[Lipopolysaccharide induces expression of macrophage inflammatory protein-1alpha in human umbilical vein endothelial cells].
OBJECTIVE
To understand whether endotoxin lipopolysaccharide (LPS) is able to induce the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA and protein in human umbilical vein endothelial cells (HUVECs).


METHODS
The expression of MIP-1alpha mRNA was determined by dot blotting analysis and by in situ hybridization using a digoxigenin-labeled MIP-1alpha cDNA probe after exposure of the cultured HUVECs to LPS at different concentrations. The expression of MIP-1alpha mRNA was determined by RT-PCR as well. In addition, the expression of MIP-1alpha protein was tested by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human monoclonal MIP-1alpha antibody.


RESULTS
Dot blotting showed that the absorbance values of the dots on the nitrocellulose membrane were 1.490 and 3.310 when exposed to LPS at the concentrations of 1 micro g/ml and 10 micro g/ml which were 1.97- and 4.38-fold over that of the control group (0.775), respectively. In situ hybridization revealed that exposure to LPS at a concentration of 1 micro g/ml led to a significant increase in the MIP-1alpha mRNA expression in HUVECs as compared to the control group (F = 142.83, P < 0.01), whereas the MIP-1alpha mRNA in HUVECs was somewhat decreased when exposed to LPS at a concentration of 10 micro g/ml. RT-PCR revealed that the expression of MIP-1alpha mRNA in HUVECs were 1.65-, 2.86- and 1.26-fold over that of the control group when exposed to LPS at the concentrations of 1 micro g/ml, 5 micro g/ml and 10 micro g/ml respectively. Cell ELISA showed that after exposure of the HUVECs to LPS at the concentrations mentioned above, the expression of MIP-1alpha protein was strongly increased, especially in the 5 micro g/ml LPS group. Analysis of variance showed that there was a significant difference between groups (F = 15.36, P < 0.05).


CONCLUSIONS
LPS may induce a high level of MIP-1alpha mRNA and protein expression in HUVECs, and it might, hereby, play an important role in the recruitment of the monocytes/macrophages into the arterial intima.
Deng
Zhong-duan
ZD
Department of Pathology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. dengzd@mails.tjmu.edu.cn
Qu
Zhi-ling
ZL
Yang
Li-min
LM
D002478:Cells, Cultured; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D017403:In Situ Hybridization; D008070:Lipopolysaccharides / Q000633:toxicity*; D019402:Macrophage Inflammatory Proteins / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014471:Umbilical Veins / Q000187:drug effects
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D008070:Lipopolysaccharides; D019402:Macrophage Inflammatory Proteins