Endomorphin-1 affecting innate immune cells in vitro

Opioid peptides belong to one of most studied groups of regulatory peptides due to exerting extremely wide range of biological activities and play an important role in regulating homeostasis. The endogenous opioid system is involved in the functioning of diverse organs and systems, including the immune system. Currently, an immunomodulatory effect of the three major families of opioid peptides such as endorphins, enkephalins, dinorphins has been thoroughly described. Much less data are available about the endomorphin-related immunomodulatory effects. The aim of this work was to assess effects of endomorphin-1 on phagocytosis, production of reactive oxygen species and IL-1β by various subsets of peripheral blood innate immune cells in vitro. The leukocytes of obtained from peripheral venous blood of healthy volunteer donors aged 22 to 40 years were used in the study. Endomorphin-1 was used at concentrations of 10-6, 10-8, 10-10, 10-12 М. Blood leukocyte fraction was isolated from heparinized venous blood settled for 2 hours in a thermostat at 37 °С. The neutrophil fraction was isolated by centrifuging in of Ficoll-Urographin (ρ = 1.077) density gradient placing the upper layer of blood plasma with leukocytes. Assessment of leukocyte oxygen-dependent microbicidal activity was carried out by using the reaction of luminol-dependent chemiluminescence (LZHL) by using opsonized zymosan at concentration of 150 μg/ml; 10-5 M luminol was used to probe reaction magnitude. The separation of the mononuclear cell fraction was carried out by centrifuging in of Ficoll-Urographin (ρ = 1.077) density gradient. The monocyte fraction was isolated mechanically. To assess IL-1β production, mononuclear cells and monocytes were cultured for 24 hours followed by measuring its level with enzyme-linked immunosorbent assay. To evaluate monocytes and neutrophil phagocyte activity, FITC-labeled St.aureus uptake method was used. Statistical processing was performed with one-way ANOVA test and paired LSD criterion for post-hoc comparison, with significance set at p 0.05. It was found that endomorphin-1 reduced leukocyte spontaneous, but not induced production of reactive oxygen species. In the neutrophil fraction, endomorphin-1 did not affect spontaneous oxygen dependent microbicidity and reduced intensity of the respiratory burst in stimulated neutrophils. In addition, it increased the percentage of monocyte phagocytosis and enhanced spontaneous IL-1β production by mononuclear cells. Thus, endomorphin-1 exhibited multidirectional effects on various parameters of innate immunity. Being typical to other groups of regulatory peptides, modality of endomorphin-1 related effects depended on cell fraction and presence of a stimulating cues.