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. Differential screening of a cDNA library prepared from a mouse macrophage-like cell line led to the identification of 11 mRNA species, designated Mig and Crg-2 encode previously undescribed members of a newly-defined family of small secreted proteins, termed chemokines, that includes platelet factor 4, melanoma growth stimulatory activity (MGSA/gro) and IL-8 among others. Using the mouse MuMig cDNA probe, a new human member of the family has been discovered. Work to date has demonstrated that the MuMig and HuMig mRNAs accumulate in monocytic cells specifically in response to gamma interferon, while Crg-2/IP-10 can respond to alpha interferon and to lipopolysaccharide as well. Following intraperitoneal injection of gamma interferon into mice, MuMig and Crg-2 are induced in a range of tissues including most prominently liver, lung, spleen and heart. Antibodies have been raised in rabbits MuMig, HuMig and Crg-2 using proteins expressed in and purified from bacteria as antigens, Chinese hamster ovary cell lines were derived that overexpress the MuMig, HuMig, Crg-2 and IP-10 proteins. As predicted, each of the proteins is secreted. Using a Western blot assay, the MuMig and HuMig proteins have been purified to near-homogeneity. By PAGE, the Mig proteins show multiple species with mobilities corresponding to 16-20 kDa for MuMig and 12-35 kDa for Humig. Ongoing work is concentrating on a more detailed analysis of the cell types that produce the MuMig/HuMig and Crg-2/IP-10 proteins; on the biochemical characterization of the purified Mig proteins; on the identification and molecular cloning of the Mig receptor(s); on determining the biological activities of MuMig/HuMig and Crg-2/IP-10; on determining the biological activities of MuMig/HuMig and Crg-2/IP-10; and on identifying additional new members of the chemokine family.
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