Our broad long-term objective is to understand the role of leukocyte integrins in inflammatory and immune responses. According to a widely accepted multistep model, chemokines displayed on endothelium bind to G protein-coupled receptors on leukocytes and very rapidly trigger dramatic increases in integrin-mediated adhesion to endothelium. This important phenomenon is poorly understood and has been difficult to model in vitro. We recently made a major advance by implementing a flow chamber system that reproduces the key features of chemokine-induced intravascular integrin activation. We completely revised the previous application to focus on using the system to study this phenomenon at the molecular level. In preliminary studies, we analyzed the effects of the CCR1 and CCR2B chemokine receptors on integrin-mediated adhesion under flow and chemotaxis. Chemokines stimulated adhesion and chemotaxis in CCR1 transfectants. In contrast, CCR2B transfectants did not increase adhesion in response to appropriate chemokines, even though chemokines induced robust chemotactic responses. This proposal has three specific aims.
Aim 1 is to use chemokine receptor-transfected leukocytes to compare the effects of CCR1 -CCR5, CXCR1, and CXCR2 on integrin- mediated adhesion. Our preliminary studies indicate that there will be important functional differences between chemokine receptors, and we aim to precisely define these differences.
Aim 2 is to analyze the basis for differences in the ability of CCR1 and CCR2B to stimulate adhesion. We will compare the ability of these two receptors to activate specific signaling pathways, and use CCR1/CCR2B chimeras to identify structural features that account for the functional differences.
Aim 3 is to analyze the roles of heterotrimeric G proteins and the monomeric G protein Rac in chemokine-induced integrin activation. Preliminary evidence points to key roles for these G proteins, and a variety of approaches will be used to define these roles. These studies should produce a detailed understanding of how chemokine-induced integrin activation contributes to the selective recruitment of specific types of leukocytes during inflammatory and immune responses.
