Cell adhesion is required for the normal function of the vertebrate immune system. The leukocyte, or beta2, integrins (LFA-1, Mac-1, P150,95 and alphad/CD18) participate in nearly all elements of immunity from phagocytosis of opsonized particles, T cell help, T cell and NK cell cytotoxicity to transendothelial migration at sites of inflammation and lymphocyte recirculation. Leukocyte adhesion deficiency (LAD), a clinical deficiency in beta2 integrins, results in severe infections in early childhood. On the other hand, beta2 integrins are also implicated in many autoimmune disease states, including rejection of organ transplants and rheumatoid arthritis (RA). Antibodies that block the interaction of beta2 integrins with their counterreceptors have allowed long term survival of organ transplants and have induced clinical remissions in patients with otherwise refractory RA. The beta2 integrins are constitutively in a low avidity state, and must be activated in order to bind efficiently their counterreceptors. Activators include antibodies to other cell surface structures such as CD3 on T cells, or chemotactic factors or phorbol esters, and result in a conformational change in the beta2 integrins. The signalling pathways by which this activation occurs are poorly understood.
The aims of this proposal are to employ established techniques of mutagenesis and selection to obtain panels of T and B lymphoblastoid cell lines that are deficient in beta2 integrin activation or de-activation. Lymphocytes were selected for study because they play a central role in the pathogenesis of RA, they express only LFA-I which simplifies the system, and activating signals are well-defined. Functional and structural analysis of the mutants will be undertaken to identify those cells that have defects in the signal transduction pathway for integrin activation. Complementation analysis by cell fusion and functional assays will allow grouping of the mutant cells into distinct classes. Analysis of these classes of mutants with known immunological and pharmacological stimulators of activation will reveal the order of the intracellular steps required for integrin activation. These mutant cell lines will be invaluable reagents for future studies which would include transfection of DNA libraries, to allow the molecular cloning of novel elements of the regulatory pathway. A greater understanding of the regulation of integrin activation may enable the selective pharmacological blockade of those beta2 integrin functions that contribute to autoimmune disease, without interfering with integrin roles in host defense.
Showing the most recent 10 out of 242 publications
