425 Activation of Murine Isolated Enteric Neurons by IgE-Antigen and Mediators Released From Mucosal Type Bone Marrow-Derived Mast Cells

Abstract

Background: Neuro-immune interaction has evolved as an integral part in the pathophysiology of gut and crosstalk between nerves andmast cells is a typical example of such interactions. We have already reported that CGRP-immunoreactive intrinsic nerve fibers are specifically increased along with the development of food allergy in the colonic mucosa of our food allergy model mice. Furthermore, our previous studies have provided vivid evidences of mucosal mast cells (MMCs) juxtaposed with CGRP-positive enteric nerve fibers in the colonic mucosa of food allergy model mice. IgE-antigen stimulation has not only documented to cause degranulation in mast cells but also, recently, reported to cause activation of neurons in superior cervical ganglion. However, no study so far employed isolated enteric neurons and MMCs to elaborate this interaction which closely mimics to the pathophysiological state of the gut in the allergic condition. In the present study, we cultured both isolated enteric neurons and MMCs and examine their interaction. Material & methods: Enteric neurons were isolated from small intestine of BALB/c (4 to 6 weeks old) and examined by anti-β3tubulin antibody (anti-pan-neuronal marker antibody). Presence of high-affinity IgE receptors (FceRIs) on myentric neurons was examined in longitudinal muscle/myenteric plexus (LMMP) preparations by immunohistochemistry (IHC). Mucosal type bone marrow-derived mast cells (mBMMCs) were prepared from the femurs of BALB/c mice using four cytokines (SCF, IL-3, IL-9 and TGF-β1) and degranulation was performed by incubation with IgEantigen (IgE-DNP) to give mast cell juice (MCJ). Isolated enteric neurons were stimulated with either IgE-DNP or MCJ and analyzed by calcium imaging using fluo-8, a fluorescent calcium indicator. Finally, isolated enteric neurons were co-incubated with IgE-pretreated mBMMCs followed by stimulation with DNP. Only isolated enteric neurons were loaded with Fluo-8. Results: IHC studies revealed the presence of FceRI on the cell body of myentric neurons. Stimulation of isolated enteric neurons with IgE-DNP demonstrated an increase in intracellular calcium concentration ([Ca2+]i). Similarly, treatment of isolated enteric neurons with MCJ also led to an increase in [Ca2+]i. Furthermore, stimulation with DNP in an isolated enteric neuron/IgE-pretreated mBMMC co-culture condition produced an elevation of [Ca2+]i in isolated enteric neurons. Conclusion: We demonstrated here for the first time the activation of isolated enteric neurons by IgE-antigen, MCJ, and activated mBMMCs via calcium imaging. Therefore, we infer both direct and indirect involvement of crosstalk between enteric neurons and mucosal mast cells in allergic and inflammatory/ functional diseases of gut like food allergy or irritable bowel syndrome.