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Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions
Published on: August 8, 2012
Lipopolysaccharide-induced differential cell surface expression of intercellular adhesion molecule-1 in cultured
1Pathobiology Branch, Naval Medical Research Institute, Bethesda, Maryland 20889, USA.
Insights
Endotoxin (lipopolysaccharide) significantly increases intercellular adhesion molecule-1 (ICAM-1) expression on human umbilical cord vein endothelial cells (HUVEC). This LPS-induced ICAM-1 expression correlates with suppressed DNA synthesis, suggesting a role in endothelial cell function.
Area of Science:
- Endothelial Cell Biology
- Immunology
- Molecular Biology
Background:
- Intercellular Adhesion Molecule-1 (ICAM-1) plays a crucial role in leukocyte adhesion to endothelial cells.
- Endotoxin, a component of Gram-negative bacteria, is known to elicit significant inflammatory responses.
- Understanding the regulation of ICAM-1 expression by endotoxin in endothelial cells is vital for comprehending inflammatory processes.
Purpose of the Study:
- To investigate the effect of lipopolysaccharide (LPS), also known as endotoxin, on ICAM-1 expression in human umbilical cord vein endothelial cells (HUVEC).
- To elucidate the mechanism underlying LPS-induced ICAM-1 expression and its relationship with endothelial cell DNA synthesis.
Main Methods:
- Solid phase radioimmunoassay, immunocytochemistry, and electron microscopy were employed to assess ICAM-1 expression.
- HUVEC were incubated with varying concentrations and durations of LPS.
- Neutralizing antibodies against IL-1 alpha, IL-1 beta, and TNF alpha were used to explore the involvement of cytokines.
Main Results:
- LPS significantly enhanced cell surface ICAM-1 expression in HUVEC in a dose- and time-dependent manner, with maximal effect at 0.1-1 µg/mL and 24 hours.
- LPS-induced ICAM-1 expression was not mediated by IL-1 or TNF alpha.
- LPS suppressed HUVEC DNA synthesis activity, and cells with high ICAM-1 expression showed reduced DNA synthesis.
- Immunoelectron microscopy revealed a polarized distribution of ICAM-1 on the luminal surface of HUVEC.
Conclusions:
- Endotoxin (LPS) potently upregulates ICAM-1 expression on HUVEC, independent of IL-1 or TNF alpha.
- The upregulation of ICAM-1 by LPS is inversely correlated with endothelial cell DNA synthesis.
- These findings suggest that LPS-induced ICAM-1 expression may modulate endothelial cell proliferation and contribute to inflammatory responses.
Abstract:
The effect of endotoxin on cell surface ICAM-1 expression in human umbilical cord vein endothelial cells (HUVEC) was examined using solid phase radioimmunoassay, immunocytochemistry and electron microscopy. At various incubation times (e.g. 3, 6, 12, 24 h), the ICAM-1 expression was enhanced by lipopolysaccharide (LPS, or endotoxin) from one ng/ml to 100 micrograms/mL with maximal enhancement at .1-1 micrograms/mL. The kinetics at 1 microgram/mL LPS showed that the maximum ICAM-1 expression occurred at 24 h. The LPS-induced ICAM-1 expression was not inhibited by the neutralizing rabbit polyclonal antibodies against human IL-1 alpha, IL-1 beta, and TNF alpha, either alone or in combination. This indicated that the mechanism of this induced expression was not an autocrine effect mediated by the LPS-induced IL-1 or TNF alpha. The LPS-induced cell surface ICAM-1 exhibited a polarized distribution shown in immunoelectron micrographs with higher density on the luminal surface. DNA synthesis activity of HUVEC was, contrary to the ICAM-1 expression, suppressed by LPS. Immunocytochemical studies indicated that ICAM-1 was not uniformly expressed in the culture, i.e., some cells expressed more surface ICAM-1 than others, and some of the ICAM-1-expressing cells had an uneven patchy distribution of this antigen. Combined immunocytochemical and [3H]thymidine incorporation studies showed that cells with strong ICAM-1 expression had little DNA synthesis activity, while those with little ICAM-1 expression synthesized DNA actively. ICAM-1 on endothelial cells serves as an anchor for the leukocytes in cell-cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)
