The chemokines are a large family of cytokines that control the recruitment of leukocytes in immune and inflammatory responses. cells transfected with CCR1, CCR3, or CCR5. Constitutive expression of MCP-5 mRNA was detected predominantly in lymph nodes, and its expression was markedly induced in macrophages activated in vitro and in vivo. Moreover, MCP-5 expression was upregulated in the lungs of mice following aerosolized antigen challenge of sensitized mice, and during the host response to contamination with These data indicate that MCP-5 is usually a novel and potent monocyte active chemokine that is involved in allergic inflammation and the host response to pathogens. The monocyte chemoattractant proteins (MCP)1 and eotaxin constitute an important subfamily of CC or -chemokines that share structural and functional features. Four human MCP proteins (-1, -2, -3, and -4) have been recognized that share 65% amino acid identity (1, 2). Of the four human MCP proteins recognized to date, only two have been TGX-221 distributor recognized in the mouse: JE (3, 4), the putative orthologue of human MCP-1, and MARC/FIC (5, 6), TGX-221 distributor the putative orthologue of human MCP-3. Human MCP-1, -2, -3, and -4 are all active on monocytes (2, 7, 8), T cells (8C 10), and basophils (2, 11, 12). In addition, human MCP-2, -3, and -4 chemoattract eosinophils (2, 8, 12, 13), and human MCP-3 is usually chemotactic for dendritic cells (14). Eotaxin, although highly related in sequence to the MCP proteins, is usually inactive on monocytes, basophils, and lymphocytes and is unique in that it specifically attracts eosinophils (15, 16). MCP-1, MCP-4, and eotaxin are similarly regulated in a variety of cells. For example, in epithelial and endothelial cells, MCP-1, MCP-4, and eotaxin are induced by TNF, IL-1, and IFN (1, 2, 15). IFN induces the secretion GU2 of MCP-2 from mononuclear cells and fibroblasts, and MARC/FIC is usually secreted from activated mast cells (1, 5). Other CC chemokines (e.g., RANTES and MIP-1/) are more distantly related in sequence to those of the MCPs and eotaxin, although they chemoattract the same spectrum of leukocyte subsets, with variable selectivity. Chemokines induce leukocyte migration and activation by binding to specific G proteinCcoupled seven transmembrane spanning cell surface receptors (17). There have been five human CC chemokine receptor (CCR) genes cloned, now being referred to as TGX-221 distributor CCR1 through CCR5. Each of these has an orthologue in the mouse. Human CCR2a and CCR2b are splice variants of the same gene. The chemokine and leukocyte selectivity of CCRs overlap extensively; a given leukocyte often expresses multiple chemokine receptors, and more than one chemokine typically binds to the same receptor. While chemokines often have overlapping activities in vitro, differences in the timing and location of chemokine production in vivo imply that the redundancy found in vitro may not be biologically relevant. This is supported by chemokine inactivation experiments conducted in animal models of contamination and inflammation, such as the targeted deletion of the macrophage inflammatory protein (MIP)- 1 gene (18). Despite the fact that all MIP-1 activities explained in vitro are shared by other -chemokines, including receptor usage, MIP-1Cdeficient mice do not mount a normal response to viral infections. To fully appreciate the role of chemokines in regulating inflammation, the entire spectrum of chemokines needs to be delineated and their functional role analyzed in the context of in vivo immune responses. In this report, we describe the cloning and functional characterization of a new member of the MCP subfamily of -chemokines, murine MCP-5. The data described below provide evidence that this novel chemokine is usually a TGX-221 distributor potent monocyte chemotactic factor that signals through CCR2. Further, we demonstrate that MCP-5 is usually a product of activated macrophages, and its expression is increased in murine models of pulmonary inflammation. Materials and Methods Isolation of the Murine MCP-5 Gene. The human MCP-4 cDNA (2) was 32P labeled and used as a probe to screen a 129SV mouse genomic library (Stratagene Inc., La Jolla, CA). Approximately 106 phages were plated, transferred to GeneScreen Plus (Dupont(Nb) model was performed as explained (23). Briefly, 12-wk-old female BALB/cJ mice were injected with 750 third stage Nb larvae and the lungs harvested at 7, 10, and 14 days for RNA extraction. Purification of Monocytes, Macrophages, Eosinophils, and Neutrophils. PBMC were obtained from 4 normal donors by density gradient centrifugation using 1.077 Histopaque (by PeproTech as the predicted mature 82C amino acid protein beginning with the NH2-terminal glycine. NH2-terminal sequence analysis of the purified recombinant MCP-5 preparation confirmed its homogeneity and the NH2terminal glycine. Cells were incubated at 37C for 30 (neutrophils), 60 (eosinophils), or 90 min.