Influence of different kinds of stress on apoptosis of CD4⁺/CD8⁺ lymphocytes upon in vivo blockage of opiate receptors

The endogenous opioid system is involved in regulation of many body functions, including immune system regulation. Endogenous opioid peptides are released under stress and communicate with opiate receptors, which are found on most immune cells. Apoptosis is one of the most important instruments in the regulation of the immune system. The aim of this study was to evaluate effects of various types of stress upon apoptosis of CD4⁺/ CD8⁺ lymphocytes under conditions of opiate receptor blockade. The objects of this study were white outbred male mice, been kept in laboratory vivarium. The following types of stress were studied: immobilization (6 h in the supine position), rotational (60 min: 10 min rotation at 78 rpm, 5 min rest), two types of acute cold (10 min or 60 min at -20 °С), chronic cold (-4 °С for 4 hours daily, for 7 days). Opiate receptors have been blocked by subcutaneous administration of naloxone hydrochloride at a dose of 0.2 mg/kg 20 minutes before stress, and again 3 hours later, with a stress duration of over 3 hours. Splenocytes were stained with PE-labeled monoclonal antibodies against murine CD4, and antibodies against mouse CD8 (BioLegent, USA); after incubation and washing, they were stained with reagents for determining apoptosis. V-FITC / 7-AAD annexin kit (Beckman Coulter, USA) according to the manufacturer's instructions. Lymphocyte apoptosis was recorded with a CytoFLEX S cytometer (Beckman Coulter, USA). Results. It was found that immobilization stress (lasting 6 h) and acute cold stress (60 min -20 °С), regardless of naloxone administration, enhance apoptosis of mouse CD8⁺ lymphocytes. Chronic cold stress (-4 °С 4 hours / 7 days) led to a decrease in apoptotic spleen lymphocyte numbers only during the blockade of opiate receptors. The effects of rotational stress and short-term acute cold stress (-20 °С, 10 min) upon apoptosis were not registered. Thus, two types of stress (immobilization and acute cold) caused intensification of CD8⁺ lymphocyte apoptosis; endogenous opioid system seems not to participate in regulation of these processes. Under conditions of chronic cold stress with naloxone injections, we have noted a decreased Annexin binding by spleen lymphocytes in mice.