The Major Birch Pollen Allergen Bet v 1 Induces Different Responses in Dendritic Cells of Birch Pollen Allergic and Healthy Individuals

<div><p>Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.</p></div

Competitive binding of allergens to iMoDCs of BP allergic (AD) and normal donors (ND).

<p>(A) Uptake of labeled Api g 1 (20 μg/ml) in the presence of unlabeled Bet v 1 (shown for donors AD3 and ND3). (B) Cross-competition of labeled Bet v 1 with Api g 1 (Bet v 1*/Api g 1) and self-competition of Bet v 1 (Bet v 1*/Bet v 1) using 20μg/ml of labeled allergen and 200 μg/ml of competitor (donor AD3). Results in (A) and (B) show uptakes after 45 minutes of chase and are representative of four independent experiments with similar outcomes for both donor groups. (C) Densitometric quantification of the competition assays measuring a 1 mm² area.</p

Gene expression induced by Bet v 1 and MFs in iMoDCs of BP allergic donors.

<p>Cells were sham-treated or treated with Bet v 1 (squares), control stimulus (MF, circles), or a combination of Bet v 1 and control stimulus (diamonds) for indicated time periods. mRNA levels of EGR-1 and-3, Th2 cytokines IL-4 and IL-13, Th1 chemokines CXCL10 and 11, and pro-inflammatory cytokines IL-1β and IL-6 were analyzed by real-time PCR. Expression levels, normalized to the average of housekeeping genes, are shown relative to unstimulated cells. Data for donor AD2 are shown, which are representative of independent experiments with mRNA from cells of three donors each performed in duplicates.</p

Phosphorylation of MAP kinases after activation by allergens.

<p>Cells of BP allergic (A) and normal donors (B) were stimulated with the allergens (each 20 μg/ml) or control stimulus (MFs) for the indicated time points and then lysed. Cell extracts were analyzed by Western blot using antibodies specific for the phosphorylated forms of PKCα, Erk1/2, and p38 MAPKs. Sample amounts were determined with antibodies to Erk1/2 and PKCα. The blots shown are representative of independent experiments with cells of three donors for each group yielding similar results (shown for donors AD2 and ND1).</p

Gene expression of MoDCs induced by Bet v 1.

<p>Cells of BP allergic (squares) and normal donors (triangles) were either sham-treated or treated with Bet v 1 for the indicated times. mRNA levels of EGR-1 and-3 and the Th2 cytokines IL-4 and IL-13 were analyzed by real-time PCR. Expression levels, normalized to the average of housekeeping genes, are shown relative to non-stimulated cells. Data are presented as mean values ± SEM (n = 7 in both groups) performed in duplicates. Significance is indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001).</p

Kinetics of allergen uptake into iMoDCs of BP allergic and normal donors.

<p>Internalization of labeled Bet v 1 (Bet v 1–488) and labeled Api g 1 (Api g 1–610) by iMoDCs of allergic (A) and normal donors (B) was followed by live-cell fluorescence imaging. Results are representative of independent experiments of three different donors for each group (shown for donors AD1 and ND3). Exocytosis of fluorescent markers (panel A) and the absence of spillover of fluorescence (panel B) are depicted by framed display details.</p

Gene expression of iMoDCs after Bet v 1 stimulation or cross-linking of cell-bound IgE.

<p>iMoDCs of BP allergic (A) and normal donors (B) were stimulated with Bet v 1 (squares), anti-IgE (diamonds), or loaded with IgE before anti-IgE treatment (IgE+anti-IgE, circles). mRNA levels of IL-4, IL-13 and the NFκB related genes IL-1β and IL-6 were analyzed by real-time PCR. Error bars indicate SD. Results are representative of two independent experiments for each donor group (shown for donors AD1 and ND1). (C) Activation of NFκB was evaluated by detection of its inhibitory protein IκBα. The Western blots shown are representative of independent experiments for three donors of each group yielding similar results (shown for donors AD2 and ND2).</p

NMR analysis of Gad m 1: scFv-gco9 complex formation.

<p>(A) Selected regions of the ¹H/¹⁵N HSQC spectra of Gad m 1 in its free form (blue) and in complex with scFv-gco9 at 40 μM or 70 μM complex concentration in red and green, respectively. (B) CSP index as a function of amino acid residue numbers was used to map the residues involved in the interaction in two complex concentrations, 40 μM (red) and 70 μM (gray). (C) Primary sequence of Gad m 1.0202 showing mapped amino acid residues (red) and amino acid residues whose NMR signals disappeared upon complex formation (yellow). (D) Same as in panel C colored in the Gad m 1 NMR solution structure (PDB:2mbx). (E) ¹⁵N backbone relaxation parameters R₂/R₁ of Gad m 1 and the Gad m 1:scFv-gco9 complex as a function of amino acid residue numbers.</p

Coomassie brilliant blue-stained SDS-PAGE analyses of the purified scFv antibodies (1 μg/lane).

<p>Coomassie brilliant blue-stained SDS-PAGE analyses of the purified scFv antibodies (1 μg/lane).</p

ELISA assay showing the sensitivity of scFv and scFv-AP antibodies.

<p>scFv-gco9, scFv-goo8 and scFv-gco9-AP were tested to recognize purified cod, carp and trout parvalbumin. Serial dilutions of purified scFv fragments were used (1 μg/ml–1 ng/ml).</p