After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2-to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by the potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-1O5MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of [35Slmethi-onine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that (g) SMCF is in fact MCP-1 and (it) MCP-1 is induced by MM-LDL.An important early event in atherogenesis is an increased recruitment of monocytes into the arterial subendothelium (1)(2)(3)(4). Previous studies have shown that endothelial cells (EC) (5, 6) and smooth muscle cells (SMC) (7,8) in culture constitutively produce a chemotactic factor that acts on monocytes but not neutrophils. Graves and colleagues (9) demonstrated that the monocyte chemotactic activity in the supernatants from a number of tumor cell lines was inhibited by an antibody made against baboon smooth muscle cell chemotactic factor (anti-SMCF) (10). Additionally they demonstrated a strong concordance with immunoprecipitation of a protein with an apparent molecular mass of 14.4 kDa. A human glioma cell line, U-1OSMG, constitutively expresses monocyte chemotactic activity. The protein responsible for this activity has been purified, sequenced, and named monocyte chemotactic protein 1 (MCP-1) (11, 12). Graves and colleagues did not test their antibody against the chemotactic activity secreted by the U-1O5MG cell line, but based on limited primary sequence data from the baboon smooth muscle chemotactic protein, they concluded that their protein was homologous to MCP-1.We have recently demonstrated that low density lipoprotein (LDL) that has been minimally modified (MM-LDL) is indistinguishable from native LDL by the LDL receptor, is not recognized by the scavenger receptor, and induces EC to secrete high levels of monocyte chemotactic activity, whereas native LDL does not (13). We have also shown that MM-LDL induces EC to produce colony-stimulating factors, including monocyte colony-stimulating factor (M-CSF), w...

By Northern analysis, freshly isolated monocytes contained no detectable mRNA for monocyte chemotactic protein-1 (MCP-1). However, after 4 hours of incubation at 37°C, MCP-1 mRNA was clearly induced in the monocytes and was found to be highly dependent and directly proportional to the monocyte density. The level of MCP-1 mRNA continued to increase, reaching a peak after 22 hours of incubation. After 3 days in culture, MCP-1 mRNA levels had declined substantially and after 8 days were undetectable in the monocytes/macrophages. The amount of MCP-1 protein secreted correlated with the density-dependent increase in MCP-1 message. We hypothesize that the migration of monocytes into inflammatory lesions may be amplified by the density and time-dependent induction of MCP-1. (Arteriosclerosis and Thrombosis 1992;12:78-82) T he infiltration of circulating blood monocytes across vascular endothelium is an important early event in the inflammatory response that is common to a variety of injurious conditions, including infection, cancer, atherogenesis, and trauma. -8 This monocyte infiltration and the subsequent events that lead to the acute and chronic presence of inflammatory macrophages within infected or diseased tissue are probably regulated by a series of rapid environmental changes.9 These changes most likely begin with a local elaboration of a chemoattractant to mediate accumulation of monocytes at the site of inflammation. Previous studies have demonstrated that endothelial cells 10 ' 11 and smooth muscle cells 12 -13 in culture constitutively express monocyte chemotactic protein-1 (MCP-1). 1415 Recently, we have shown that MCP-1 accounts for virtually all of the monocyte chemotactic activity produced by endothelial cells and smooth muscle cells, alone and in coculture. 16 The results of this study suggest that after the initial recruitment by cells of the vessel wall, monocytes may be involved in the amplification of their own recruitment into inflammatory lesions by inducing MCP-1. Methods Cell CultureHuman monocytes and lymphocytes were obtained by a modification of the Recalde method. 17The monocytes were suspended in Iscove's modified Dulbecco's medium containing 30% autologous serum, 1% glutamine, 1% penicillin/streptomycin/ fungizone, and 0.0022% insulin (supplemented media) and then plated at varying densities in plastic culture dishes or treated lipopolysaccharide-free Teflon pots. The cells were incubated at 37°C from 4 hours to 8 days. The media were endotoxin free 18 ' 19 and were changed every 3-4 days. Northern Blot AnalysisTotal RNA was isolated from the monocytes/macrophages by the guanidinium isothiocyanate method of Chomczynski and Sacchi. 20 The alcohol-precipitated RNA was electrophoresed on formaldehyde/1% agarose gels and transblotted to Biotrans nylon membranes (Bio-Rad, Richmond, Calif.). The blots were hybridized with a 32 P-end-labeled 35-mer oligonucleotide probe. The probe was complementary to nucleotides 257-291 of the published cDNA sequence for MCP-1. 21 The sequence of the p...

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