Immunologic responses to infection depend on adhesive interactions between leukocytes, endothelium, and platelets. However, in some situations, leukocyte behavior results in damage to tissues, particularly following ischemia; and has also been shown to play a role in adult respiratory distress syndrome and septic shock associated multi.organ failure. Consequently, inhibition of leukocyte adhesion to the vessel wall has been proposed as a means to limit tissue damage due to vascular injury and ischemia. Therefore, a mechanistic understanding of the factors that regulate leukocyte adhesion to the vessel wall and emigration into tissue is important-to define optimal targets for therapeutic intervention strategies. It has recently become appreciated that vascular hemodynamics exert a profound effect on leukocyte adhesion to the endothelial lining of the blood vessel wall. A recently described family of leukocyte adhesion receptors, the selectins, appear to mediate the distinctive rolling of neutrophils on the blood vessel wall that marks the early stages of acute inflammation. Neutrophil adhesion to endothelium apparently proceeds by a sequential interaction of selectins with their ligands followed by binding of CD18 integrins to their endothelial cell ligands, ultimately leading to arrest and emigration of the rolling neutrophils. A number of elements of the sequential rolling and arrest model for neutrophils are presently not understood. In particular, it is unclear how adhesion receptor expression and cellular regulation of binding to specific ligands cooperate in the presence of shear flow to direct neutrophils to sites of inflammation. For instance, neutrophils roll whenever a selectin is expressed on endothelium, but arrest and transendothelial migration occurs only alter a signal is received by the neutrophil to activate CD 18 adhesiveness. How neutrophils are signaled to arrest once they have begun rolling on the vessel wall is unknown. One function of selectins may be to bring neutrophils into contact with chemoattractants bound to the endothelium, which then activate CD18 adhesiveness. Near the vessel wall soluble chemoattractants would face significant dilution effects at physiologic flowrates, which would limit their effIcacy. It is also possible that selectin ligation may by itself activate CD18 integrin adhesiveness and thereby arrest rolling neutrophils. Since endothelial cell selectins may bind to more than one type of ligand on the neutrophil surface depending on the level of shear forces, understanding of the signaling role of selectin ligation would depend on defining the specific structures mediating discrete stages of attachment and rolling. It is also not understood how or if the sequential model of selectin and integrin interactions applies to neutrophil adhesion to platelets, which express high levels of P-selectin and may be important for recruiting neutrophils to sites of vascular damage. The investigators propose to test the sequential model of neutrophil adhesion in flow using a parallel plate chamber which generates a well defined shear field. Neutrophils will be perfused at defined shear rates over substrates containing purified selectins and CD18 integrin ligands co-immobilized with chemoattractants. Using biochemically purified adhesion receptors allows specific components of highly complex cell/cell interactions to be studied in Isolation. Neutrophil interactions with cultured endothelium and platelet monolayers in flow will be studied to extend and test hypotheses generated in the reconstitution assay.
