Piecemeal degranulation (PMD) of basophils and mast cells plays a central role in the secretion of histamine which is effected by the transport of histamine-loaded vesicles from secretory granules to plasma membrane. In contrast to anaphylactic degranulation, which represents the central cellular event in acute disorders of immediate hypersensitivity, such as anaphylaxis, PMD permits the slow and/or sustained release of mediators. Accordingly, PMD may contribute to the persistence of allergic inflammation associated with IgE-dependent reactions as well as to the sustained inflammation associated with asthma and chronic allergic disorders. Basophils and mast cells that have emptied their granules by PMD also replenish these containers in the absence of expansion of traditional synthetic organelles, such as Golgi structures and rough endoplasmic reticulum. The mechanism(s) for this renewal which serves to extend the functional capabilities of these cells in allergic inflammation is (are) currently unknown. Augmented vascular permeability accompanies allergic inflammation in settings characterized by PMD of basophils and mast cells. A new endothelial cell permeability organelle, the vesiculo-vacuolar organelle (VVO) is the major trans-endothelial cell route by which macromolecules leak from venules exposed to basophil and mast cell mediators. Cellular diapedesis (of neutrophils) in inflammation similarly takes a transcellular route. We propose experiments which rely heavily on sophisticated ultranstructural analyses to test hypotheses generated by our discovery that PMD of basophils and mast cells is associated with histamine secretion in small vesicles and recovery of histamine in granules and that basophil and mast cell mediators (histamine, serotonin, VPF/VEGF) enhance vascular permeability through VVO. Specifically, we will: 1. assess the spatial distribution of RNA in mast cells and basophils; 2. assess the spatial distribution of specific mRNA (TNF-alpha,b-FGF, SCF, IL-4, VPF/VEGF) in human mast cells and basophils undergoing secretion and recovery; 3. assess the formation and function of endothelial cell VVO in allergic inflammation; 4. define the pathway(s) of endothelial cell diapedesis of basophils and eosinophils.
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