The recruitment and activation of immune effector cells is essential for host defenses. The chemokine, MIP-1b, is a protein with proinflammatory and myelopoietic properties, and which is expressed in response to mitogenic stimulation of T cells and nonproliferative stimuli to other hematopoietic cells. MIP-1b is likely to have a unique role in immune and inflammatory responses, probably by enhancing neutrophil chemotaxis and the production of antimicrobial oxidants, and by mediating the chemotaxis and endothelial adherence of activated T cells. In addition, because MIP-1b antagonizes the stem cell inhibitory function of MIP-1a, a related chemokine, MIP-1b expression can promote hematopoiesis in conjunction with other cytokines. As a number of non-infectious, inflammatory diseases are associated with neutrophil and T cell infiltration into affected tissues and/or neutrophil hyperactivity, MIP-1b expression may be involved in the pathogenesis of such diseases, possibly as a result of an autoimmune or another aberrant stimulus to cytokine producing cells. Using a limited region of the hMIP-1b promoter, the signalling requirements of the endogenous gene can be recapitulated and localized primarily to two adjacent elements that act cooperatively to confer inducibility to promoter constructs. Each of these elements, a CRE and an NK-kB-like element (CLE- 1; CK-1), is found in a number of promoters and is known to mediate inducible expression in a wide array of cell types. In this proposal, we will determine the mechanism by which tissue specific inducible expression results from the recognition of these elements by a subset of proteins from those predicted by the sequences of the binding sites. We will also analyze the cooperativity which results from the configuration of these elements in the hMIP-1b promoter, and the contribution of additional regulatory elements. The proposed experimental system is based on the expression pattern of MIP- 1b by agents which initiate or perturb signal transduction pathways. Based on detailed mutational and deletion analysis, and in vitro binding assays, we will make predictions regarding the effect of transduced signals on individual regulatory proteins. These predictions will be tested in functional assays. After the recognition of individual elements by regulatory proteins has been defined, the importance of their spatial orientation in the MIP-1b promoter will be examined. This study will define mechanisms by which this cytokine is normally regulated - a necessary basis for further studies concerning the inappropriate expression of MIP-1b in autoimmune and other pathological states. It will also help clarify the level at which T cell specific and nonspecific and nonspecific regulatory sequelae are mediated. In addition to elucidating specific mechanisms underlying hMIP-1b expression, it is hoped that the conclusions reached in this study will address the question of combinatorial interactions between regulatory proteins 0 a fundamental issue in eukaryotic gene regulation.
