Stimulated human monocytes release several proteins thought to play a role in inflammation, including interleukin 1, tumor necrosis factor, and plasminogen activator. We have purified another proinflammatory protein that is chemotactic for human neutrophils from conditioned medium of lipopolysaccharide-stimulated monocytes. After a series of steps that included anion-exchange chromatography, gel filtration, and HPLC on cation-exchange and reverse-phase columns, an apparently pure protein was obtained that migrated as a single 7-kDa band on NaDodSO4/polyacrylamide gels under reducing or nonreducing conditions. The amino acid composition of this monocyte-derived neutrophil chemotactic factor was different from that of interleukin 1 and tumor necrosis factor. N-terminal amino acid sequence of the first 42 residues was determined. This portion of the molecule has up to 56% sequence similarity with several proteins that may be involved in host responses to infection or tissue inJury. It is identical to a portion of a sequence deduced from an mRNA induced by staphylococcal enterotoxin treatment of human leukocytes. At the optimal concentration of 10 nM, 50% of neutrophils added to chemotaxis assay wells migrated toward the pure attractant. Potency and efficacy are comparable to that of fMet-Leu-Phe, which is often used as a reference. In contrast to many attractants, the protein was not chemotactic for human monocytes.Accumulation of leukocytes at sites of inflammation is the result of many steps that include vasodilation, leukocyte adherence to vascular endothelium, and directed migration caused by elaboration of chemotactic factors at the initiating locus. The type of leukocyte in the inflammatory infiltrate differs according to the nature of the stimulus and the temporal stage of the response. Therefore, characteristics of a proinflammatory chemoattractant should include (i) production and release in response to the inflammatory stimulus and (ii) at least in some cases the capacity to attract specific types of leukocytes.It was reported that interleukin 1 (IL-1), which is produced by monocytes in response to the inflammatory agent lipopolysaccharide (LPS), is chemotactic for human neutrophils and monocytes (1, 2). Thus, IL-1 appeared to fulfill the first of the above requirements for a proinflammatory attractant. However, we showed that highly purified or recombinant IL-1 has no chemotactic activity for neutrophils and that the neutrophil chemotactic activity in culture fluids of LPSstimulated human monocytes could be separated from IL-1.The activity is due to a basic protein with a molecular mass of about 10 kDa determined by gel filtration (3). We now report the purification to homogeneity of this monocyte-derived neutrophil chemotactic factor (MDNCF), present evidence that the molecule is distinct from monocyte IL-1 and tumor necrosis factor (TNF), and show that it has peptide sequence similarity to several other host-defense cytokines. MDNCF is potentially a mediator of a leukocytespecific inflammatory ...

The recruitment of monocyte-macrophages into the artery wall is one of the earliest events in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte chemoattractant secreted by many cells in vitro, including vascular smooth muscle and endothelial cells. To test whether it is expressed in the artery in vivo, we used Northern blot analysis, in situ hybridization, and immunocytochemistry to study the expression of MCP-1 in normal and atherosclerotic human and rabbit arteries. Northern blot analysis showed that MCP-1 mRNA could be isolated from rabbit atherosclerotic lesions but not from the intima media of normal animals. Furthermore, MCP-1 mRNA was extracted from macrophage-derived foam cells isolated from arterial lesions of ballooned cholesterol-fed rabbits, whereas alveolar macrophages isolated simultaneously from the same rabbits did not express MCP-1 mRNA. MCP-1 mRNA was detected by in situ hybridization in macrophage-rich regions of both human and rabbit atherosclerotic lesions. No MCP-1 mRNA was found in sublesional medial smooth muscle cells or in normal arteries. By using immunocytochemistry, protein was demonstrated in human lesions, again only in macrophage-rich regions. Immunostaining of the serial sections with an antiserum against malondialdehyde-modified low density lipoprotein indicated the presence of oxidized low density lipoprotein and/or other oxidation-specific lipidprotein adducts in the same areas that contained macrophages and MCP-1. We conclude that (a) MCP-1 is strongly expressed in a small subset of cells in macrophage-rich regions of human and rabbit atherosclerotic lesions and (ii) MCP-1 may, therefore, play an important role in the ongoing recruitment of monocyte-macrophages into developing lesions in vivo.The earliest grossly visible atherosclerotic lesion is the fatty streak, characterized by the accumulation of lipid-loaded foam cells in the subendothelial space (1). Many of these foam cells are derived from circulating monocytes (2-4) that have penetrated into the subendothelial space and presumably taken up excess native and/or oxidized low density lipoprotein (LDL) (5-8). Thus, one of the important early events in the pathogenesis of atherosclerosis is the adherence of monocytes to the endothelium, followed by their migration into the subendothelial space (1, 3). The entry between endothelial cells presumably is in response to a gradient of one or more chemotactic factors. Several monocyte chemotactic factors have been described-produced by endothelial cells, by smooth muscle cells, or by macrophages (9-14)-but there is almost no information on which of these are important in vivo. Chemotactic activity may also be derived from the extracellular components of the artery wall. For example, proteolytic peptide fragments from several connective tissue matrix proteins are chemotactic for leukocytes (15)(16)(17). In addition, in vitro-oxidized LDL is chemotactic for circulating monocytes (18) and oxidatively modified LDL isolated ...

Activated monocytes produce a variety of cytokines that are involved in inflammation, such as IL-1, TNF, chemotactic factors, transforming growth factor R, platelet-derived growth factor, and IFN-a and -Q (1). Chemotactic factors released at foci of injury or bacterial invasion are thought to mediate directed migration of leukocytes into inflammatory sites. Since the leukocyte composition of the inflammatory infiltrate depends on the temporal stage of the lesion (2) and the nature of the stimulus (3), it follows that some chemoattractants should be specific for a given type of leukocyte. We recently showed that LPS-stimulated monocytes produce a chemotactic factor that attracts neutrophils, but not monocytes (4). We purified this factor to homogeneity and described the N1-12-terminal sequence of the first 42 amino acids (5). We now report molecular cloning and sequencing ofthe full-length cDNA for this monocyte-derived neutrophil chemotactic factor (MDNCF)' and the deduced amino acid sequence of the entire molecule. Specific cDNA probes also enabled us to test the capacity of a number of cytokines to induce MDNCF mRNA expression in human PBMC. The stimulation of MDNCF mRNA expression by IL-1 and TNF suggests that the local pro-inflammatory action of these cytokines may be mediated by induction of chemotactic factor secretion. Volume 167 June 1988 1883-1893 Materials and Methods cDNA Cloning of MDNCF and Nucleotide Sequence . Normal human PBMC were first fractionated by Ficoll-Hypaque and plastic adherent cells (> 90% nonspecific esterase-positive monocytes), were cultured in RPMI-1640 medium supplemented with 1% FCS and 10 ug/ml LPS (Serotype 055:1155; Difco Laboratories Inc., Detroit, MI) for 6 h at 37°C . Total RNA

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The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-l (MCP-I), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-l, but not by IL-2, TNF, or IFN-y. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggests that MCP-1 is the human homologue of the mouse competence gene JE.