreign or (altered) self-structures. CLRs on myeloid dendritic cells (DCs) have been well characterized as patternrecognition receptors (PRRs) combining ligand internalization with complex signaling events. Much less is known about CLR expression and function in human plasmacytoid DCs (pDCs), the major type I interferon (IFN) producers. In this study, we demonstrate that, next to the CLR BDCA-2, human pDCs express DC immunoreceptor (DCIR), a CLR with putative immune-inhibitory function, but not dectin-1, mannose receptor, or DC-specific ICAM-3-grabbing nonintegrin. DCIR surface levels are reduced on pDC maturation after TLR9 triggering. Interestingly, DCIR triggering inhibits TLR9-induced IFN-␣ production while leaving up-regulation of costimulatory molecule expression unaffected. Furthermore, DCIR is readily internalized into pDCs after receptor triggering. We show that DCIR internalization is clathrin-dependent because it can be inhibited by hypertonic shock and dominant-negative dynamin. Importantly, antigens targeted to pDCs via DCIR are presented to T cells. These findings indicate that targeting DCIR on pDCs not only results in efficient antigen presentation but also affects TLR9-induced IFN-␣ production. Collectively, the data show that targeting of DCIR can modulate human pDC function and may be applied in disease preven-tion and treatment. (Blood. 2008;111:4245-4253)© 2008 by The American Society of Hematology Introduction C-type lectin receptors (CLRs) recognize defined carbohydrate moieties, the "C" indicating Ca 2ϩ dependence of carbohydrate binding. Ligands may be of endogenous or exogenous nature. 1 The prototypic CLR, dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), has been shown to bind to high-mannose N-linked carbohydrates. DC-SIGN ligands are expressed by various pathogens, such as the fungus Candida albicans, HIV-1, bacteria, and protozoa. 2 In addition, endogenous ligands such as intercellular adhesion molecule (ICAM)-2 and ICAM-3 have been identified. 1 In antigen-presenting cells (APCs), CLRs and Toll-like receptors (TLRs) play an important role as pattern-recognition receptors (PRRs) by recognizing conserved pathogen-associated molecular patterns. 3 Interestingly, because most CLRs are rapidly internalized after ligand-binding monoclonal antibodies (mAbs) directed against CLRs are currently exploited to target APCs. [4][5][6] Plasmacytoid DCs (pDCs), also known as natural interferonproducing cells, are APCs playing an important role in the immune balance. 7,8 Resting pDCs induce mostly regulatory responses, whereas activated pDCs secrete large amounts of interferon-␣ (IFN-␣) and IFN-␤ as well as chemokines. 7,9 To identify microbial intruders, they predominantly express TLR7 and TLR9, allowing them to respond to single-stranded RNA and DNA viruses. 10 PDCs induce Th1 polarization 11 and are crucial for initiating and sustaining antiviral immune respo...

Renal excretion and reabsorption of organic cations are mediated by electrogenic and electroneutral organic cation transporters, which belong to a recently discovered family of polyspecific transporters. These transporters are electrogenic and exhibit differences in substrate specificity. In rat, the renal expression of the polyspecific cation transporters rOCT1 and rOCT2 was investigated. By in situ hybridization, significant amounts of both rOCT1 and rOCT2 mRNA were detected in S1, S2, and S3 segments of proximal tubules. By immunohistochemistry, expression of the rOCT1 protein was mainly observed in S1 and S2 segments of proximal tubules, with lower expression levels in the S3 segments. At variance, rOCT2 protein was mainly expressed in the S2 and S3 segments. Both transporters were localized to the basolateral cell membrane. Neither rOCT1 nor rOCT2 was detected in the vasculature, the glomeruli, and nephron segments other than proximal tubules. The data suggest that rOCT1 and rOCT2 are responsible for basolateral cation uptake in the proximal tubule, which represents the first step in cation secretion.