Functional interaction of common allergens and a C-type lectin receptor, dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), on human dendritic cells

Abstract

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 alpha1,3-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-alpha 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-alpha expression in MDDCs via, in part, Raf-1 signaling pathways.

FIGURE 1.

Representative binding analyses of purified allergens and soluble CLRs (n = 4–8 experiments). A serial 3-fold dilution of purified allergens, including natural (nCor a11 and nDer p2) or recombinant (rCor a11 and rDer p2) allergens and MOXF₃-BSA was probed with soluble DC-SIGN-Fc (A and C) and soluble L-SIGN-Fc (B and D), with binding activity expressed as optical density (OD).

FIGURE 2.

Representative inhibition assays (n = 2–3 experiments). A, shown are competitive inhibition assays with europium-labeled Man₅₁-BSA and purified allergens, BG60 and Der p2, as competitors. A line at 50% inhibition intercepting the inhibition curves is indicated. B, shown is inhibition of DC-SIGN-Fc binding to MOXF₃-BSA and purified allergens by various inhibitors as indicated. *, p < 0.05 versus bindings without the addition of inhibitors.

FIGURE 3.

Analysis of BG60-induced response in THP-1 cells and PMA/IL-4-stimulated THP-1 cells. A, shown is analysis of DC-SIGN (CD209) expression. B, shown is BG60-induced TNF-α expression. LPS, lipopolysaccharide. *, p < 0.05; NS, not significant. C, flow analysis of DC-SIGN expression is measured as mean fluorescence intensity (MFI) in cells transduced with recombinant lentivirus expressing mock control (LV-mock) or two independent DC-SIGN miRNA sequences, LV-DC-SIGN3 and LV-DC-SIGN4. Ig ctr, isotype control Abs. D, analysis of TNF-α expression in cells with DC-SIGN knockdown is shown.

FIGURE 4.

Allergen-induced response in human MDDCs. BG60-induced TNF-α (A) and IL-12p70 (B) levels in MDDCs (n = 8 subjects) are shown. C, Der p2-induced TNF-α expression is shown. D, flow analysis of DC-SIGN expression is measured as mean fluorescence intensity (MFI) in MDDCs with DC-SIGN knockdown as in Fig. 3C. Ig ctr, isotype control Abs. E, shown is BG60-induced TNF-α expression in DC-SIGN-silenced MDDCs.

FIGURE 5.

Analysis of signaling events in MDDCs. A, shown is a Western blot analysis of BG60-induced activation of Raf-1 (pRaf-1 Ser-338) and pERK as described under “Experimental Procedures.” Shown are relative levels of phosphorylated Raf-1 Ser-338 (n = 5 subjects) in BG60- (B) and Der p2-stimulated MDDCs (C). *, p < 0.05. BG60 (D)- and Der p2 (E)-induced TNF-α expression in MDDCs treated with a Raf-1 inhibitor is shown. *, p < 0.05.