Abstract

Induction of adhesion molecules by cytokines and LPS is an important mechanism of regulating leukocyte migration into tissue. Expression and regulation of E-selectin may be differentially influenced by the stimuli involved with effects on mRNA or surface protein kinetics. Surface protein and mRNA expression kinetics of bovine E-selectin were measured and compared in primary cultures of bovine aortic endothelial cells (BAEC) stimulated for various periods of time with recombinant bovine tumor necrosis factor alpha (rbTNF-α) or Escherichia coli lipopolysaccharide (LPS). E-selectin mRNA expression was measured via quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) using a construct that contained multiple synthetic oligonucleotides for several bovine adhesion molecules and cytokines. Surface expression of E-selectin was measured by flow cytometry. Unstimulated BAECs expressed minimum or no E-selectin on the surface. A low number of endothelial cells expressed surface E-selectin as early as 1 h post-stimulation and surface expression was sustained after both stimuli for 24–72 h. Mean fluorescence intensity (MFI) indicated peak surface concentration of E-selectin at 6 h post-stimulation after LPS followed by a gradual decrease to 72 h without returning to baseline values. Mean fluorescence intensity following stimulation with TNF-α increased slightly between 0 and 72 h. The pattern of mRNA expression differed between stimuli. LPS-stimulated BAECs expressed peak amounts of E-selectin mRNA at 6 h, followed by a decline to baseline by 24 h. Conversely, BAECs stimulated with rbTNF-α expressed significantly (p£ 0.05) higher amounts of mRNA at 1 h than compared to unstimulated controls (0 h), but this decreased to below baseline levels by 6 h; followed by a gradual increase and eventually a sharp increase between 18 and 72 h. To account for the lack of correlation between mRNA and protein expression, it was hypothesized that shedding of surface E-selectin accounted at least in part, for the large increase in mRNA expression seen at 18–72 h. Culture supernatants from rbTNF-α-treated BAECs were harvested, and tested for the presence of shed E-selectin using ELISA. Unstimulated culture supernatants contained little or no E-selectin. Between 6 and 48 h, the concentration of E-selectin in culture supernatants from rbTNF-α-stimulated BAECs increased approximately two-fold, suggesting that the sharp increase in E-selectin mRNA expression around 18 h may be related to significant loss of surface E-selectin during this period.

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