Allergen-IgE complexes trigger CD23-dependent CCL20 release from human intestinal epithelial cells

Abstract

Background & aims: In food allergic individuals, exposure to food allergens by the oral route can trigger immediate (within minutes) local hypersensitivity reactions in the intestine followed by a late-phase inflammatory response. Previous work has shown that CD23 is constitutively expressed by human intestinal epithelial cells and mediates the uptake of allergen-IgE complexes. We hypothesized that allergen-IgE complexes could also signal via CD23 to trigger an inflammatory cascade in the local environment.

Methods: Caco-2 monolayers were stimulated with human IgE-antigen (Ag) complexes. IL-8 and CCL20 mRNA and protein were determined by RT-PCR and ELISA, respectively. Signaling pathways were assessed by immunoblotting. Endogenous CD23 expression was knocked down by stable transfection with CD23 shRNA retroviral plasmid. Migration assays were performed using human monocyte-derived dendritic cells.

Results: Stimulation of Caco-2 cells with IgE-Ag complexes triggered upregulation of IL-8 and CCL20 at the mRNA and protein level. Allergen complexes induced phosphorylation of ERK and JNK, but not p38 MAP kinase or NK-kappaB, and resulted in AP-1 activation. Cross-linking of CD23 replicated the findings with IgE-Ag complexes, and silencing of CD23 expression abrogated the response to allergen-IgE complexes. Supernatant from IgE-Ag-stimulated epithelial cells induced migration of dendritic cells in a CCL20-dependent manner. Finally, immunostaining of duodenal biopsies demonstrated that CCL20 was constitutively expressed by epithelial cells in vivo.

Conclusions: Signaling via epithelial CD23 may participate in the late-phase inflammatory response by the release of chemokines capable of recruiting antigen presenting cells and effector cells of allergic inflammation.

Figure 1. Polarized responsiveness of intestinal epithelial cells to IgE-Ag

Caco-2 cells were polarized on filter supports and stimulated with anti-NP IgE (IgE), NP-BSA (Ag), or IgE-Ag complexes. Cells were stimulated either on the apical or basal side of the transwell, and IL-8 (A) and CCL20 (B) mRNA expression was assessed by real-time PCR after 6 hours. Data are expressed as fold increase compared to control, mean + SEM, n = 4/group. IL-8 and CCL20 chemokine secretion into the apical or basal transwell was measured 24 h after stimulation with IgE-Ag (C).

Figure 2. Signaling in intestinal epithelial cells in response to IgE-Ag

Caco-2 cells were polarized on filter supports and stimulated with anti-NP IgE plus NP-BSA (Ag-IgE) on the basolateral side. A. Immunoblotting for phospho (p−) or total ERK 1/2, JNK, p38, and IκBα. Cell extracts were prepared from unstimulated cells or cells stimulated for 20 or 60 minutes with Ag-IgE. B. EMSA showing NF-κB binding activity in nuclear extracts prepared from unstimulated cells (media), or cells stimulated with TNFα (20 ng/ml) for 1 or 3 h, or IgE-Ag complexes for 1–4 hours. C. AP-1 binding in nuclear extracts prepared from cells that were untreated (media), treated with epidermal growth factor as positive control (EGF), or treated with Ag-IgE for 1 h or 3 h. AP-1 binding in nuclear extracts was determined by an ELISA-based technique.

Figure 3. Effect of JNK and ERK inhibitors on IL-8 and CCL20 release by IgE-Ag-stimulated intestinal epithelial cells

Caco-2 cells were polarized on filter supports and pre-treated with the MEK inhibitor U0126 or its negative control U0124, or JNK inhibitor II or its negative control prior to stimulation with IgE-Ag complexes (IgE-Ag). A. Cell extracts were obtained 60 min after stimulation and immunoblotting performed for phospho-ERK, phospho-JNK, or actin as loading control. B. Chemokine secretion was measured by ELISA using the basolateral culture supernatant obtained 24 h after stimulation. Data are mean + SEM, n = 3–4/group.

Figure 4. CD23 cross-linking induces epithelial chemokine secretion

Caco-2 cells were polarized and stimulated with TNFα (20 ng/ml), Ag-IgE complexes, or anti-CD23 antibodies + anti-IgG to cross-link. IL-8 (A) and CCL20 (B) release into the basolateral supernatant was measured by ELISA. ND = none detected.

Figure 5. CD23 is required for IgE-Ag-induced activation of epithelial cells

Caco-2 cells were stably transfected with shRNA targeting CD23, or with control shRNA or were left untransfected. Knockdown of CD23 expression was confirmed by western blotting and RT-PCR (A). Cells were stimulated with EGF and immunoblotting for phospho-ERK and phospho-JNK was performed to show that CD23 RNAi did not interfere with these pathways (B). Cells were then polarized on filter supports and stimulated with IgE-Ag complexes on the basolateral side as above. C. Immunoblotting for phospho (p−) ERK 1/2 and JNK or actin as a loading control. Lysates were prepared 60 min after stimulation. D. IL-8 and CCL20 secretion as measured in the basolateral supernatant 24 h after stimulation with IgE-Ag complexes. E. AP-1 binding activity as measured in cell extracts obtained 60 min after stimulation. F. AP-1 luciferase reporter assay.

Figure 6. Epithelial Cells Induce CCL20-Dependent Migration of Human Dendritic Cells after Stimulation with IgE-Ag

Dendritic cells (DCs) were generated from peripheral blood monocytes by culture with IL-4 and GM-CSF for 6 days. DCs were added to the top chamber of chemotaxis chambers, and supernatant from epithelial cell cultures was added to the basolateral chamber. A. Supernatant was derived from Caco-2 cells stimulated with nothing (media), IgE or antigen (Ag) alone, or IgE-Ag complexes (IgE+Ag). Data are mean + SEM of 6/group. ** p < 0.01 compared to control. B. Supernatant was obtained from Caco-2 cells or cells with CD23 knocked down (siCD23) 24 h after stimulation with Ag-IgE. Supernatant was pre-incubated with neutralizing anti-CCL20 antibody (α-CCL20) or isotype control (IgG). Pertussis toxin (PTX, 200 ng/ml) was added to DCs for 2 h prior to addition of cells to chemotaxis chambers. Data are mean + SEM of 4/group. *** p < 0.001 compared to control.

Figure 7. Expression of CCL20 by Human Duodenal Epithelial Cells In Vivo

Sections from duodenal biopsies were stained with anti-CCL20 (A,C–F) or isotype control (B). A: Section from a subject with allergic eosinophilic gastroenteritis (AEG). Note the villous blunting, and the positive (brown) immunostaining in the epithelium and the crypts. Original magnification 100x. B: Section from the same biopsy as A, stained with isotype control (100x). C: Section from a non-inflamed control showing a complete absence of CCL20 staining (100x). D: Section from a non-inflamed control showing abundant CCL20 immunoreactivity in villous and crypt epithelium (100x). E: Higher power magnification of the section shown in A, showing detail of positive immunostaining in the surface epithelium (400x). F: Higher power magnification of section shown in A, showing detail of positive immunostaining in the crypt epithelium (200x).