An investigation into IgE-facilitated allergen recognition and presentation by human dendritic cells

Abstract

Background: Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcεRI and FcεRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs.

Objectives: This study aims at understanding the extent to which IgE- and IgG-facilitated Der p 1 uptake by DCs influence T cell polarisation and in particular potential bias in favour of Th2. We have addressed this issue by using two chimaeric monoclonal antibodies produced in our laboratory and directed against a previously defined epitope on Der p 1, namely human IgE 2C7 and IgG1 2C7.

Results: Flow cytometry was used to establish the expression patterns of IgE (FcεRI and FcεRII) and IgG (FcγRI) receptors in relation to MR on DCs. The impact of FcεRI, FcεRII, FcγRI and mannose receptor mediated allergen uptake on Th1/Th2 cell differentiation was investigated using DC/T cell co-culture experiments. Myeloid DCs showed high levels of FcεRI and FcγRI expression, but low levels of CD23 and MR, and this has therefore enabled us to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal interference by CD23 and MR. Our data demonstrate that DCs that have taken up Der p 1 via surface IgE support a Th2 response. However, no such effect was demonstrable via surface IgG.

Conclusions: IgE bound to its high affinity receptor plays an important role in Der p 1 uptake and processing by peripheral blood DCs and in Th2 polarisation of T cells.

Figure 1

Time-related FcϵRI, FcϵRII, FcγRI and MR expressions. Receptor expression was detected during the course of generating DCs starting from day 0 (monocyte) through to days 2, 4, 6 (immature DC) and 8 (mature DC). The data presented show the average of six independent experiments for FcϵRI, CD23 and MR, and three independent experiments for FcγRI, all expressed as mean ± SEM.

Figure 2

Inhibition of IgE-FITC binding by Mo-DCs. Immature dendritic cells (10⁵) were treated with IgE-FITC (7 μg/ml) plus blocking IgE or IgG antibodies (20 μg/ml) for 20 minutes at 4°C and then washed and fixed. Analysis was done by flow cytometry. The results show a significant decrease in IgE-FITC binding when mixed with unlabelled IgE, but not control IgG (*: p value ≤0.05). The data presented represent the average of three independent experiments expressed as mean ± SEM.

Figure 3

Effect of mannan on the binding of IgE-FITC. Immature Mo-DCs (10⁵) were treated with IgE-FITC (7 μg/ml) after pre-incubation with and without mannan (200 μg/ml) for 20 minutes at 4°C, and then washed and fixed. Analysis was done by flow cytometry. The data presented represent the average of two independent experiments expressed as mean ± SEM.

Figure 4

The effect of sodium periodate on Der p 1. Der p 1 was exposed to periodate for one hour and then immunoblotted alongside natural Der p 1 (as a control) against the anti-mannose sugar (GNA). The blot shows a very clear band with natural Der p 1, while no band can be seen with the deglycosylated Der p 1.

Figure 5

ELISA results showing the reactivity of Der p 1 with monoclonal anti Der p 1 antibodies. Wells were coated with 5 μg/ml of Der p 1 or a cat allergen (Fel d 1). IgE 2C7 (A) or IgG 2C7 (B) antibodies were used in different concentrations and binding detected using a goat anti-human κ chain HRP-conjugated secondary antibody. The data presented represent the average of two (A) or three (B) independent experiments expressed as mean ± SEM. In further experiments, wells were coated with 5 μg/ml of deglycosylated Der p 1 or Fel d 1. IgE 2C7 (C) or IgG 2C7 (D) antibodies were used in different concentrations and binding detected using a goat anti-human κ chain HRP-conjugated secondary antibody. The data presented represent the average of two independent experiments expressed as mean ± SEM. Analysis by ELISA of the binding of anti-Der p 1 5H8 antibody with periodate-treated Der p 1 (E) shows retention of Der p 1 protein structure. The data presented represent the average of three independent experiments expressed as mean ± SEM.

Figure 6

Uptake of Der p 1 by Mo-DCs is mediated by IgE receptors. The median fluorescent index for the uptake of Der p 1 (0.26 μg/ml Cy5 labelled), with and without human IgE (10 μg/ml), IgE 2C7 (1 μg/ml) or 200 μg/ml mannan, by immature Mo-DCs at 37˚. **: p ≤ 0.01; ***: p ≤ 0.001. The data presented represent the average of three independent experiments expressed as mean ± SEM.

Figure 7

FcϵRI, FcϵRII, FcγRI and MR expressions on myeloid DCs. Peripheral blood myeloid DCs were stained with various monoclonal antibodies to detect FcϵRI, CD23, FcγRI and MR. The data presented are the average of four independent experiments expressed as mean ± SEM.

Figure 8

Th1 and Th2 cytokine responses to stimulated myeloid DCs, demonstrating the effect of Der p 1 taken up via IgE or IgG 2C7 uptake on naïve T cells polarisation. Myeloid DCs were pre-loaded with Der p 1 or deglycosylated Der p 1, both with and without IgE 2C7, prior to establishing DC-naïve T cell co-cultures. T cells were re-stimulated at day 10 with PMA (15 ng/ml) and Ionomycin (1ug/ml). The following conditions were used: cells only and Der p 1 or deglycosylated Der p 1, with and without IgE 2C7 (n = 3, except for deglycosylated Der p 1 only condition which was done twice). The results were statistically significant for IL-4 when myeloid DCs were pre-loaded with Der p 1 and IgE 2C7 (*:p ≤ 0.05).

Figure 9

Th1 and Th2 cytokine responses to stimulated myeloid DCs, demonstrating the effect of Der p 1 taken up via IgE 2C7, and using non-specific human IgE antibodies as controls, on naïve T cell polarisation. Myeloid DCs were pre-loaded with Der p 1, with and without IgE 2C7 antibody, prior to establishing DC-naïve T cell co-cultures. T cells were re-stimulated at day 10 with PMA (15 ng/ml) and Ionomycin (1 ug/ml). The following conditions were used: cells only and Der p 1, with and without IgE 2C7 or IgE control (n =4). The data presented represent the average of four independent experiments expressed as mean ± SEM.

Figure 10

Th1 and Th2 cytokine responses to stimulated myeloid DCs, demonstrating the effect of Der p 1 taken up via IgG 2C7, and using non-specific human IgG antibodies as controls, on naïve T cell polarisation. Myeloid DCs were pre-loaded with Der p 1 or deglycosylated Der p 1, both with and without IgG 2C7 antibody, prior to establishing DC-naïve T cell co-cultures. T cells were re-stimulated at day 10 with PMA (15 ng/ml) and Ionomycin (1ug/ml). The following conditions were used: cells only, Der p 1 or deglycosylated Der p 1, with and without IgG 2C7 (n = 4 and n = 5, respectively), and Der p 1 or deglycosylated Der p 1, with IgG controls (n =3). The results were statistically significant for IFN-γ when myeloid DCs were pre-loaded with Der p 1 and IgG 2C7 (*:p ≤ 0.05).