Endogenous morphine levels are increased in sepsis: a partial implication of neutrophils.
BACKGROUND
Mammalian cells synthesize morphine and the respective biosynthetic pathway has been elucidated. Human neutrophils release this alkaloid into the media after exposure to morphine precursors. However, the exact role of endogenous morphine in inflammatory processes remains unclear. We postulate that morphine is released during infection and can be determined in the serum of patients with severe infection such as sepsis.


METHODS
The presence and subcellular immunolocalization of endogenous morphine was investigated by ELISA, mass spectrometry analysis and laser confocal microscopy. Neutrophils were activated with Interleukin-8 (IL-8) or lipopolysaccharide (LPS). Morphine secretion was determined by a morphine-specific ELISA. mu opioid receptor expression was assessed with flow cytometry. Serum morphine concentrations of septic patients were determined with a morphine-specific ELISA and morphine identity was confirmed in human neutrophils and serum of septic patients by mass spectrometry analysis. The effects of the concentration of morphine found in serum of septic patients on LPS-induced release of IL-8 by human neutrophils were tested.


RESULTS
We confirmed the presence of morphine in human neutrophil extracts and showed its colocalisation with lactoferrin within the secondary granules of neutrophils. Morphine secretion was quantified in the supernatant of activated human polymorphonuclear neutrophils in the presence and absence of Ca(2+). LPS and IL-8 were able to induce a significant release of morphine only in presence of Ca(2+). LPS treatment increased mu opioid receptor expression on neutrophils. Low concentration of morphine (8 nM) significantly inhibited the release of IL-8 from neutrophils when coincubated with LPS. This effect was reversed by naloxone. Patients with sepsis, severe sepsis and septic shock had significant higher circulating morphine levels compared to patients with systemic inflammatory response syndrome and healthy controls. Mass spectrometry analysis showed that endogenous morphine from serum of patient with sepsis was identical to poppy-derived morphine.


CONCLUSIONS
Our results indicate that morphine concentrations are increased significantly in the serum of patients with systemic infection and that morphine is, at least in part, secreted from neutrophils during sepsis. Morphine concentrations equivalent to those found in the serum of septic patients significantly inhibited LPS-induced IL-8 secretion in neutrophils.
Glattard
Elise
E
Inserm, U575, Physiopathologie du Système Nerveux and Nociception and Pain Department, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique, Université de Strasbourg, Strasbourg, France.
Welters
Ingeborg D
ID
Lavaux
Thomas
T
Muller
Arnaud H
AH
Laux
Alexis
A
Zhang
Dan
D
Schmidt
Alexander R
AR
Delalande
François
F
Laventie
Benoît-Joseph
BJ
Dirrig-Grosch
Sylvie
S
Colin
Didier A
DA
Van Dorsselaer
Alain
A
Aunis
Dominique
D
Metz-Boutigue
Marie-Hélène
MH
Schneider
Francis
F
Goumon
Yannick
Y
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D016209:Interleukin-8 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D018613:Microscopy, Confocal; D009020:Morphine / Q000097:blood*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D017450:Receptors, Opioid, mu / Q000378:metabolism; D018805:Sepsis / Q000097:blood*; D053719:Tandem Mass Spectrometry
D016209:Interleukin-8; D008070:Lipopolysaccharides; D017450:Receptors, Opioid, mu; D009020:Morphine