In this study we investigated the effect of dobutamine and norepinephrine on cytokine production after simulation of whole blood with 500 pg of LPS ex-vivo. Ten male healthy volunteers served as blood donors. Blood was collected and processed under specific protocol. All of them were male residents of the urban area of Athens (mean age 33.6 ± 1.5 years). Medical history, physical examination, hematological and biochemical screening and electrocardiogram were all normal. The participants did not smoke, did not take any medication and did not have any febrile illness in the month before the study. Blood samples were collected via a peripheral vein at the same time (8.00 am) after the subjects were acclimatized for 60 min at room temperature and were placed in tubes containing EDTA and immediately transferred to the lab for processing. Briefly, heparinized blood from healthy volunteers’ donors was diluted 1:10 in RPMI 1640 culture medium. One hundred μL of blood sample were added in 900 μL of RPMI 1640 for final volume 1 ml and placed to plastic culture dishes. Samples were added to wells and maintained at 37o C in a 5% CO2 atmosphere and LPS was added at different doses. Norepinephrine and dobutamine were added in concentrations 10-4, 10-5 και 10-6 Μ. Metoprolol, a β1 adrenergic blocker was added in concentration 10-5 Μ. After 4 hours incubation period the culture plate was briefly centrifuged (1800 rpm, 5 min) and supernatants were collected and stored at - 20o C until measurements. Subsequently, cytokines levels in the supernatant were evaluated by the ELISA. In this study, we have demonstrated that in the blood of healthy volunteers norepinephrine and dobutamine lead to a dose-dependent reduction of the production of TNF-α and IL-6 (proinflammatory cytokines) and this reduction is associated with β1-adrenergic mechanism, because it is reversed by metoprolol (β1-adrenergic inhibitor).
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