Ethylene, an early marker of systemic inflammation in humans

Abstract

Ethylene is a major plant hormone mediating developmental processes and stress responses to stimuli such as infection. We show here that ethylene is also produced during systemic inflammation in humans and is released in exhaled breath. Traces of ethylene were detected by laser spectroscopy both in vitro in isolated blood leukocytes exposed to bacterial lipopolysaccharide (LPS) as well as in vivo following LPS administration in healthy volunteers. Exposure to LPS triggers formation of ethylene as a product of lipid peroxidation induced by the respiratory burst. In humans, ethylene was detected prior to the increase of blood levels of inflammatory cytokines and stress-related hormones. Our results highlight that ethylene release is an early and integral component of in vivo lipid peroxidation with important clinical implications as a breath biomarker of bacterial infection.

Figure 1

Ethylene forms upon peroxidation of unsaturated lipids. (a) Ethylene released from pig brain L-α-phosphatidylethanolamine (PE) suspended in milliQ H₂O with 0.1% Triton X-100 and in the presence or absence of 500 µM Fe(II)SO₄ after 30 minutes incubation (n = 3). (b) Ethylene released from cis-4,7,10,13,16,19-docosahexaeinoic acid (22:6 (n-3)), cis-9-Octadecenoic (18:1 (n-9)) or palmitic acid (16:0) fatty acids suspended in milliQ H₂O with 0.1% Triton X-100 and 500 µM Fe(II)SO₄ after 30 minutes incubation (n = 3). Triton X-100: detergent control with 0.1% Triton X-100 (n = 3). (c) Ratio of ethylene released per molecule of fatty acid (FA) for cis-4,7,10,13,16,19-docosahexaeinoic acid (22:6 (n-3)) and cis-9-Octadecenoic acid (18:1 (n-9)). Bar graphs show mean with SEM, asterisks indicate significance compared to untreated samples.

Figure 2

LPS triggers lipid peroxidation and ethylene formation in leukocytes. (a) Lipid peroxidation in moDCs as measured by blue-shift of Bodipy581/591-C11 fluorescence. MoDCs were treated with 1 µg/ml LPS or 100 µM Fe(II)SO₄ (n = 4). (b) Lipid peroxidation in moDCs measured by EIA assay for 8-isoprostane. Cells were treated with either LPS or 100 µM Fe(II)SO₄ in serum-free RPMI-1640 (n = 3). (c) Total ethylene released during 1 hour stimulation of leukocytes with either 1 µg/ml LPS or 100 µM Fe(II)SO₄, as detected by laser-based photoacoustic spectroscopy (n = 11). Data points show mean with SEM (panels a and b) or geometric mean with 95% CI (panel c), asterisks indicate significance compared to untreated samples.

Figure 3

Ethylene and 8-isoprostane release during human endotoxemia. (a) Time course of ethylene levels in breath of healthy volunteers after intravenous administration of a single dose of LPS at t = 0. (b) Time course of 8-isoprostane levels as determined by EIA in serum samples of healthy volunteers after intravenous administration of a single dose of LPS (present study, n = 8) or phosphate buffered saline (PBS) (previous study, n = 6) at t = 0. Data points show median with interquartile range, asterisks indicate significance compared to baseline (t = −30 for ethylene, t = 0/−50 for 8-isoprostane PBS/LPS).

Figure 4

Systemic inflammation and stress response during human endotoxemia. Time course of cytokine levels as determined in plasma for (a) pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), (b) anti-inflammatory cytokines interleukin-1 receptor antagonist (IL-1RA) and interleukin-10 (IL-10) and (c) chemotactic cytokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) (n = 8). (d) Time dependent changes in plasma levels of epinephrine, norepinephrine and cortisol. Data points (n = 8) show median with interquartile range, asterisks indicate significance compared to baseline (t = −50).