The effects of LPS and probucol on interleukin 1 (IL-1) and platelet-derived growth factor (PDGF) gene expression in the human monocytic cell line U-937

Abstract

We have investigated the effects of lipopolysaccharide (LPS) and probucol (a lipid soluble antioxidant) on the gene expression of interleukin 1 alpha and beta (IL-1 alpha and IL-1 beta), and platelet-derived growth factor A chain and B chain (PDGF-A and PDGF-B) in the human monocytic cell line U-937. Steady-state mRNA levels were measured quantitatively by the reverse transcription-polymerase chain reaction (RT-PCR) using a non-radioactive label. Cells were incubated with LPS, in the presence or absence of probucol for 20 h. The cells were harvested and RNA was then prepared, reverse-transcribed in the presence of an internal standard and subsequently amplified and labelled with digoxigenin-11-dUTP by the PCR reaction. The PCR products were subjected to agarose gel electrophoresis, blotted onto nylon membranes and visualised by immunological detection of digoxigenin followed by a chemiluminescent reaction. We found that: (1) LPS treatment of U-937 cells caused an up-regulation of gene expression of IL-1 beta and PDGF-A chain by approximately 250% and 100% respectively, although it did not stimulate the expression of IL-1 alpha nor PDGF-B chain mRNA. (2) Probucol treatment in vitro had no effect on the basal or LPS-stimulated mRNA levels of IL-1 alpha, IL-1 beta, PDGF-A and PDGF-B despite its reported activity in vivo. (3) PDGF-A and PDGF-B were expressed at a similar level in unstimulated U-937 cells approximately 10-50 copies/ng total RNA, whereas the expression of IL-1 beta mRNA was approximately 2-4 times higher than IL-1 alpha mRNA. (4) Finally, in U-937 monocytic cells the expression of IL-1 alpha and IL-1 beta, and PDGF-A and PDGF-B appear to be independently regulated.