mimmunМедицинская иммунологияMedical Immunology (Russia)1563-06252313-741XSPb RAACI10.15789/1563-0625-2019-4-689-702mimmun-1652Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESЗАВИСИМОСТЬ ФЕНОТИПА ДЕНДРИТНЫХ КЛЕТОК ОТ СОДЕРЖАНИЯ ПРОВОСПАЛИТЕЛЬНЫХ МОНОЦИТОВ КРОВИ У БОЛЬНЫХ РАКОМ ПОЧКИINTERDEPENDENCE BETWEEN THE PHENOTYPE OF DENDRITIC CELLS AND AMOUNTS OF BLOOD PROINFLAMMATORY MONOCYTES IN PATIENTS WITH KIDNEY CANCERСавченкоА. А.SavchenkoA. A.

д.м.н., профессор, руководитель лаборатории клеточно-молекулярной физиологии и патологии

Красноярск

PhD, MD (Medicine), Professor, Head, Laboratory of Molecular and Cellular Physiology and Pathology

Krasnoyarsk

aasavchenko@yandex.ruБорисовА. Г.BorisovA. G.

к.м.н., ведущий научный сотрудник лаборатории клеточно-молекулярной физиологии и патологии

Красноярск

PhD (Medicine), Leading Research Associate, Laboratory of Molecular and Cellular Physiology and Pathology

Krasnoyarsk

2712939@mail.ruКудрявцевИ. В.KudryavtsevI. V.

к.б.н., с.н.с. отдела иммунологии; доцент кафедры иммунологии

197376, Россия, Санкт-Петербург, ул. Акад. Павлова, 12

Тел.: 8 (812) 234-16-69

PhD (Biology), Senior Research Associate, Department of Immunology, ; Assistant Professor, Department of Immunology

197376, Russian Federation, St. Petersburg, Acad. Pavlov str., 12

Phone: 7 (812) 234-16-69

igorek1981@yandex.ruМошевА. В.MoshevA. V.

младший научный сотрудник лаборатории клеточно-молекулярной физиологии и патологии

Красноярск

Junior Research Associate, Laboratory of Molecular and Cellular Physiology and Pathology

Krasnoyarsk

kinger1@mail2000.ruФГБНУ Федеральный исследовательский центр «Красноярский научный центр Сибирского отделения Российской академии наук», обособленное подразделение «НИИ медицинских проблем Севера»РоссияResearch Institute of Medical Problems of the North, Krasnoyarsk Science Center, Siberian Branch, Russian Academy of SciencesRussian FederationФГБНУ "Институт экспериментальной медицины"; ГБОУ ВПО «Первый Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова» Министерства здравоохранения РФРоссияInstitute of Experimental Medicine, First St. Petersburg State I. Pavlov Medical UniversityRussian Federation201911032019214препринт689702Copyright © Савченко А.А., Борисов А.Г., Кудрявцев И.В., Мошев А.В., 20192019Савченко А.А., Борисов А.Г., Кудрявцев И.В., Мошев А.В.Savchenko A.A., Borisov A.G., Kudryavtsev I.V., Moshev A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.mimmun.ru/mimmun/article/view/1652

Целью исследования явилось изучение зависимости фенотипа дендритных клеток (ДК), дифференцированных из моноцитов, от количества провоспалительных моноцитов в крови у больных раком почки (РП). Обследовано 28 больных РП (Т3N0М0, светлоклеточный тип) в возрасте 40-55 лет до хирургического лечения. Диагноз верифицирован гистологически. В качестве контрольной группы проведено обследование 31 практически здоровых людей аналогичного возрастного диапазона. Мононуклеарные клетки выделяли из гепаринизированной венозной крови центрифугированием в градиенте плотности Histopaque®-1077 с последующей адсорбцией на пластике в среде RPMI-1640 в присутствии 10% аутологичной сыворотки. Незрелые ДК (нДК) генерировали из моноцитов крови путем культивирования в течение 5 суток с GM-CSF и IFNα. Активацию ДК (аДК) индуцировали внесением в среду инкубации лизата опухолевых клеток и TNFa с последующей инкубацией в течение 48 часов. Для приготовления лизата аутологичных опухолевых клеток использовали фрагмент опухоли. Фенотипирование моноцитов крови и ДК различной степени зрелости проводили методом проточной цитометрии. У больных РП в периферической крови снижается количество CD14+CD16+-моноцитов (до 42% от уровня общих моноцитов) относительно контрольного диапазона. В связи с этим, анализ зависимости фенотипа ДК, дифференцированных из моноцитов, от количества провоспалительных моноцитов в крови был проведен путем сравнения показателей с высоким содержанием провоспалительных моноцитов в крови у больных РП (>42%, приближается к уровню контрольного диапазона) и низким (<42%). Установлено, что у больных РП с низким количеством в крови провоспалительных моноцитов (<42%) повышается содержание толерогенных нДК в клеточной культуре. Особенностью фенотипа нДК у пациентов с высоким содержанием провоспалительных моноцитов в крови (>42%) является относительное увеличение экспрессии молекул, осуществляющих антигенпрезентацию и костимуляцию. При созревании/активации фенотип дендритных клеток у больных РП с различным содержанием провоспалительных моноцитов различается сильнее. У больных с низким уровнем провоспалительных моноцитов в крови в клеточной культуре формируется пул зрелых ДК с низким уровнем экспрессии CD86- и HLA-DR-рецепторов, что, соответственно характеризует слабую костимулирующую и антигенпрезентирующую активность. У больных с высоким уровнем провоспалительных моноцитов в клеточной культуре формируется пул активированных ДК с высоким уровнем функциональной активности. Выявленные различия фенотипа ДК от субпопуляционного состава моноцитов крови у больных РП характеризуют механизмы программирования клеточной дифференцировки в зависимости от микроокружения, в том числе и патогенного характера (на фоне опухолевого роста).

The aim of the study was to investigate an interdependence between the phenotype of dendritic cells (DC) differentiated from monocytes and the number of pro-inflammatory monocytes in peripheral blood of patients with kidney cancer (KC). The study involved 28 patients at the age of 40-55 years suffering with KC (Т3N0М0, clear cell type) before surgical treatment. The diagnosis was verified histologically. 31 healthy agematched persons were examined as a control group. Mononuclear cells were isolated from heparinized venous blood by centrifugation in a Histopaque®-1077 density gradient followed by plastic adsorption in RPMI 1640 medium supplied with 10% autologous serum. Immature DCs (iDCs) were generated from blood monocytes by culturing for 5 days with GM-CSF and IFNα. Activation of DCs (mDCs) was induced by incubation with the tumor cell lysate and TNFα, followed by incubation for 48 hours. A tumor fragment was used to prepare the lysate of autologous tumor cells. Phenotyping of blood monocytes and DC at various maturation stages was performed by flow cytometry. The numbers of CD14+CD16+ monocytes in peripheral blood of KC patients were decreased (up to 42% of the total monocyte level) against the control ranges. In this regard, the analysis of the dependence between the phenotype of DCs differentiated from monocytes and the number of pro-inflammatory blood monocytes was carried out by comparing the groups with a high content of pro-inflammatory monocytes in the blood in KC patients (> 42%, near-control range) and low content (resp., < 42%). We have found that the contents of tolerogenic iDC in cell culture are increased in KC patients with low amounts of pro-inflammatory monocytes in blood (< 42%). A relatively increased expression of antigen-presenting and co-stimulatory molecules proved to be the specific feature of iDC phenotype in patients with high contents (> 42%) of proinflammatory monocytes in blood. The phenotype of dendritic cells in KC patients with different content of proinflammatory monocytes during maturation/activation showed more differences. In the patients with low levels of pro-inflammatory monocytes, the cell pool of in vitro maturing DCs was characterized by low level of CD86 and HLA-DR receptor expression, thus reflecting a weak co-stimulating and antigen-presenting activity. In the patients with high levels of pro-inflammatory monocytes in blood, the in vitro activated DCs showed higher level of functional activity using the above markers. The revealed differences in the DC phenotype and interrelations with amounts of blood monocyte subpopulations in KC patients may presume the programmed cell differentiation mechanisms depending on the microenvironment, under pathogenic conditions (i.e., in presence of malignant tumor growth).

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The authors declare that there are no conflicts of interest present.