MOLECULAR-CLONING OF THE MCP-3 CHEMOKINE GENE AND REGULATION OF ITS EXPRESSION

We have isolated a cDNA (NC28) transcribed from a mRNA which is transi ently induced in U937 promonocytic cells by PMA and super-induced by c ycloheximide. NC28 cDNA encodes a new member of the chemokine family, MCP-3, recently purified from MG-63 osteosarcoma cells by Van Damme et al. [1]. The MCP-3 protein sequence shows 74% identity with human mon ocyte chemoattractant protein 1 (MCP-1) and, like MCP-1, recombinant M CP-3 protein shows chemotactic activity for monocytes but not for neut rophils. However the secreted MCP-3 protein differs from MCP-1 in bein g N-glycosylated . The 3' non-coding regions of MCP-3 and MCP-1 mRNAs are more diverged (44 %), allowing specific cDNA probes to be made, an d indicating that the two genes are evolutionarily distant. Sequence c omparisons of the 3' non-coding regions suggest that MCP-3 may be the human homologue of the mouse MARC gene [2], and that MCP-1 and MCP-3 g enes arose by a gene duplication event before the mammalian radiation. Both MCP-1 and MCP-3 mRNAs are expressed by PBMC, principally by mono cytes, with MCP-1 mRNA being expressed at levels 2 - 4 times that of M CP-3 mRNA . However, while MCP-1 mRNA is also expressed at high levels in fibroblast or astrocytoma cell lines after IL-1 and TNF stimulatio n , MCP-3 mRNA is expressed only at very low levels in these cells. Th e cellular origin of MCP-3 is thus more restricted than that of MCP-1. In our experiments on PBMC, LPS is not a consistent inducer of MCP-1 and MCP-3 mRNAs. In some experiments, it actually decreases levels of these two mRNAs, while concomitantly increasing IL-6 and TNF-alpha mRN A levels. Levels of MCP-1 and MCP-3 mRNAs in PBMC are both increased b y IFN-gamma, although IL-6 mRNA is not induced. They are also increase d by PHA-P and are decreased, in most cases, by IL-13 [3]. MCP-1 and M CP-3 mRNAs are thus co-ordinately regulated in monocytes in response t o a number of inducing or inhibitory agents, in a manner differing in several respects from that of other monokines such as IL-6.