Activation and homologous desensitization of human endothelial cells by CD40 ligand, tumor necrosis factor, and interleukin 1.

Abstract

SummaryWe have reported previously that activation of human umbilical vein endothelial cells (HUVECs) through CD40, using a recombinant soluble form oftrimerized CD40 ligand, leads to induction of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Here, we compare the effects of CD40 ligand with those of tumor necrosis factor (TNF) and interleukin 1 (IL-1). All three ligands induce transient increases in E-selectin (peak 4 h) and VCAM-1 (peak 8-24 h), as well as sustained increases in ICAM-1 (plateau 24 h). Quantitatively, TNF is more potent than 1L-1, which is much more potent than CD40 ligand. The same hierarchy is observed for transcriptional activation of an E-selectin promoter reporter gene construct in transiently transfected HUVECs. TNF and CD40 ligand each induced activation of the transcription factors NF-KB, IRF-1, and ATF-2/c-Jun, measured by electrophoretic mobility shift assays, but this response appeared quantitatively similar. All three agents transiently (peak 30 rain) activatedJun NH2-terminal kinase (]NK), which has been implicated in transcription of E-selectin through its actions on ATF-2/c-Jun. Activation of JNK again showed a hierarchy of potency (TNF> IL-I>> CD40 ligand), although the time course of induction was similar for all three agents. After 44 h ofpretreatment, TNF, IL-1, and CD40 ligand each display homologous desensitization for reinduction of surface expression of E-selectin. A similar pattern of homologous desensitization for reactivation of JNK was observed. We conclude that TNF, IL-1, and CD40 ligand all activate similar responses in ECs, and that homologous desensitization of JNK may explain the inability of individual cytokines to reinduce E-selectin expression.

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

“…However, in our experiments with primary endothelial cells, we used Fc-CD40L at similar concentrations as in our other experiments where the concentrations of Fc-CD40L were fully sufficient to induce strong responses and which were also sufficient to drive maturation of dendritic cells (data not shown). Of note, primary endothelial cells were often cotreated with other proinflammatory triggers (IL-4, INF-g, CMV infection) to increase CD40 expression and CD40 responses (53)(54)(55)(56). Conventional cultivated primary endothelial cells might thus not represent a suitable model to analyze the relevance of the CD40-Fn14 crosstalk in this type of cells in vitro.…”

“…However, reports demonstrating robust effects of CD40 activation on endothelial cells are often based on in vivo studies. Indeed, studies analyzing endothelial CD40 functions in vitro typically report modest responses that only reach a very small percentage of the same response induced by TNF (53)(54)(55)(56). Furthermore, quite strong CD40L stimulation protocols have been used in these studies (10-20 mg/ml soluble CD40L or membrane CD40L transfectants) to see the comparable weak effects on CD40 activity (53)(54)(55)(56).…”

“…Indeed, studies analyzing endothelial CD40 functions in vitro typically report modest responses that only reach a very small percentage of the same response induced by TNF (53)(54)(55)(56). Furthermore, quite strong CD40L stimulation protocols have been used in these studies (10-20 mg/ml soluble CD40L or membrane CD40L transfectants) to see the comparable weak effects on CD40 activity (53)(54)(55)(56). However, in our experiments with primary endothelial cells, we used Fc-CD40L at similar concentrations as in our other experiments where the concentrations of Fc-CD40L were fully sufficient to induce strong responses and which were also sufficient to drive maturation of dendritic cells (data not shown).…”

TRAIL Induces Apoptosis and Inflammatory Gene Expression in Human Endothelial Cells

“…We transiently transfected HUVEC cells with NF-B promoter-firefly luciferase and ␤-actin promoter-Renilla luciferase reporter plasmids using a DEAE-dextran protocol (30). The transfected HUVEC were incubated with 50 ng/ml TRAIL or TNF for 7 h, and their luciferase activity was determined using a Promega (Madison, WI) Dual-Luciferase reporter assay system according to the manufacturer's instruction, and the activity of the NF-B promoter-reporter was normalized to that of the ␤-actin promoter-reporter.…”

Kinases: positive and negative regulators of apoptosis

“…These pathways are known to be activated by ligation of a variety of receptors such as the TNF or FAS receptor. 108 In addition, the physical environment in which the cell exists may also activate the Jnk and p38 signaling pathways. Both of these pathways can be activated following cellular stress.…”