ABSTRACT
The Notch-1 signaling pathway is responsible for homeostatic tight junction expression in vitro, and promotes barrier function in vivo in the RAG1-adoptive transfer model of colitis. In this study, we sought to determine the role of colonic Notch-1 in the lymphoepithelial crosstalk in health and disease. We utilized in vivo and in vitro knockdown to target the expression of Notch-1. We identified that epithelial Notch-1 is required for appropriate activation of intestinal epithelial cells at steady state and upon inflammatory stimulus. Notch-1 expression modulates mucosal chemokine and cytokine secretion, and FoxP3 and effector T-cell responses. We showed that epithelial Notch-1 controls the immune function of the epithelium through crosstalk with the nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathways that, in turn, elicits T-cell responses. Overall, epithelial Notch-1 bridges innate and adaptive immunity in the gut. Our findings highlight an indispensable role for Notch-1-mediated signaling in the intricate epithelial-immune crosstalk, and validate that epithelial Notch-1 is necessary and sufficient to support protective epithelial proinflammatory responses.
Subject(s)
Immunity, Mucosal/physiology , Mucous Membrane/immunology , Mucous Membrane/metabolism , Receptor, Notch1/metabolism , Animals , Cell Line , Chemokines/genetics , Colitis/genetics , Colitis/immunology , Colitis/metabolism , Colon/immunology , Colon/metabolism , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Inflammation Mediators/metabolism , Mice , Mucous Membrane/pathology , Severity of Illness Index , Signal Transduction , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Tight Junction Proteins/genetics , Tight Junction Proteins/metabolismABSTRACT
Normal intestinal epithelial cells (IECs) could act as non-professional antigen-presenting cells, selectively activating CD8(+)-suppressor T cells. An epithelial cell surface glycoprotein, gp180, recognized by monoclonal antibodies B9 and L12 was determined to be critical in this process. Purification and sequence analysis of mAb B9 reactive material revealed amino-acid sequence homology with CEACAM5. We demonstrate that CEACAM5 has properties attributed to gp180, such as CD8α binding and activation of CD8-associated Lck. CEACAM5 is the only CEACAM member interacting with CD1d through the B3 domain. Its N domain (recognized by B9) is required for CD8α binding. Removal of the N-domain glycosylated residues reduces B9 recognition, CD8α binding affinity, and activation of LcK. Therefore, conformational changes in CEACAM5 glycosylation site are critical for its interaction with CD8α. CEACAM5-activated CD8(+) T cells acquire the ability to suppress the proliferation of CD4(+) T cells in vitro in the presence of interleukin (IL)-15 or IL-7. We provide new insights into the role of CEACAM5 and define its specific immunoregulatory properties among the CEACAMs expressed on IECs. We suggest that unique set of interactions between CEACAM5, CD1d, and CD8 render CD1d more class I-like molecule, facilitating antigen presentation and activation of CD8(+)-suppressor regulatory T cells.
Subject(s)
Antigens, CD1d/metabolism , CD8 Antigens/metabolism , Carcinoembryonic Antigen/metabolism , Homeostasis , Intestinal Mucosa/metabolism , Intestines/immunology , Amino Acid Sequence , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Carcinoembryonic Antigen/chemistry , Carcinoembryonic Antigen/genetics , Cell Line , Epitopes/chemistry , Epitopes/immunology , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Glycosylation , Humans , Lymphocyte Activation/immunology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Models, Biological , Molecular Sequence Data , Multigene Family , Phosphorylation , Protein Interaction Domains and Motifs , Sequence AlignmentABSTRACT
BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.
