Влияние глутамата на миграцию Т-клеток здоровых доноров и больных рассеянным склерозом в модели in vitro

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Introduction. The primary excitatory neurotransmitter of the nervous system, glutamate, was also observed in cells of the immune system. T cells express functional glutamate receptors and secrete glutamate, affecting neighboring cells. Glutamate is able to activate lymphocytes inducing their key functions. Migration of autoreactive T lymphocytes from blood vessels to the central nervous system is an important step in the pathogenesis of multiple sclerosis (MS). According to the literature, elevated glutamate levels affect T cells, which penetrate across blood brain barrier, leading to destruction of neuronal myelin sheath in MS [1]. The process of cell migration mediated by glutamate is controlled by many factors, which are not fully understood. Materials and methods. PBMCs were purified from blood of 15 patients with relapsing-remitting MS in relapse phase and 15 healthy donors by density gradient centrifugation of ficoll. T cell migration was studied in vitro using transwells. Resting or activated by phorbol myristate acetate (РМА, 10 ng/mL) PBMCs (2 · 105 cells per sample) were loaded in transwells with fibronectin-coated (10 μg/mL) membrane. Low chamber contained either glutamate, AMPA or NMDA (100 μM for each) in complete RPMI medium. After migration assay cells were collected, incubated with monoclonal antibodies against CD3 marker and labeled with secondary antibodies Alexa Fluor 488. Cell migration was determined by flow cytometry while analyzing the number of events per 1 minute. Results and discussion. The number of CD3+ cells in healthy donors and MS patients was consistent and did not change during 2 hours incubation with agonists of glutamate receptors. In presence of glutamate there was a tendency to inhibition of resting T cells migration activity in both groups of donors. NMDA causes a 25% decrease in T-cell chemotaxis of healthy donors, but not MS patients. Upon PMA activation of T-lymphocytes a reduction in number of migrated cells by 17% (p < 0.01) was observed in both groups of donors. In presence of glutamate there is a tendency to an increase of PMA-stimulated T cells chemotaxis in MS patients, and to a decrease with AMPA, when compared with the control group. Activated T-lymphocytes from healthy donors migrated more readily toward the AMPA and NMDA. Thus, we conclude that glutamate and selective agonists of its receptors do not possess noticeable chemoattractant effect. Moreover, there is a tendency to a decrease in the intensity of T-lymphocyte migration in the gradient of their concentration, both at rest and activation. This effect was observed with all studied ligands in MS patients. It should be noted that cell migration might be associated not only with chemoattractant properties of receptor ligands (directed migration) but also with an increase in motility and adhesive properties of cells (non-directional migration), which is probably observed in relation to glutamate and T-lymphocytes. Table The effect of glutamate receptor agonists on T cell migration in healthy donors and MS patients in vitro Healthy MS Rest PMA Rest PMA Control 59.15 ± 10.57 43.98 ± 17.36 66.79 ± 11.55 44.24 ± 22.19 Glutamate 54.15 ± 4.78 45.44 ± 14.84 49.36 ± 11.82 50.27 ± 9.80 AMPA 55.51 ± 5.24 49.48 ± 13.69 59.27 ± 19.13 38.73 ± 8.12 NMDA 44.59 ± 20.63 53.86 ± 11.05 60.46 ± 13.23 46.48 ± 9.62 Note. The data is presented as the mean ± standard deviation.

Об авторах

М А Максимова

ФГБУН «Институт биохимии и генетики» Уфимского научного центра РАН

У Ш Кузьмина

ФГБУН «Институт биохимии и генетики» Уфимского научного центра РАН

К З Бахтиярова

ФГБОУ ВО «Башкирский государственный медицинский университет» Минздрава РФ

Ю В Вахитова

ФГБУН «Институт биохимии и генетики» Уфимского научного центра РАН

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