RUDN Journal of MedicineRUDN Journal of MedicineВестник Российского университета дружбы народов. Серия: Медицина2313-02452313-0261Peoples’ Friendship University of Russia named after Patrice Lumumba (RUDN University)2502510.22363/2313-0245-2020-24-4-304-314EXPERIMENTAL PHYSIOLOGYЭКСПЕРИМЕНТАЛЬНАЯ ФИЗИОЛОГИЯResearch ArticleRegulatory cytokine effects in vitro on the phenotype of subpopulations CD62L+CD63-, CD62L+CD63+ and microbicidal activity of neutrophilic granulocytes in patients with colorectal cancerОсобенности влияний in vitro регуляторных цитокинов на фенотип субпопуляций CD62L+CD63-, CD62L+CD63+ и микробицидную активность нейтрофильных гранулоцитов пациентов с колоректальным раком2092-6412ChudilovaG. A.ЧудиловаГ. А.chudilova2015@yandex.ru4714-2488NesterovaI. V.НестероваИ. В.chudilova2015@yandex.ru8289-5342KovalevaS. V.КовалеваС. В.chudilova2015@yandex.ru2060-9316LomtatidzeL. V.ЛомтатидзеЛ. В.chudilova2015@yandex.ruKuban State Medical UniversityКубанский государственный медицинский университет15122020244EXPERIMENTAL AND CLINICAL PHYSIOLOGYЭКСПЕРИМЕНТАЛЬНАЯ И КЛИНИЧЕСКАЯ ФИЗИОЛОГИЯ30431423112020Copyright ©; 2020, Chudilova G.A., Nesterova I.V., Kovaleva S.V., Lomtatidze L.V.Copyright ©; 2020, Чудилова Г.А., Нестерова И.В., Ковалева С.В., Ломтатидзе Л.В.2020Chudilova G.A., Nesterova I.V., Kovaleva S.V., Lomtatidze L.V.Чудилова Г.А., Нестерова И.В., Ковалева С.В., Ломтатидзе Л.В.http://creativecommons.org/licenses/by/4.0https://journals.rudn.ru/medicine/article/view/25025

Relevance. Neutrophilic granulocytes (NG) are the first cells of the immune system to migrate to the tumor and are actively involved in the implementation of a full-fledged antitumor response through the mechanisms of direct killing of tumor cells, extracellular lysis (NET), and through the activation of antibody-dependent cellular cytotoxicity (ADCC), inhibition of angiogenesis, initiation of other cells with antitumor activity. The aim of the study was to study the effect of cytokines IFNα, IFNγ, G-CSF on the CD62L+CD63- and CD62L+CD63+ subsets and the microbicidal activity of NGs in patients with colorectal cancer (CRC) in vitro. Materials and methods. We studied samples of peripheral blood (PB) of 10 patients of both sexes 38-70 years old with newly diagnosed untreated CRC stage II-III (study group) and 10 healthy volunteers (comparison group). The subsets CD62L+CD63+ NG, CD62L+CD63- NG were assessed by flow cytometry (CYTOMICS FC500, Beckman Coulter, USA), the microbicidal functions of NG were tested by cytochemical methods: activity of NADPH - oxidases, myeloperoxidase (MP), level of cationic protein (CP) in spontaneous tests and under additional stress of S. aureus . The effect of IFNα, IFNγ, G-CSF cytokines on subsets and the microbicidal activity of NG in vitro was studied in both study groups. Microsoft Exel 2016 and StatPlus 2010 were used for statistical processing of the obtained data using nonparametric tests: Me (Q1; Q3), Mann-Whitney U-test and Wilcoxon test . Results . The features of transformation of CD62L+ CD63-NG and CD62L+ CD63+ NG subsets of PB in CRC have been established, that allows to get an idea of the NG ability to roll and readiness to activate the microbicidal arsenal, various defects of spontaneous and induced microbicidal activity of oxygendependent and oxygen-independent mechanisms of NG. The effects of cytokine influence on NG in CRC in vitro have been shown, which indicates the possibility of regulating the receptor and microbicidal functions of NG, and, on the other hand, suggests defects in NG perception of regulatory stimuli, that is confirmed by the progression of tumor growth.

Актуальность. Нейтрофильные гранулоциты (НГ) первыми из клеток иммунной системы мигрируют к опухоли и активно вовлекаются в реализацию полноценного противоопухолевого ответа через механизмы прямого киллинга клеток опухоли, внеклеточного лизиса (NET), так и через активацию антителозависимой клеточной цитотоксичности (АЗКЦ), ингибирование ангиогенеза, инициации других клеток, обладающих противоопухолевой активностью. Цель: изучить влияние цитокинов IFNa, IFNg, G-CSF на субпопуляции CD62L+CD63- и CD62L+CD63+ и микробицидную активность НГ пациентов с колоректальным раком (КРР) в системе in vitro. Материалы и методы . Исследованы образцы периферической крови (ПК) 10 пациентов обоего пола 38-70 лет с впервые выявленным нелеченым КРР II-III стадии (группа исследования) и 10 условно-здоровых добровольцев (группа сравнения). Проведена оценка субпопуляций CD62L+CD63+НГ, CD62L+CD63-НГ методом проточной цитометрии (CYTOMICS FC500, Beckman Coulter, США), цитохимическими методами тестировались микробицидные функции НГ: активность NADPH-оксидаз, миелопероксидазы (МП), уровень катионного белка (КБ) в спонтанных тестах и при дополнительной нагрузке S.aureus . В обеих исследуемых группах изучалось влияние цитокинов IFNα, IFNγ, G-CSF на субпопуляции и микробицидную активность НГ системе in vitro. Данные подвергали обработке непараметрическими методами статистики с помощью программ Microsoft Exel 2016 и StatPlus 2010, результаты представлены как медиана с интерквартильным размахом - Ме (Q1; Q3). Значимость различий между группами рассчитывали с использованием U-критерия Манна-Уитни и критерия Вилкоксона. Результаты . Установлены особенности трансформации субпопуляций CD62L+CD63-НГ и CD62L+CD63+НГ ПК при КРР, позволяющие получить представление о способности НГ к роллингу и готовности активировать микробицидный арсенал, различные дефекты спонтанной и индуцированной микробицидной активности кислород зависимых и кислород независимых механизмов НГ. В эксперименте in vitro показаны эффекты влияния цитокинов на НГ при КРР, что свидетельствуют о возможности регулирования рецепторных и микробицидных функций НГ, с другой стороны, позволяют говорить о дефектах восприятия НГ регулирующих стимулов, что подтверждается прогрессированием опухолевого роста.

colorectal cancerneutrophilic granulocytessubsetcytokinesmicrobicidal functionsколоректальный ракнейтрофильные гранулоцитысубпопуляциицитокинымикробицидные функцииThe study was funded by the Ministry of health of the Russian Federation (project no. AAAA-A18–118122690053–0).Исследование выполнено в рамках государственного задания Министерства здравоохранения Российской Федерации (проект № АААА-А18–118122690053–0).Cancer Research UK. Cancer statistics. available at: www. cancerresearchuk.org. Access date: 10/08/2020.Cancer Research UK. Cancer statistics. available at: www. cancerresearchuk.org. Дата доступа: 10/08/2020.Mantovani A., Romero P., Palucka A.K., Marincola F.M. Tumour immunity: effector response to tumor and role of the microenvironment. Lancet. 2008; 371:771–783.Mantovani A., Romero P., Palucka A.K., Marincola F.M. Tumour immunity: effector response to tumour and role of the microenvironment. Lancet. 2008. V. 371. P. 771-783.McLean M.H., Murray G.I., Stewart K.N., Norrie G., Mayer C., Hold G.L., Thomson J., Fyfe H., Mowat N.A., Drew E.J., El-Omar M.E. The inflammatory microenvironment in colorectal neoplasia. PLoS One. 2011;6(1): e15366. doi:10.1371/journal.pone.0015366.McLean M.H., Murray G.I., Stewart K.N., Norrie G., Mayer C., Hold G.L., Thomson J., Fyfe H., Mowat N.A., Drew E.J., ElOmar M.E. The inflammatory microenvironment in colorectal neoplasia. PLoS One. 2011. V.6(1): e15366. doi:10.1371/journal. pone.0015366.Swierczak A., Mouchemore K.A., Hamilton J.A., Anderson R.L. Neutrophils: important contributors to tumor progression and metastasis. Cancer Metastasis. 2015;34:735–751. doi:10.1007/ s10555–015–9594–9Swierczak A., Mouchemore K.A., Hamilton J.A., Anderson R.L. Neutrophils: important contributors to tumor progression and metastasis. Cancer Metastasis. 2015. V. 34. P. 735-751. doi:10.1007/s10555-015-9594-9Sionov R.V., Fridlender Z.G., Granot Z. The multifaceted roles neutrophils play in the tumor microenvironment. Cancer Microenviron. 2015;8:125–158. doi:10.1007/s12307–014–0147–5Sionov R.V., Fridlender Z.G., Granot Z. The multifaceted roles neutrophils play in the tumor microenvironment. Cancer Microenviron. 2015. V. 8. P. 125-158. doi:10.1007/s12307- 014-0147-5Coffelt S.B., Wellenstein M.D., de Visser K.E. Neutrophils in cancer: neutral no more. Nat. Rev. Cancer. 2016;16:431–446. doi:10.1038/nrc.2016.52Coffelt S.B., Wellenstein M.D., de Visser K.E. Neutrophils in cancer: neutral no more. Nat. Rev. Cancer. 2016. V. 16. P. 431-446. doi:10.1038/nrc.2016.52Mishalian I., Granot Z., Fridlender Z.G. The diversity of circulating neutrophils in cancer. Immunobiology. 2017;222:82–88. doi:10.1016/j.imbio.2016.02.001Mishalian I., Granot Z., Fridlender Z.G. The diversity of circulating neutrophils in cancer. Immunobiology. 2017. V. 222. P. 82-88. doi:10.1016/j.imbio.2016.02.001Kabayashi Y. The role of chemokines in neutrophil biology Frontiers in bioscience: a journal and virtual library. 2008;13:2400–2407.Kabayashi Y. The role of chemokines in neutrophil biology. Frontiers in bioscience: a journal and virtual library. 2008. V. 13. P. 2400-2407.Khajah M., Millen B., Cara D.C., Waterhouse C., Mc Cafferty D. Granulocyte-macrophage colony-stimulating factor (GM–CSF): a chemoattractive agent for murine leukocytes in vivo. Journal of Leukocyte Biology. 2011; 89 (6):945–953.Khajah M., Millen B., Cara D.C., Waterhouse C., Mc Cafferty D. Granulocyte-macrophage colony-stimulating factor (GM-CSF): a chemoattractive agent for murine leukocytes in vivo. Journal of Leukocyte Biology. 2011. V. 89 (6). P. 945-953.Dumitru C.A., Lang S., Brandau S. Modulation of neutrophil granulocytes in the tumor microenvironment: Mechanisms and consequences for tumor progression. Seminars in Cancer Biology. 2013;23:141–148.Dumitru C.A., Lang S., Brandau S. Modulation of neutrophil granulocytes in the tumor microenvironment: Mechanisms and consequences for tumor progression. Seminars in Cancer Biology. 2013. Vol. 23. P. 141-148.Mishalian I., Granot Z., Fridlender Z.G. The diversity of circulating neutrophils in cancer.Immunobiology. 2017;222 (1):82–88.Mishalian I., Granot Z., Fridlender Z.G. The diversity of circulating neutrophils in cancer. Immunobiology. 2017. V. 222 (1). P. 82-88.Zhang X., Zhang W., Yuan X., Fu M., Qian H., Xu W. Neutrophils in cancer development and progression: Roles, mechanisms, and implications (Review). International journal of oncology. 2016; 49(3):857–867.Zhang X., Zhang W., Yuan X., Fu M., Qian H., Xu W. Neutrophils in cancer development and progression: Roles, mechanisms, and implications (Review). International journal of oncology. 2016. V. 49(3). P. 857-867.Lau D., Mollnau H., Eiserich J.P., Freeman B.A., Daiber A., Gehling U.M., Brümmer J., Rudolph V., Münzel T., Heitzer T., Meinertz T., Baldus S. Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins. Proceedings of the National Academy of Sciences. 2005;102 (2):431–436.Lau D., Mollnau H., Eiserich J.P., Freeman B.A., Daiber A., Gehling U.M., Brümmer J., Rudolph V., Münzel T., Heitzer T., Meinertz T., Baldus S. Myeloperoxidase mediates neutrophil activation by association with CD11b/CD18 integrins. Proceedings of the National Academy of Sciences. 2005. V.102 (2). Р.431-436.Mantovani А. The yin-yang of tumor-associated neutrophils. Cancer Cell. 2009;8:173–174.Mantovani А. The yin-yang of tumor-associated neutrophils. Cancer Cell. 2009. V. 8. Р.173-174.Levy O. Innate immunity of the newborn: basic mechanisms clinical correlates. Nature reviews Immunology. 2007;7(5): 379–390.Levy O. Innate immunity of the newborn: basic mechanisms clinical correlates. Nature reviews Immunology. 2007. V. 7(5). P. 379-390.Lin A., Lore K. Granulocytes: New Members of the AntigenPresenting Cell Family. Frontiers in Immunology. 2017;11. doi:10.3389/fimmu.2017.01781Lin A., Lore K. Granulocytes: New Members of the AntigenPresenting Cell Family. Frontiers in Immunology. 2017. V. 11. doi:10.3389/fimmu.2017.01781Dallegri F., Ottonello L., Ballesterro A., Patrizia Dapino, F. Ferrando, F. Patrone, C. Sacchetti Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen. Inflammation. 1991; 1(15):15–30.Dallegri F., Ottonello L., Ballesterro A., Patrizia Dapino, F. Ferrando, F. Patrone, C. Sacchetti Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen. Inflammation. 1991. V. 1. № 15. P. 15-30.Lai X., Gu Q., Zhou X., Feng W., Lin X., He Y., Cao J., Liu P., Zhang H., Zheng X. Decreased expression of CD63 tetraspanin protein predicts elevated malignant potential in human esophageal cancer. Oncol Lett. 2017;13(6):4245–4251. DOI: 10.3892/ol.2017.6023.Lai X., Gu Q., Zhou X., Feng W., Lin X., He Y., Cao J., Liu P., Zhang H., Zheng X. Decreased expression of CD63 tetraspanin protein predicts elevated malignant potential in human esophageal cancer. Oncol Lett. 2017. V. 13(6). Р. 4245-4251. doi: 10.3892/ ol.2017.6023.Liu W.H., Li X., Zhu X.L., Hou M.L., Zhao W. CD63 inhibits the cell migration and invasion ability of tongue squamous cell carcinoma. Oncol Lett. 2018;(6): 9033–9042. DOI: 10.3892 / ol.2018.8499.Liu W.H., Li X., Zhu X.L., Hou M.L., Zhao W. CD63 inhibits the cell migration and invasion ability of tongue squamous cell carcinoma. Oncol Lett. 2018. V.(6). P. 9033-9042. doi: 10.3892 / ol.2018.8499.Beinert T., Munzing S., Possinger K., Krombach F. Increased expression of the tetraspanins CD53 and CD63 on apoptotic human neutrophils.Journal of Leukocyte Biology. 2000; 67:369–373.Beinert T., Munzing S., Possinger K., Krombach F. Increased expression of the tetraspanins CD53 and CD63 on apoptotic human neutrophils. Journal of Leukocyte Biology. 2000. V. 67. P. 369-373.Droeser R.A., Hirt C., Eppenberger-Castori S., Zlobec I., Viehl C.T., Frey D.M., Nebiker C.A., Rosso R., Zuber M., Amicarella F. High myeloperoxidase positive cell infiltration in colorectal cancer is an independent favorable prognostic factor. PLoS One. 2013; 8: e64814.Droeser R.A., Hirt C., Eppenberger-Castori S., Zlobec I., Viehl C.T., Frey D.M., Nebiker C.A., Rosso R., Zuber M., Amicarella F. High myeloperoxidase positive cell infiltration in colorectal cancer is an independent favorable prognostic factor. PLoS One. 2013. V. 8. e64814.Roncucci L., Mora E., Mariani F., Bursi S., Pezzi A., Rossi G., Pedroni M., Luppi D., Santoro L., Monni S. Myeloperoxidasepositive cell infiltration in colorectal carcinogenesis as indicator of colorectal cancer risk. Cancer Epidemiol Biomarkers Prev. 2008; 17: 2291–2297.Roncucci L., Mora E., Mariani F., Bursi S., Pezzi A., Rossi G., Pedroni M., Luppi D., Santoro L., Monni S. Myeloperoxidasepositive cell infiltration in colorectal carcinogenesis as indicator of colorectal cancer risk. Cancer Epidemiol Biomarkers Prev. 2008. V. 17. P. 2291-2297.Rao H.L., Chen J.W., Li M., Xiao Y.B., Fu J., Zeng Y.X., Cai M.Y., Xie D. Increased intratumoral neutrophil in colorectal carcinomas correlates closely with malignant phenotype and predicts patients’ adverse prognosis. PLoS One. 2012;7: e30806Rao H.L., Chen J.W., Li M., Xiao Y.B., Fu J., Zeng Y.X., Cai M.Y., Xie D. Increased intratumoral neutrophil in colorectal carcinomas correlates closely with malignant phenotype and predicts patients’ adverse prognosis. PLoS One. 2012. V. 7. e30806.Gustafson M.P., Lin Y., Maas M.L., Van Keulen V.P., Johnston P.B., Peikert T., Gastineau D.A., Dietz A.B. A method for identification and analysis of non-overlapping myeloid immunophenotypes in humans. PLoS One. 2015; 10: e0121546.Gustafson M.P., Lin Y., Maas M.L., Van Keulen V.P., Johnston P.B., Peikert T., Gastineau D.A., Dietz A.B. A method for identification and analysis of non-overlapping myeloid immunophenotypes in humans. PLoS One. 2015. V. 10. e0121546.