Using Mast Cells To Probe Regulation Of Plasma Membrane Heterogeneity, Membrane Trafficking And Host-Pathogen Interactions

Abstract

Cells of the immune system have many unique functions, but underlying similarities in signaling mechanisms and pathways make a well-defined model system useful. We utilize RBL mast cells to probe a variety of questions pertinent to immune cell function and infection. Mast cells serve as innate immune responders as well as the mediators of allergic disease. A major signaling cascade in mast cells is initiated by multivalent antigen crosslinking of immunoglobulin E (IgE)-Fc[epsilon]RI receptor complexes. Membrane reorganization and signal transduction involving multiple players leads to Ca2+ mobilization and protein kinase C activation that ultimately triggers the release of preformed mediators from secretory lysosomes and, additionally, to outward trafficking of recycling endosomes (RE). The first study investigates the dynamic lipid environment surrounding crosslinked IgE-Fc[epsilon]RI complexes. By isolating detergent-resistant membranes and performing detailed mass spectrometry analysis, cytoskeletal interactions are found to be important for regulating membrane lipid reorganization resulting from antigen stimulation. Next, we characterized the molecular basis of sphingosine inhibition of cell function. We find that sphingosine derivatives electrostatically interfere with phosphoinositide-dependent membrane processes, including both Ca2+ mobilization and exocytic processes such as degranulation and RE outward trafficking. The function and regulation of RE trafficking in immune cells, especially as it pertains to cytokine secretion, is of great interest. We provide evidence, by immunofluorescence microscopy and cytokine secretion assays, that mast cells secrete both IL-4 and TNF[alpha] in a manner that is consistent with a role for RE in their secretion. The dynamic trafficking of RE in RBL mast cells makes them likely candidates in many processes, including interactions with pathogens. We provide strong evidence for a role for RE in the formation of the parasitophorous vacuole (PV) of the obligate intracellular parasite, Toxoplasma gondii. In addition, we find that Toxoplasma gondii infection of mast cells results in suppression of IgE-Fc[epsilon]RI signaling by interfering with the activation of phospholipase C[gamma]. Colllectively, these studies show RBL mast cells as a versatile model of cellular immune function in both health and disease.