ISSN 0012�4966, Doklady Biological Sciences, 2012, Vol. 444, pp. 141–143. © Pleiades Publishing, Ltd., 2012.Original Russian Text © S.V. Gein, S.G. Gileva, 2012, published in Doklady Akademii Nauk, 2012, Vol. 444, No. 2, pp. 221–223.

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Opioid peptides are a large group of physiologicallyactive peptides with a pronounced affinity for thereceptors of the opioid (morphine) type (μ, δ, and κ).It has been so far demonstrated that the three maintypes of opioid receptors are expressed in all cell pop�ulations of the immune system [1]. Endogenous opi�oid peptides, in particular, β�endorphin, are secretedinto the blood during stresses, traumas, and psychoe�motional states, as well as locally produced by cells ofvarious organs and tissues, including the immune sys�tem cells, in response to an antigen, thereby providinga wide range of immunoregulatory effects mediated byparacrine and autocrine mechanisms.

It is well known that functioning of the immunesystem changes with age, in particular, displaying adecrease in the functional activity of immunocytes [2].On the other hand, the age�related dependence of theimmunomodulatory effect of opioid peptides onimmunocytes has been insufficiently studied. It is cur�rently known that the secretion of β�endorphin, inparticular, its production by lymphocytes in the nor�mal state and stress, considerably changes during thelife [3], whereas no changes in its concentration in theperipheral blood with age have been observed [4].However, the content of β�endorphin in unstimulatedmononuclear cells is increased in elderly subjects.When cells are stimulated with lectins, the concentra�tion of intracellular β�endorphin in elderly subjectsdecreases more than in young ones [5]. We have earlierdemonstrated that β�endorphin stimulates the lym�phocyte proliferative response to phytohemagglutinin(PHA) in healthy volunteer donors at an age of 20–30 years [6, 7] and elevates the splenocyte prolifera�tion in young BALB/c mice [8].

The goal of this work was to study the effect of β�endorphin on the lymphocyte proliferative response inhealthy male volunteer donors of different ages.

The examined group contained somatically healthymale volunteer donors at ages of 20–29 (n = 10), 30–39 (n = 13), 40–49 (n = 12), and 50–59 (n = 9) years.Heparinized venous blood was placed into a thermo�stat at a temperature of 37°C for 60–90 min. After set�tling, the upper layer of plasma containing leukocyteswas removed and centrifuged at 1500 rpm for 15 min.After centrifugation, the pellet was suspended in 2 mLof complete culture medium. Lymphocytes weremicrocultivated in plastic round�bottomed 96�wellplates (Medpolimer, St. Petersburg, Russia). Each wellcontained 2 × 10 cells per 0.2 mL of complete culturemedium. This medium was prepared ex tempore usingmedium 199 supplemented with 2 mM L�glutamine,10 mM HEPES, 100 μg/mL gentamicin sulfate, and10% autoplasma. PHA�P (Sigma) at concentrationsof 2.5 and 20 μg/mL was used as a T�cell mitogen. Thecultivation was performed in a humid medium con�taining 5% CO2 at 37°C for 72 h. Each well, 18 h beforecompletion of cultivation, was supplemented with 2 μCiof 3H�methyl thymidine in a volume of 10 μL. Thecontent of each well was successively sedimented onfilter paper at a vacuum pump pressure of 0.5–1.0 atm;each well was washed with 0.9% sodium chloride atleast four or five times. The filters were washed toremove unbound thymidine with 5 mL of 0.9% NaCl,5 mL of 5% trichloroacetic acid cooled to 4°C, and5 mL of 0.9% NaCl. The radioactivity of samples wasmeasured after drying the filters with a Guardianliquid scintillation counter (Wallac, Finland) usingZhS�107 scintillation liquid.

β�Endorphin (Scytek Laboratories, United States)at concentrations of 10–7 to 10–10 was used in thein vitro experiments. The data were statistically pro�cessed using one�way ANOVA and Fisher’s LSD testfor intergroup comparison.

β�Endorphin Regulation of Lymphocyte Proliferative Response in Men of Different Age Groups

S. V. Gein and S. G. GilevaPresented by Academician V.A. Chereshnev February 3, 2012

Received February 3, 2012

DOI: 10.1134/S0012496612030064

Institute of Ecology and Genetics of Microorganisms, Ural Division, Russian Academy of Sciences, Perm, RussiaPerm State University, Perm, Russia

PHYSIOLOGY

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GEIN, GILEVA

It was demonstrated that β�endorphin at a concen�tration of 10–7 M increased the lymphocyte prolifera�tive response in the group of 20� to 29�year�old donorsin the presence of suboptimal mitogen concentrationand inhibited it at a concentration of 10–10 M. At anoptimal mitogen concentration, β�endorphin inhib�ited lymphocyte proliferation at a concentration of10–10 M. In the same cohort of donors, β�endorphinincreased spontaneous lymphocyte proliferative activ�ity at concentrations of 10–8 and 10–10 M.

Assessment of the proliferation dynamics in thegroup of 30� to 39�year�old donors demonstrated thatβ�endorphin at concentrations of 10–8 and 10–9 Mstimulated the lymphocyte proliferative response inthe presence of a suboptimal PHA concentration.On the other hand, similarly to the group of 20� to29�year�old donors, β�endorphin in the presence ofan optimal mitogen concentration inhibited lympho�cyte proliferation at a concentration of 10–10 M. In this

group of donors, the peptide had no effect on the lym�phocyte spontaneous proliferative activity.

In the group of 40� to 49�year�old donors, β�endorphin also inhibited proliferation in the presenceof an optimal PHA concentration and did not influ�ence the intensity of 3H�thymidine incorporation inspontaneous cultures in a statistically significant man�ner. On the other hand, in the presence of a subopti�mal PHA concentration, β�endorphin displayed onlya tendency towards proliferation stimulation in thisage group.

In the group of 50� to 59�year�old donors, β�endorphin did not modulate the lymphocyte prolifer�ative response in either spontaneous cultures or thosestimulated with 2.5 μg/mL PHA. On the other hand,the peptide at a concentration of 10–9 stimulated pro�liferative response in the cultures with 20 μg/mL PHA.

Thus, these results demonstrate that β�endorphinmodulates the lymphocyte proliferative response pre�dominantly in young donors, and this effect reduceswith age, almost disappearing in the group of 50� to60�year�old donors. It is natural that the functions ofthe immune system decrease with age, and a decrease

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Fig. 1. The effect of β�endorphin on the lymphocyte pro�liferative response in the cohorts of (a) 20� to 29� and (b)30� to 39�year�old donors. Here and in Fig. 2: (1) cultureswithout mitogen; (2) 2.5 μg/mL PHA; and (3) 20 μg/mLPHA; the abscissa shows the concentration of β�endor�phin (M); * p < 0.05; ** p < 0.01; and *** p < 0.001.

Fig. 2. The effect of β�endorphin on the lymphocyte pro�liferative response in the cohorts of (a) 40� to 49� and(b) 50� to 59�year�old donors. For designations, see Fig. 1.

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β�ENDORPHIN REGULATION OF LYMPHOCYTE PROLIFERATIVE RESPONSE 143

in the ability of neuropeptides to modulate theimmune response may be one of the reasons.

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