The overall objective of this grant proposal is to investigate how perivascular mast cells modulate microvascular endothelial interactions with circulating eosinophils. The ability of anti-IgE activated mast cells in human lung, nasa mucosa, or skin to express a 'TH2' cytokine profile (IL-4+, IL-5+, GM-CSF+, gammaIFN, IL-2) able to influence endothelial cell adhesion molecule expression (IL-4) and eosinophil function (IL-5, GM-CSF) will be assessed by combining in situ hybridization to identify cytokine mRNA with immunostaining (tryptase or chymase) to identify T or TC mast cells. In selected experiments, purified populations of mast cells will be in situ hybridized with both a non-radioactively labelled digoxigenin-11-UTP gammaIFN RNA probe and an S labelled IL-4 RNA probe to determine whether individual mast cells co-express IL-4 and/or gamma interferon. The ability of stimulated mast cell supernatant applied to mesenteric blood vessels to effect the rolling, adhesion and transmigration across endothelium of fluorescently labelled eosinophils in vivo in a rabbit mesentery model will be studied utilizing intravital video microscopy. The relative importance to eosinophil adhesion of individual mediators in mast cell supernatant will be studied using defined mast cell mediators (IL-4, histamine, LTC), as well as mast cell lysates, and supernatant studies in the presence of specific mediator and cytokine antagonists. Unidentified factors in stimulated mast cell supernatant (which induce eosinophil adhesion) will be purified to homogeneity to determine if they are novel cytokines. Eosinophils which have rolled, adhered and transmigrated in vivo will be recovered and studied by in situ hybridization for IL-5 and GM-CSF mRNA expression. In in vitro studies, peripheral blood eosinophils will be purified utilizing a MACS (magnetic cell separator) and separated according to density on Pecoll gradients. hypodense and normodense eosinophils will be stimulated with either the calcium ionophore A23187 or mast cell supernatant for varying time periods. Cytokine (IL-5, GM-CSF) mRNA expression by hypodense and normodenses eosinophils will be assessed by combining staining with carbol chromotrope 2R (to identify eosinophils) and in situ hybridization to identify cytokine mRNA. Supernatant will be assessed for LTC as well as GM-CSF and IL-5 protein using bioassays and an immunoassay. Overall, these studies will define whether mediators derived from perivascular mast cells communicate directly or indirectly (via activation of endothelium) with circulating eosinophils to influence their adhesion to influence their adhesion and state of activation.
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