Вплив ліпополісахариду <i>Shigella sonnei</i> на систему циклічних нуклеотидів Т-лімфоцитів крові людини in vitro
The article reveals the results of study of cyclic nucleotides in human T-limphocytes been under influence of lipopolysaccharide from Shigella sonnei.Introduction. Bacterial lypopolysaccharides (LPS) can modulate concentrations of cyclic nucleotides in target cells. Cyclic nucleotides are universal intracellular bioregulators of metabolic processes [5]. For instance, in patients with acute shigellosis cAMP concentrations in the mucous coat of large intestine, blood plasma and urine rise dramatically [3]. It is also known that apoptic program is induced and realized with the help of different signal systems; it should be noted, though, that calcium ion and cAMP concentrations in the cells involved in apoptosis briefly increase [2]. The system of cyclic nucleotides is extremely labile: it can, for example, cause some immune reactions to intensify and simultaneously cause others to subside [2, 3]. There is no doubt that changing cyclic nucleotide concentrations in lymphocytes can lead to modulation of different reactions. It should also be taken into consideration that T-lymphocyte populations vary according to their basal concentration of both cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) [3].The effect of Shigella sonnei LPS on cyclic nucleotides in human T-lymphocytes has not been studied before.The aim of this study is to analyze how Shigella sonnei LPS affect cyclic nucleotides in human T-lymphocytes in vitro.Material and methods. The researchers studied the values of cyclic nucleotides using T-lymphocyte cultures extracted from peripheral blood of 35 fairly healthy donors in the age group of 19 - 24 y.o. (average age - 22.6±1.2 y.o.). The research was carried out in accordance with all principles of bioethics (Strasburg, 1985).Lymphocytes were extracted on fikoll-verografin density gradient (р=1,076) by modified Böyum method [6]. T-lymphocytes were separated from NK-lymphocytes, B-lymphocytes, monocytes, and neutrophil populations with the help of monoclonal antibodies CD14, CD16 and CD22 (produced by scientific and manufacturing center«Medbiospectrum», Moscow, RF). The listed antibodies were introduced into a lymphocyte suspension with the following dilution proportions: 0.1-0.2mcg/ml in 0.025ml. After 40 minutes a guinea pig complement diluted 1:1 with sodium chloride isotonic solution was added to the suspension. The mixture was incubated in thermostat during 60 minutes, after which T-lymphocytes were thrice washed by centrifuging in medium 199. The effective concentration of T-lymphocyte suspensions was 2*(9 lg)/l. Supravital staining of extracted lymphocytes with trypan blue showed that 98-99 % of the cells were viable. T-helper/inductor and T-suppressor/cytotoxic subpoplulations were separated with monoclonal antibodies CD4 и CD8 (produced by scientific and manufacturing center «Medbiospectrum», Moscow, RF) similarly to the procedure described above.Shigella bacteria were identified with diagnostic sets «Enterotest 24» manufactured by Micro-La-Test jointstock company «Lachema», Czech Republic.LPS preparations were produced from Shigella sonnei cultures by water-phenol extraction [1, 6]. To stimulate T-lymphocytes in vitro we used LPS in concentrations of 1-10-100 mcg/ml.Concentration of cAMPH and cGMPH in T-lymphocytes was measured by immunoenzyme method with test-systems manufactured by R&D Systems. USA and commercial test-systems manufactured by «Gen-Probe Diaclone» (France). in compliance with official protocols on investigation procedures for each of the above mentioned mediators. CD4+ и CD8+ lymphocyte cultures were lysed with distilled water before performing investigations.Statistical processing of the data was performed with Student criterion.Results and discussion. It is known that main immunoregulatory human T-lymphocyte subpopulations vary according to their basal concentrations of intracellular cAMP and cGMP. So, in the CD4+-lymphocyte (T- helpers/inductors) subpopulation intracellular cAMP was much lower while cGmp was higher than those values in the subpopulation of CD8+-lymphocytes (T-suppressors/cytotoxics).The study also showed that during in vitro incubation of these subpopulations of intact T-lymphocytes, i.e. T-lymphocytes not stimulated by Shigella sonnei LPS, intracellular concentrations of cyclic nucleotide fractions gradually change. An increase in cAMP and a decrease in cGMP were recorded for CD4+-lymphocyte cultures, unlike for CD8+-lymphocytes, where both cAMP and cGMP concentrations increased. Naturally, these shifts resulted in change of cAMP/cGMP ratio, which rose in CD4+-lymphocyte subpopulation, and fell in CD8+-lymphocyte subpopulation. These dynamics of changes that took place in cyclic nucleotide system can probably be explained by the fact that T-lymphocytes in vitro suffer from hypoxia.Direct exposure of T-lymphocytes to Shigella sonnei LPS had a dose- and time-dependent effect on cyclic nucleotides (see tables 1, and 2). Namely, in CD4+-lymphocyte subpopulation intracellalr cAMP concentration increased, cGMP concentration decreased, and cAMP/cGMP ratio increased in direct proportion to effective concentration of LPS and incubation period. The most significant changes in cyclic nucleotides were recorded after 96 hours of incubation, when CD4+-lymphocytes were exposed to Shigella sonnei LPS in the effective concentration of 100mcg/ml. Moderate changes in cAMP/cGMp system were observed when CD4+-lymphocytes were exposed to Shigella sonnei LPS in the effective concentration of 10mcg/ml, while the least significant changes in cyclic nucleotides resulted from exposure to LPS in the effective concentration of 1mcg/ml.For example, exposure of CD4+-lymphocytes to Shigella sonnei LPS in the effective concentration of 1mcg/ml during 96 hours of incubation caused the value of intracellular cAMP concentration to rise 1.63timesagainst this value for intact CD4+-lymphocytes; with effective concentration of 10mcg/ml it increased 2.26times; effective concentration of 100mcg/ml saw an increase of 3.89 times (р<0.001 for all comparisons). As for the value ofintracellular cGMP concentration, the experimenters recorded a decrease of 1.32 times against that of intact CD4+- lymphocytes after 96 hours of exposure to effective LPS concentration of 1mcg/ml; 1.57 times with effective LPS concentration of 10mcg/ml, and 1.76 times when the effective LPS concentration was 100mcg/ml.The experiment also showed dose- and time-dependent effect of Shigella LPS on cyclic nucleotides in CD8+-lymphocyte subpopulation (see table 2).Similarly to CD4+ group, the most significant changes in intracellular concentrations of cyclic nucleotides in CD8+-lymphocyte subpopulation were registered with effective Shigella sonnei LPS concentration of 100mcg/ml and the incubation lasted 96 hours. The least significant changes in cyclic nucleotides were recorded during experiments with effective LPS concentration of 1mcg/ml and incubation period of 24 hours.After 24 hours, the mean value of cAMP/cGMP ratio for intact CD8+-lymphocyte cultures was 4.59±0.23 s.u., while for CD8+-lymphocyte cultures exposed to Shigella LPS in the concentration of 1, 10, and 100mcg/ml this value was respectively1.07, 1.18, and 1.88 times lower (р>0.05, р<0.05 и р<0.001).The findings of this study show that Shigella sonnei LPS are a biologically active substance which can affect cyclic nucleotide concentrations in subpopulations of immunoregulatory T-lymphocytes.Conclusion. Direct exposure in vitro to Shigella sonnei LPS has a dose- and time-dependent effect on cyclic nucleotides in subpopulations of T-lymphocytes. These LPS cause an increase in intracellular cAMP and a decrease in cGMP concentrations in CD4+-lymphocyte subpopulations, while both cAMP and cGMP values increase in CD8+-lymphocyte subpopulation.