Ketotifen is a microglial stabilizer by inhibiting secretory vesicle acidification.

AIMS: Microglia survey the brain environment by sensing alarm signals to provide the first line of defense against injury or infection after which they acquire an activated phenotype, but they also respond to chemical signals sent from brain mast cells, sentinels of the immune system, when these are degranulated in response to noxious agents. Nevertheless, excessive microglia activation damages the surrounding healthy neural tissue causing progressive loss of neurons and inducing chronic inflammation. Thus, it would be of intense interest the development and application of agents which prevent mast cell mediator release and inhibit the actions of such mediators once released on microglia. MAIN METHODS: Fluorescence measurements of fura-2 and quinacrine were used to measure intracellular Ca2+ signaling and exocytotic vesicle fusion in resting and activated microglia. KEY FINDINGS: We show that treatment of microglia with a cocktail of mast cell mediators induces microglia activation, phagocytosis, and exocytosis, and reveal by the first-time microglia undergo a phase of vesicular acidification just before the exocytotic fusion occurs. This acidification is an important process for vesicular maturation and contributes with ∼25 % to the content that the vesicle can store and later release by exocytosis. Pre-incubation with ketotifen, a mast cell stabilizer and H1R antagonist completely abolished histamine-mediated calcium signaling and acidification of microglial organelles, and concomitantly reduced the discharge of vesicle contents. SIGNIFICANCE: These results highlight a key role for vesicle acidification in microglial physiology and provide a potential therapeutic target for diseases related to mast cell and microglia-mediated neuroinflammation.

Mast Cell Changes the Phenotype of Microglia via Histamine and ATP.

BACKGROUND/AIMS: Microglia are the dynamic motile phagocytes of the brain considered the first line of defense against threats or disturbances to the Central Nervous System (CNS). Microglia help orchestrate the immunological response by interacting with others immune cells. Mast cells (MCs) are effector cells of the innate immune system distributed in all organs and vascularized tissues, brain included. Several molecular mechanisms for potential interactions between MCs and microglia have been determined. However, the effect of MCs on regulated exocytosis and phagocytic clearance in microglia has not been explored. METHODS: Cocktails of MCs mediators (MCM) obtained at 37°C and 53°C were used to induce microglia activation. Changes in intracellular calcium [Ca2+]i and ATP release were studied by calcium and quinacrine fluorescence imaging. Fluorescent latex beads were used to assay phagocytosis in microglia after MCM treatment and compared to that measured in the presence of histamine, ATP and lipopolysaccharide (LPS). Iba-1 expression and area were quantified by immunofluorescence and histamine levels evaluated by ELISA techniques. RESULTS: Local application onto microglia of the MC mediator cocktail elicited Ca2+ transients and exocytotic release associated with quinacrine dye de-staining. Ca2+ signals were mimicked by histamine and blocked by the H1 receptor (H1R) antagonist, cetirizine. Hydrolysis of ATP by apyrase also affected Ca2+ transients to a lesser extent. Iba-1 fluorescence, cell area and phagocytosis were enhanced by histamine through H1R. However, ATP prevented iba-1 expression and microglial phagocytosis. MCM showed combined effects of histamine and ATP, increasing the number of internalized microbeads per cell and area without raising iba1 expression. CONCLUSION: Our results highlight the relevance of MC-derived histamine and ATP in the modulation of secretory and phagocytic activities that would explain the heterogeneity of microglial responses in different pathological contexts.

Proteoglycans involved in bidirectional communication between mast cells and hippocampal neurons.

BACKGROUND: Mast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions. METHODS: Calcium imaging was used to simultaneously record changes in intracellular calcium [Ca2+]i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat. RESULTS: Bidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca2+]i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca2+]i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS). CONCLUSIONS: MC-hippocampal neuron interaction affects neuronal [Ca2+]i and exocytosis signaling through a NMDAR-dependent mechanism.