Interleukin-1β, tumor necrosis factor-α and lipopolysaccharide induce expression of monocyte chemoattractant protein-1 in calf aortic smooth muscle cells

Abstract

To investigate whether interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) induce expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and protein in calf aortic smooth muscle cells (SMCs), calf aortic SMCs were cultured by a substrate-attached expiant method. The cultured SMCs were used between the third to the fifth passage. After the cells became confluent, the SMCs were exposed to 2 ng/ml IL- 1β, 20 ng/ml TNF-1α and 100 ng/ml LPS respectively, and the total RNA of SMCs which were incubated for 4 h at 37 C were extracted from the cells by using guanidinium isothiocyanate method. The expression of MCP-1 mRNA in SMCs was detected by using dot blotting analysis using a probe of γ-³²P-end-labelled 35-mer oligonucleotide. After a 24-h incubation, the media conditioned by the cultured SMCs were collected. The MCP-1 protein content in the conditioned media was determined by using sandwich ELISA. The results were as follows: Dot blotting analysis showed that the cultured SMCs could express MCP-1 mRNA. After a 4-h exposure to IL-1β, TNF-α and LPS, the MCP-1 mRNA expression in SMCs was increased (3.6-fold, 2.3-fold and 1.6-fold, respectively). ELISA showed that the levels of MCP-1 protein in the conditioned media were also increased (2. 9-fold, 1.7-fold and 1.1-fold, respectively). The results suggest that calf aortic SMCs could express MCP-1 mRNA and protein. IL-1β and TNF-α can induce strong expression of MCP- 1 mRNA and protein, and the former is more effective than the latter.

Keywords

interleukin-1β / tumor necrosis factor α / lipopolysaccaride / monocyte chemoattractant protein 1 / muscle, smooth, vascular

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