Effects of beta-endorphin and dynorphin A on in vitro apoptosis in human peripheral blood lymphocytes

Abstract

Along with functioning in the nerve system, the endogenous opioid peptides as the ligands of mu-, delta-, and kappa-opioid receptors exert multiple effects on immune cells. Moreover, experimental evidence showed that morphine as an exogenous agonist of mu-opioid receptors affects immune cell viability. Such effects were discovered in experiments with cultured cells and laboratory animals. Hence, we studied effects of endogenous opioid peptides dynorphin A and beta-endorphin on viability of human peripheral blood mononuclear cells in vitro. For this, we used samples of peripheral blood cells collected from the fourteen healthy volunteers, who provided with signed informed consent and might request any information regarding the research. Mononuclear cells were collected from the heparinized blood samples according to standard protocol and cultivated in the humid atmosphere for 72 hours. Two μCi 3H-Methyl-thymidine was added into each test tube at 18 hours before the end of the cultivation period. Scintillation counting was performed by using Guardian liquid scintillation analyzer (Wallac, Finland) expressing the data as count per minute. To assess apoptosis, the cells were cultured for 24 hours in similar conditions except for adding radioactive probe. Next, the cells were stained with Annexin V-FITC/7-AAD kit (Beckman Coulter, USA) according to the manufacturer’s instructions for further recording apoptotic cells in flow cytometers BD FACSCalibur (Becton Dickinson, USA) or CytoFLEX S (Beckman Coulter, USA). The lymphocyte gate set by light scatter parameters was shown in typical Annexin V-FITC vs 7-AAD plot followed by counting Annexin V+/7-AADcells. All data were expressed as means ± S.E. Statistical significance was assessed by using Student’s t-test. It was found that physiologic concentrations of mu- and kappa-opioid receptor agonists beta-endorphin and dynorphin A exerted multidirectional effects on proliferation of human peripheral blood lymphocytes. In particular, dynorphin A increased basal proliferation and proliferation in response to suboptimal mitogen stimulation. Moreover, beta-endorphin enhanced effects of mitogen stimulation at suboptimal concentration but profoundly suppressed proliferation in maximally activated cells. The modulating effects of beta-endorphin and dynorphin A on in vitro proliferation are not associated with augmented cell apoptosis.