ROLE OF BETA-2 INTEGRINS IN LEUKOCYTE ADHESION AND SIGNALING

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The attraction of leukocytes to a site of inflammation is an essential homeostatic response. Inappropriate attraction and activation of myeloid leukocytes, however, is a mechanism underlying a variety of diseases characterized by marked inflammation. Precise regulation of the recruitment process is necessary both to respond appropriately and to prevent excessive tissue damage. Many of the cellular and molecular events involved in this response have been described. The Beta2 integrins are a family of adhesion proteins on the surface of PMNs and macrophages that are crucial in supporting a normal inflammatory response. For example, patients with an inherited deficiency of these proteins suffer recurrent life-strengthening infections and have poor wound healing. Conversely, antibodies that block the adhesive function of Beta2 integrins ameliorate the tissue damage in some animal models of inflammation. The Beta2 integrins are dimers comprised of a common Beta chain and one of three alpha chains. Although their primary structures are known and many functional responses have been characterized, it is not clear whether each of the heterodimers makes unique contributions to adhesion and signaling. We propose to make permanent cell lines from HL60 cells, a promyelocytic leukemia line that can be differentiated in vitro to PMN- or macrophage- like cells. Each line will have a deficiency of one subunit (i.e. the Beta, or one of the alpha's) created by targeted gene disruption through homologous recombination. We will create homozygous lines and use them to determine the contribution of each individual heterodimer to adhesion to candidate counter-receptors on other cells and in extracellular matrix. We will use the same cells to assess the role of each integrin in ligand-induced responses including changes in intra cellular calcium concentration, the oxidant burst, synthesis of lipid mediators, and gene expression. The proposed studies will dissect the functions of the Beta2 integrins at the molecular level, and will provide permanent reagents for studies of leukocyte function, integrins, and inflammation.