The overall goal of the project is to identify and characterize macrophage products of potential importance in immune and inflammatory responses in order to manipulate these responses for clinical benefit. This laboratory identified mouse and human Mig and mouse Crg-2, previously undescribed members of a family of small secreted proteins, termed chemokines. Crg-2 is the murine analogue of the human chemokine IP-10. Mig and Crg-2/IP- 10 are inducible in macrophage and other cells by IFN- and target activated T cells and NK cells through the CXCR3 receptor. The chemokines are members of an expanding family of chemotactic factors, now numbering more than 30, that are thought to be critical for the trafficking of leukocytes in both homeostasis and in inflammatory reactions. Additionally, the chemokines have been found to be active as reversible inhibitors of hematopoiesis. Most recently, chemokines and their receptors have been found to be of central importance in HIV disease, since some chemokine receptors are used by HIV to enter cells and some chemokines are able to suppress HIV infection by inhibiting HIV entry. We have expanded this project beyond Mig and Crg-2/IP-10 to include studies of chemokines generally in their activities on lymphocytes and hematopoiesis, and studies of signalling by HIV envelope glycoproteins through chemokine receptors. In one part of this project, we have refined a flow cytometric assay for calcium fluxes that allows for simultaneous phenotypic analysis of responding cells, and we have used this assay to investigate responses of lymphocytes to a large collection of chemokines. We have correlated signalling with chemokine receptor expression using anti-receptor antibodies. We have investigated signalling and receptor expression on lymphocyte subsets for both freshly-isolated cells and cells activated in vitro, determining specific patterns that provide new information relevant to the roles of the chemokines and their receptors in lymphocyte biology and pathophysiology. We have applied the flow cytometer assay to studies of signalling by HIV envelope glycoproteins (Envs) through chemokine receptors, in collaboration with members of Anthony Fauci?s laboratory. Following our novel observations that Envs could produce a calcium signal through the chemokine receptors, we have been analyzing signalling and receptor usage by a variety of Envs derived from T cell line tropic and macrophage tropic HIV strains. There is evidence that different Envs can interact differently with the same chemokine receptor and that these interactions may effect the outcome of infection. We are interested in determining if signalling by the Envs correlates with and is related to the Env-dependent effects. In collaborative studies with Gretchen Schwartz and Ronald Gress of the NCI, we have investigated the roles of chemokines, particularly Mig and IP-10, on colony formation by hematopoetic progenitors. After demonstrating an inhibitory effect of the chemokines on the enhancement of CFU formation by insulin-like growth factor II, we are investigating mechanisms for the inhibition by analysis of chemokine signaling, and possible inhibition of growth factor signaling on hematopoietic cell lines and primary bone marrow-derived cells.
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