We have previously shown that the uptake of a number of glycoallergens such as Der p 1 is mediated through MR; however, MR seems to be responsible for only approximately 60% of this uptake (15
). Previous work (14
) suggested that the residual Der p 1 uptake does not seem to be mediated through macropinocytosis, which is in line with our own observations (data not shown). Almost all endocytic pathways are sensitive to cholesterol perturbation (28
), and clathrin-dependent (35
) and -independent pathways (36
) are both inhibited by the removal of cholesterol. In this study, using cyclodextran (27
), we depleted the DC membrane from cholesterol thereby disrupting all receptor-mediated endocytosis (28
). This led to almost complete abrogation of Der p 1 uptake by DCs compared with control cultures which were not treated by cyclodextran. These data clearly suggest the presence of other putative receptors on DCs that are capable of allergen uptake.
To identify such receptors we used retagging, which is mainly based on labeling the protein of interest with a multifunctional cross-linker composed of a biotin tag disulfide bonded to a photoreactive group. Thus, allowing the labeled allergen to interact with the cells and then subjecting the cells to UV irradiation will enable the photoreactive group to form a covalent bond with structures in the immediate vicinity. The bound receptor could then be extracted and identified by mass spectroscopy (16
). Using this technique, we identified DC-SIGN as a novel receptor for the major house dust mite and dog allergens, Der p 1 and Can f 1, respectively.
Having identified DC-SIGN as a potential receptor for Der p 1 on DCs, we set out to investigate its role in Der p 1 uptake by DCs. The functional importance and contribution of DC-SIGN to Der p 1 uptake by human DCs were assessed using different, but complementary approaches. First, we investigated the binding of DC-SIGN to different allergens using ELISA. Der p 1, Can f 1, and Ara h 1 were all found to bind to DC-SIGN in contrast to the cat allergen Fel d 1 which showed no binding. Another, perhaps more physiologically relevant, approach is to study the binding of Der p 1 to DC-SIGN using 3T3/DC-SIGN cells (20
). This showed that Cy5 Der p 1 binds to 3T3 fibroblasts expressing DC-SIGN, but not to mock cells as detected by flow cytometry. Incubation with EDTA completely abrogated the binding, indicating that binding is Ca2+
The above binding data prompted us to investigate whether DC-SIGN is involved in allergen uptake by human DCs. Based on the carbohydrate specificity of Lewis-x for DC-SIGN (30
), we therefore sought to target DC-SIGN on DCs using a natural ligand (Lewis-x antigen) to investigate further the impact of DC-SIGN engagement on Der p 1 uptake by DCs. Results obtained demonstrated dose-dependent and significant inhibition of Der p 1 uptake by Lewis-x with nearly 20% inhibition with highest concentration (20 mg/ml) of Lewis-x.
DC-SIGN has also been shown to mediate the uptake of various allergens such as the major peanut allergen (Ara h 1) (29
), as well as the Bermuda grass pollen allergen (BG-60) and the major group 2 allergen from house dust mite (Der p 2) (38
). As such, this and other reports clearly underscore the potential of CLRs (in particular MR and DC-SIGN), which are highly expressed on the surface of antigen-presenting cells, to serve as common receptors for recognition of a wide range of glycosylated allergens. Intriguingly, we have also shown for the first time that down-regulation of DC-SIGN expression through siRNA leads to a bias toward Th2 polarization in autologous DC-T cell co-cultures. This is in contrast with our earlier work showing that down-regulation of MR, a major Der p 1 receptor expressed on DCs, leads to an opposite effect, i.e.
bias toward Th1 polarization (15
). This could be partly explained by data showing that differentiation of naive T cells toward Th2 occurs upon co-stimulation through ICAM-1 and ICAM-2 instead of ICAM-3, which is thought to be the main DC-SIGN counterstructure on T cells (39
). Within this context, it is interesting to note that we have shown previously (37
) that Der p 1, a cysteine protease, in its enzymatically active form, can cleave DC-SIGN (its “Th1-promoting” receptor) but not MR (its “Th2-promoting” receptor), and this will further amplify its alleregenicity. Although the exact mechanism of polarized Th2 cell differentiation in the absence of DC-SIGN is yet to be determined, these data clearly indicate that glycoallergen uptake by DCs and events leading to downstream Th2 polarization are complex and involve at least two receptors, MR and DC-SIGN, whose engagement by allergen leads to distinct and possibly antagonistic signaling events. Therefore, it is reasonable to assume that the overall fate of T cells in response to allergen exposure in different individuals could at least be partly determined by differential level of MR and DC-SIGN expression on DC subsets. In this connection, it is interesting to note that MR expression has been shown to be higher in atopic individuals (14
Early events at the interface of DCs and allergens are clearly of great importance in determining T cell polarization. In this study, we have identified a novel receptor for two major allergens from house dust mite and dog, Der p 1 and Can f 1, respectively. This new insight could lead to better understanding of the molecular basis of allergen-induced Th2 cell polarization, and this should hopefully pave the way for the rational design of novel intervention strategies.