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 two mRNA species, designated Mig and Crg-2 that encode previously undescribed members of a newly-defined family of small secreted proteins, termed chemokines. Crg-2 is likely the murine homologue of the human chemokine IP-10. Using the mouse MuMig cDNA probe, HuMig a new human member of the family was discovered. Our mapping studies have placed the human mig and IP- 10 genes to within 14 kilobases of each other on chromosome 4q 21.21 at a site distant from the cluster of other chemokine genes. 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. By PAGE, the Mig proteins show multiple species with mobilities corresponding to 16-20 kDa for MuMig and 9.5-14.3 kDa for HuMig. HuMig was purified from transfected CHO cells. N-terminal sequencing and mass spectrometry have revealed that HuMig's heterogeneity is due to carboxy-terminal truncations. Functional studies have revealed that HuMig targets activated T cells, causing a rise in intracellular calcium and chemotaxis. Resting T cells, neutrophils, monocytes, and EBV-transformed B cells do not respond to HuMig. In addition, we found that HuMig has suppressive effects on the production of CFU-GM by CD34+ cells. As part of our studies of the actions of chemokines on lymphocytes, we have begun to use multi-parameter flow cytometry to identify lymphocyte subsets that flux calcium in response to specific chemokines. These studies have demonostrated overlapping but different responses among lymphocyte subsets to different chemokines.
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