Adherence of circulating eosinophils to the vascular endothelium and their accumulation at extravascular sites in inflamed tissues is a hallmark of allergic inflammation. However, the molecular mechanisms mediating eosinophil adhesion under conditions of flow in vivo are not well understood. Recent studies on neutrophils have shown that intravascular adhesion of these cells is a multi-step process involving distinct adhesion and activation steps. Adhesion molecules include members of the selectin family and beta2 integrins which promote neutrophil rolling and subsequent extravasation. We have recently observed that eosinophils from atopic patients similarly roll and adhere to IL-1 stimulated endothelial cells at shear rates. We have found that a beta1 integrin, VLA-4, in addition to L-selectin promotes early events of human eosinophil adhesion to endothelial cells in vivo.
The aim of the proposed research is to elucidate the functional role of adhesion receptors in mediating human eosinophil interaction with vascular adhesion molecules in vivo. Using intravital microscopy we will (1) examine the role of L-selectin and VLA-4 integrin in promoting human eosinophil rolling along IL-1 or IL-4 stimulated endothelial cells in vivo. Using anti-VLA-4 and L-selectin antibodies and cell lines transfected with cDNA for human VLA-4 we will determine if both receptors function sequentially or in parallel to promote eosinophil rolling; (2) determine the effects of eosinophil-active cytokines, IL-5 and GM-CSF, on receptor activation and in vivo adhesion of circulating eosinophils (3) examine the effect of chemokines, RANTES, GM- CSF and PAF on receptor mediated firm adhesion (sticking) and subsequent extravasation of normodense and hypodense subpopulations across cytokine stimulated mesenteric EC in vivo and (4) determine the role of histamine, GM-CSF, IL-1, IL-4 and IL-5 on expression of inducible vascular endothelial surface ligands and their role in promoting rolling, sticking and transendothelial migration of activated eosinophils at physiological shear rates in vivo. The surface expression of vascular adhesion molecules in the mesentery under conditions of flow will be correlated with the in vivo function of human eosinophils. These proposed studies are intended to give a better understanding of molecular mechanisms mediating cell adhesion and provide a basis for examining therapeutic strategies for the treatment of allergic inflammation.
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