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. 2017 Sep;10(5):1190-1201.
doi: 10.1038/mi.2016.120. Epub 2017 Jan 4.

Cadherin 26 Is an Alpha Integrin-Binding Epithelial Receptor Regulated During Allergic Inflammation

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Free PMC article

Cadherin 26 Is an Alpha Integrin-Binding Epithelial Receptor Regulated During Allergic Inflammation

J M Caldwell et al. Mucosal Immunol. .
Free PMC article

Abstract

Cadherins (CDH) mediate diverse processes critical in inflammation, including cell adhesion, migration, and differentiation. Herein, we report that the uncharacterized cadherin 26 (CDH26) is highly expressed by epithelial cells in human allergic gastrointestinal tissue. In vitro, CDH26 promotes calcium-dependent cellular adhesion of cells lacking endogenous CDHs by a mechanism involving homotypic binding and interaction with catenin family members (alpha, beta, and p120), as assessed by biochemical assays. Additionally, CDH26 enhances cellular adhesion to recombinant integrin α4β7 in vitro; conversely, recombinant CDH26 binds αE and α4 integrins in biochemical and cellular functional assays, respectively. Interestingly, CDH26-Fc inhibits activation of human CD4+ T cells in vitro including secretion of IL-2. Taken together, we have identified a novel functional CDH regulated during allergic responses with unique immunomodulatory properties, as it binds α4 and αE integrins and regulates leukocyte adhesion and activation, and may thus represent a novel checkpoint for immune regulation and therapy via CDH26-Fc.

Figures

Figure 1
Figure 1
CDH26 expression in allergic tissue. (a) Relative esophageal tissue CDH26 transcript levels were determined (n = 66 control [CTL], n = 77 eosinophilic esophagitis [EoE] patients). (b and c). The mean raw expression value for each cadherin probe in which any patient sample exhibited a signal intensity greater than 100 is graphed for (b) the gastric tissue of 5 CTL and 5 eosinophilic gastritis (EG) patients or for (c) the esophageal tissue of 14 CTL and 18 EoE patients characterized previously. For each cadherin, only the probe with the highest raw signal is shown. (d) Relative CDH26, CCL26 (eotaxin-3), and C3 transcript levels from the gastric antrum tissue of CTL patients (n = 5), EG patients (n = 5), and H. pylori gastritis patients (n = 3) were determined. For (a) and (d), data were analyzed by Mann-Whitney test.
Figure 2
Figure 2
CDH26 expression and localization in epithelial cells in allergic gastrointestinal tissue. (a) Representative control (CTL) and eosinophilic gastritis (EG) patient gastric biopsy specimen (200X) stained with anti-CDH26 antibody or control IgG. (b) CTL and EG biopsy specimen (800X) stained with anti-CDH26 antibody. (c) Left: Gastric antrum protein lysates were subjected to SDS-PAGE and western blot analysis. Right: The ratio of CDH26 to beta-actin signal was graphed. (d) Immunohistochemical staining for CDH26 was performed on esophageal biopsy specimens (n = 7 CTL, n = 3 EoE). Representative CTL and EoE biopsies are shown (200X, 800X inset). (e) Left: Esophageal protein lysates (n = 4 CTL, n = 4 EoE) were subjected to SDS-PAGE and western blot analysis. Right: The ratio of CDH26 to beta-actin signal was graphed. For (c) and (e), data were analyzed by unpaired t test. For (a), (b), and (d), the location of the lumen is denoted to facilitate interpretation of the orientation of the tissue section.
Figure 3
Figure 3
Biochemical and functional properties of CDH26. (a) Schematic representing human CDH26 domain structure. S: signal peptide, EC1-EC5: extracellular cadherin repeat 1–5, T: transmembrane domain, JMD: juxtamembrane domain, C: CBD, catenin binding domain. (b) Surface biotinylation of TE-7 cells. Cell surface proteins were labeled with biotin and pulled down with streptavidin beads. Total cell lysates (input) and proteins bound to the streptavidin beads were subjected to SDS-PAGE and western blot analysis. Predicted CDH26 molecular weight: 92.4 kDa. (c) Immunoprecipitates from transiently transfected HEK 293T cells were treated with either peptide: N-glycosidase F (PNGase F) (+) or heat-inactivated PNGase F (−). Inputs (1/10 of amount used for IP) and treated immunoprecipitates were subjected to SDS-PAGE and western blot analysis. Each blot shown is representative of three independent experiments. (dg) Immunoprecipitates from transiently transfected HEK 293T cells and inputs (1/10 of amount used for IP) were subjected to SDS-PAGE and western blot analysis. Each blot shown is representative of 3 independent experiments. (h) Transduced L929 cells were dispersed, incubated in buffer either containing or lacking 1 mM CaCl2, and assessed for the degree of aggregation. Data show 1 experiment representative of 3 and were analyzed by one-way ANOVA followed by Tukey post-test.
Figure 4
Figure 4
Binding of CDH26 to integrins. (a) Pairwise structure alignment of CDH26 with known integrin ligands. CDH26 structure was modeled (blue) and aligned to the resolved structures of MAdCAM-1 (PDB ID 1BQS), ICAM-1 (PDB ID 1IC1), CDH1 (PDB ID 1EDH), and fibronectin (PDB ID 1FNF) (gray). For each pair, integrin binding amino acids and the corresponding CDH26 residues are labeled (arrows) and rendered using a stick representation. (b) Transduced L929 cell clones were dispersed and added to wells coated with either BSA or recombinant α4β7. The percentage of adherent cells remaining after wells were washed is shown. The graph represents seven experiments combined that each involved separate control and CDH26-overexpressing clones. (c) Pictures of Giemsa-stained wells from b were taken (magnification = 4X), with 1 control and 1 CDH26-overexpressing clone shown. (d) Wells were coated with or without recombinant α4β7 and then blocked with BSA, followed by addition of either hIgG1 or CDH26-hIgG1-Fc (CDH26-Fc). Bound antibody or fusion protein was then detected and expressed as A450nm–A900nm. Each condition was performed in triplicate. This graph shows 1 experiment representative of 3. (eh) Inputs (1/10 of amount used for IP) and immunoprecipitates from transiently transfected HEK 293T cells were subjected to SDS-PAGE and western blot analysis. Each blot shown is representative of 3 independent experiments. (ij) Fluorescently labeled Jurkat cells (untreated [i] or incubated with TS2/16 integrin β1-activating antibodies [j]) pre-incubated with the indicated amount of either control mIgG1, anti-integrin α4 (HP2/1), or anti-CD32 antibodies were added to wells coated with control hIgG1, CDH26-hIgG1-Fc (CDH26-Fc), or CDH1-hIgG1-Fc (CDH1-Fc), as indicated. The graph indicates the percentage of fluorescence remaining after wells were washed. For (b), (d), (i), and (j), data were analyzed by one-way ANOVA followed by Tukey post-test.
Figure 5
Figure 5
Effect of CDH26-Fc and CDH1-Fc on CD4+ T cell activation. Human peripheral blood CD4+ T cells were isolated and cultured for 48 h in wells coated with the indicated amounts of proteins (IgG, anti-CD3, CDH26-Fc, and/or CDH1-Fc). Cells were stained for flow cytometry analysis to detect CD4 and CD25, and supernatants were analyzed to detect IL-2 levels by ELISA. For (a) and (c), the percent of live CD4+ cells that are CD25+ are shown, and for (b) and (d), the amount of IL-2 detected in the supernatant is shown. The dotted lines represent the detection limit for the ELISA. Data were analyzed by one-way ANOVA followed by Tukey post-test. Data are results from one subject representative of those from five individual subjects for (a) and (b), and from one subject representative of those from four individual subjects for (c) and (d).
Figure 6
Figure 6
Model of CDH26 expression and function in allergic inflammation. CDH26 is expressed by GI epithelial cells in allergic GI inflammatory conditions. CDH26 dimerizes, interacts with beta-, alpha-, and p120-catenins, and mediates calcium-dependent cell adhesion. CDH26 additionally interacts with integrin α4 (ITGA4) and integrin αE (ITGAE), which may impact leukocyte migration, localization, or activation status in allergic tissue.

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