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.
PLoS One. 2010 Jan 20;5(1):e8791. doi: 10.1371/journal.pone.0008791.
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.
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.
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.
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.
哺乳动物细胞合成吗啡，其相应的生物合成途径已被阐明。人类中性粒细胞在暴露于吗啡前体后会将这种生物碱释放到培养基中。然而，内源性吗啡在炎症过程中的确切作用仍不清楚。我们假设吗啡在感染期间释放，并可以在严重感染（如败血症）患者的血清中确定。
通过 ELISA、质谱分析和激光共聚焦显微镜研究内源性吗啡的存在和亚细胞免疫定位。用白细胞介素-8 (IL-8) 或脂多糖 (LPS) 激活中性粒细胞。通过吗啡特异性 ELISA 测定吗啡分泌。用流式细胞术评估 μ 阿片受体表达。用吗啡特异性 ELISA 测定败血症患者的血清吗啡浓度，并通过质谱分析在人中性粒细胞和败血症患者的血清中确认吗啡的存在。测试在败血症患者血清中发现的吗啡浓度对 LPS 诱导的人中性粒细胞释放 IL-8 的影响。
我们证实了吗啡存在于人中性粒细胞提取物中，并显示其与中性粒细胞次级颗粒中的乳铁蛋白共定位。在存在和不存在 Ca(2+) 的情况下，定量测定激活的人多形核中性粒细胞上清液中的吗啡分泌。只有在 Ca(2+) 存在的情况下，LPS 和 IL-8 才能诱导吗啡的显著释放。LPS 处理增加了中性粒细胞上的 μ 阿片受体表达。当与 LPS 共孵育时，低浓度吗啡 (8 nM) 可显著抑制中性粒细胞释放 IL-8。这种作用被纳洛酮逆转。败血症、严重败血症和感染性休克患者的循环吗啡水平明显高于全身炎症反应综合征患者和健康对照者。质谱分析表明，来自败血症患者血清的内源性吗啡与罂粟衍生的吗啡相同。
我们的结果表明，全身性感染患者的血清中吗啡浓度显著升高，并且至少部分吗啡是在败血症期间从中性粒细胞中分泌的。相当于败血症患者血清中发现的吗啡浓度可显著抑制 LPS 诱导的中性粒细胞中 IL-8 的分泌。
