Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factors

Abstract

Interindividual differences of endothelial cells in response to endotoxins might contribute to the diversity in clinical outcome among septic patients. The present study was conducted to test the hypothesis that endothelial cells (EC) with high and low proinflammatory potential exist and to dissect the molecular basis underlying this phenomenon. Thirty human umbilical vein endothelial cell (HUVEC) lines were stimulated for 24 h with lipopolysaccharide (LPS) and screened for interleukin (IL)-8 production. Based on IL-8 production five low and five high producers, tentatively called types I and II responders, respectively, were selected for genome-wide gene expression profiling. From the 74 genes that were modulated by LPS in all type II responders, 33 genes were not influenced in type I responders. Among the 41 genes that were increased in both responders, 17 were expressed significantly stronger in type II responders. Apart from IL-8, significant differences in the expression of proinflammatory related genes between types I and II responders were found for adhesion molecules [intercellular adhesion molecule (ICAM-1), E-selectin)], chemokines [monocyte chemoattractant protein (MCP-1), granulocyte chemotactic protein (GCP-2)], cytokines (IL-6) and the transcription factor CCAAT/enhancer binding protein-delta (C/EBP-delta). Type I responders also displayed a low response towards tumour necrosis factor (TNF)-alpha. In general, maximal activation of nuclear factor (NF)-kappaB was achieved in type I responders at higher concentrations of LPS compared to type II responders. In the present study we demonstrate that LPS-mediated gene expression differs quantitatively and qualitatively in types I and II responders. Our results suggest a pivotal role for common transcription factors as a low inflammatory response was also observed after TNF-alpha stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients.

Fig. 1

Heterogeneity in lipopolysaccharide (LPS)-mediated interleukin (IL)-8 production among 30 human umbilical vein endothelial cell (HUVEC) lines. (a) Endothelial cells were stimulated for 24 h with 1 µg/ml of LPS. Supernatants were collected and assessed for IL-8 production. The results are expressed as mean IL-8 production ±s.d. of triplicate cultures. (b) Mean IL-8 production was calculated from the data set obtained in (a) for each of the different genotypes. The results are expressed as mean IL-8 production ± s.d.

Fig. 2

Differences in gene expression between types I and II responders. (a) Significant differences in lipopolysaccharide (LPS)-mediated gene expression between type I and type II responders for genes implicated in inflammatory processes. Filled bars: type II responders, open bars: type I responders. The results are expressed as mean signal log ratio (SLR) (n = 5 for each responder type) ± s.d. (b) Validation of gene expression profiling was performed by reverse transcription–reverse polymerase chain reaction (RT-PCR) for interleukin (IL)-8, IL-6, monocyte chemoattractant protein (MCP)-1, E-selectin and intercellular adhesion molecule (ICAM)-1. Total RNA was isolated from unstimulated (−) or LPS (1 µg/ml, 24 h)-stimulated (+) cells. A representative experiment using two different types I and II responders is depicted. Similar findings were found in all types I and II responders.

Fig. 3

Susceptibility to tumour necrosis factor (TNF)-α stimulation in type I and type II responders. (a) Type I and type II responder cell lines were stimulated for 24 h with different concentrations of TNF-α. The supernatants were collected and assessed for interleukin (IL)-8 production by enzyme-linked immunosorbent assay (ELISA). The results are expressed as mean IL-8 ± s.d. production for each responder type (n = 5 for both types I and II). Hatched bars: type II responders, open bars: type I responders, *P < 0·05 compared to type I. (b) Constitutive (thin lines) and TNF-α mediated (bold lines) intercellular adhesion molecule (ICAM)-1 expression in type I (histograms in black) and type II (histograms in grey) responders. The negative control is depicted as filled histogram. The results of a representative experiment of types I and II responders is depicted. Similar findings were found in all types I and II responders. (c) Endothelial cells of types I and II responders were pretreated with 125 ng/ml of interferon (IFN)-γ (+) or left untreated (−) for 24 h. Hereafter the cells were washed and stimulated with lipopolysaccharide (LPS) (1 µg/ml) (+) or not (−). Twenty-four h hereafter supernatants were collected and assessed for IL-8 production. The results are expressed as mean IL-8 ± s.d. production for each responder type (n = 5 for both types I and II). Hatched bars: type II responders, open bars: type I responders, *P < 0·05 compared to type I.

Fig. 4

Electrophoresis mobility shift assay (EMSA) for NF-κB. (a) Susceptibility towards lipopolysaccharide (LPS) stimulation as measured by NF-κB activation in types I and II responders. The data of representative experiment are depicted. Similar findings were observed in all types I and II responders. Endothelial cells were stimulated for 4 h using different concentrations of LPS. (b) Time response of NF-κB activation in types I and II responders. Endothelial cells were stimulated with 1 µg/ml of LPS. At different time-points the cells were harvested for preparation of nuclear extracts. The data of representative experiment are depicted. Similar findings were observed in all types I and II responders. Endothelial cells were stimulated for 4 h using different concentrations of LPS. (c) Kinetics of LPS-mediated interleukin (IL)-8 production in types I and II responders. Human umbilical vein endothelial cell (HUVEC) were stimulated for different time-periods with 1 µg/ml of LPS. Hereafter the supernatants were collected and assessed for IL-8 production. The results are expressed as mean IL-8 ± s.d. production for each responder type (n = 5 for both types I and II). Dotted line: type II responders, bold line: type I responders, *P < 0·05 compared to type I. (d) The specificity of the shifted bands was demonstrated by incubating a positive sample either with an excess of cold consensus (cons.) or mutated (mut.) NF-κB oligonucleotides before adding labelled consensus NF-κB oligonucleotides. Note that the NF-κB shifted band (arrow) consisted of both p50 and p65 as demonstrated by super-shift (dotted arrow).

Fig. 5

Lipopolysaccharide (LPS) mediated intercellular adhesion molecule (ICAM)-1 expression in types I and II responders. Endothelial cells were stimulated for 24 h with different concentrations of LPS. ICAM-1 expression was measured by fluorescence activated cell sorter (FACS) analysis. The data of a representative experiment are depicted. Similar results were obtained for all types I and II responders. The negative control is depicted as grey dotted histogram.