Retagging identifies dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN) protein as a novel receptor for a major allergen from house dust mite

Dendritic cells (DCs) have been shown to play a key role in the initiation and maintenance of immune responses to microbial pathogens as well as to allergens, but the exact mechanisms of their involvement in allergic responses and Th2 cell differentiation have remained elusive. Using retagging, we identified DC-SIGN as a novel receptor involved in the initial recognition and uptake of the major house dust mite and dog allergens Der p 1 and Can f 1, respectively. To confirm this, we used gene silencing to specifically inhibit DC-SIGN expression by DCs followed by allergen uptake studies. Binding and uptake of Der p 1 and Can f 1 allergens was assessed by ELISA and flow cytometry. Intriguingly, our data showed that silencing DC-SIGN on DCs promotes a Th2 phenotype in DC/T cell co-cultures. These findings should lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization and in doing so paves the way for the rational design of novel intervention strategies by targeting allergen receptors on innate immune cells or their carbohydrate counterstructures on allergens.     

Uptake of Proteins Modified with 3-Deoxyglucosone,a Maillard Reaction Intermediate,by the Type I Macrophage Scavenger Receptor

We investigated the mechanism of recognition by murine peritoneal macrophages of bovine serum albumin (BSA) modified with glucose and 3-deoxyglucosone (3DG), the main intermediate compound in the Maillard reaction of proteins with glucose. A scatchard analysis of the binding data of BSA incubated with 3DG for 17 days (3DG-BSA 17d) and of glycated BSA indicated two binding sites, each with a different binding affinity, in both cases. The apparent K_d values for the higher affinity sites of 3DG-BSA 17d and of glycated BSA were 8.5 nM and 4.6 nM, and those for the lower affinity ones were 0.95 μM and 1.3 μM, respectively. 3DG-BSA 17d and glycated BSA competed with each other in the specific binding to macrophages, this binding being most effectively inhibited by maleylated BSA and polyinosinic acid. The binding of 3DG-BSA 17d and glycated BSA to the type I macrophage scavenger receptor [Kodama et al. Nature, 343, 531–535 (1990)] was investigated, because these modified BSAs share binding sites with maleylated BSA and polyinosinic acid. It was found that 3DG-BSA 17d and glycated BSA could be recognized by the type I macrophage scavenger receptor expressed on COS cells, and that the affinity of 3DG-modified BSA for the receptor increased with increasing time of incubation for BSA with 3DG. We suggest that this can be explained by the lower pI values for 3DG-BSA 17d and glycated BSA than that for the control BSA.     

Functional Interaction of Common Allergens and a C-type Lectin Receptor, Dendritic Cell-specific ICAM3-grabbing Non-integrin (DC-SIGN), on Human Dendritic Cells

Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the α1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le^x). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-α expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-α expression in MDDCs via, in part, Raf-1 signaling pathways.