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Mast cells in the CNS participate in the pathophysiology of chronic neurodegenerative inflammatory diseases. This study aimed to investigate the signaling pathway of mast cells activated in an environment cocultured with astrocytes and to explore the role of their colocalization in brain of experimental allergic encephalomyelitis. Human mast cell line-1 cells and human U87 glioblastoma cell lines (U87) or mouse bone marrow-derived mast cells and mouse cerebral cortices-derived astrocytes were cocultured. Intracellular Ca(2+) was measured by confocal microscopy; histamine by fluorometric analyzer; leukotrienes by ELISA; small GTPases, protein kinase Cs, MAPK, c-kit, CD40, and CD40L by Western blot; NF-kappaB and AP-1 by EMSA; cytokines by RT-PCR; and colocalization of mast cells and astrocytes in brain by immunohistochemistry. Mast cells cocultured with astrocytes showed time-dependent increases in intracellular Ca(2+) levels, release of histamine and leukotrienes, and cytokine production. Mast cells or astrocytes showed enhanced surface expression of CD40L and CD40, respectively, during coculture. Mast cells cocultured with astrocytes induced small GTPases (Rac1/2, cdc42), protein kinase Cs, MAPK, NF-kappaB, and AP-1 activities. These changes were blocked by anti-CD40 Ab pretreatment or CD40 small interfering RNA. Mast cells increased in the thalamus of experimental allergic encephalomyelitis model, particularly colocalized with astrocytes in the thalamic border region of the habenula. In conclusion, the data suggest that activation of mast cells cocultured with astrocytes induces release of mediators by small GTPases/Ca(2+) influx through CD40-CD40L interactions to participate in the pathophysiology of chronic neurodegenerative inflammatory diseases, such as multiple sclerosis.

the journal of immunology/the journal of immunology

Signaling Pathways in the Activation of Mast Cells Cocultured with Astrocytes and Colocalization of Both Cells in Experimental Allergic Encephalomyelitis