ISSN 16076729, Doklady Biochemistry and Biophysics, 2015, Vol. 462, pp. 163–165. © Pleiades Publishing, Ltd., 2015. Original Russian Text © I.S. Starskaya, I.V. Kudryavtsev, V.V. Guselnikova, M.K. Serebriakova, A.V. Polevshchikov, 2015, published in Doklady Akademii Nauk, 2015, Vol. 462, No. 2, pp. 238–240.
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
Apoptosis Level in Developing T Cells in the Thymus I. S. Starskayaa, I. V. Kudryavtseva, V. V. Guselnikovab, M. K. Serebriakovaa, and A. V. Polevshchikovc Presented by Academician E.A. Korneva November 21, 2014 Received November 25, 2014
DOI: 10.1134/S1607672915030060
The thymus as the central organ of the immune system is responsible for the maturation of T cells. It is assumed that the formation of the Tcell receptor (TCR) is accompanied by the destruction of over 95% of CD4+CD8+ cells. This hypothesis is based on the results of studies obtained in 1960–1970 in vitro, which were extrapolated to the processes in vivo with out experimental confirmation [1, 2]. The mathemat ical models of the processes developed to date, which are based on the existing hypothesis, predict the death of 75% of thymocytes in the course of selection [3, 4]. Solving the problem on the level of apoptosis in maturing T cells is essential for the validation of the model of their intrathymic maturation, including pos itive and negative selection as a prerequisite. The morphological analysis of intact mammalian thymus does not reveal the whole picture of massscale apoptosis of maturing cells [5]. The use of flow cytom etry also showed that the level of apoptosis in rat thy mus reaches its maximum on prenatal day 18 (but accounts for only 25% of thymocytes), after which it drops to 5% and remains at this level in the postnatal period [6]. Due to the heterogeneity of published data and a great theoretical importance, the purpose of our work was to estimate the level of apoptosis in intact thymus by comparing the results of immunohistochemical analysis and the data obtained by flow cytometry. The study was performed with 6–10weekold out bred male mice and male CBA/JRap mice weighing 18–20 g. The animals were housed under standard vivarium conditions and received water and feed ad libitum. Work with animals were performed in accor
a
Institute of Experimental Medicine, Russian Academy of Medical Sciences, ul. akademika Pavlova 12, St. Petersburg, 197376 Russia email: [email protected] b St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg, 199034 Russia c Far Eastern Federal University, Vladivostok, Russia
dance with the rules of the Commission on Bioethics of the Research Institute of Experimental Medicine. The level of apoptosis was assessed using panspe cific caspase inhibitors FAM FLICA Poly Caspase Assay Kit conjugated with fluorochromes [7] (Immu nochemistry, United States); the results were expressed as percentage of FLICA+ cells. The level of proliferating cells in the thymus was determined using the antibodies to the nuclear protein PCNA (BioLeg end, United States); the results were expressed as per centage of PCNA+ cells. The main populations of thy mocytes were identified using the monoclonal anti bodies against mouse CD4, CD8, and CD45, which were conjugated with PE/Cy5, PE/Cy7, and APC/Cy7, respectively (BioLegend). All experiments were performed according to the protocols provided by the manufacturers. Samples were analyzed using a Navios flow cytometer (Beckman Coulter, United States). Paraffin sections of the thymus were prepared as described in [8]. To determine the localization of apo ptotic cells in the thymus, we performed immunohis tochemical staining of sections with the antibodies against caspase3 in accordance with the protocol provided by the manufacturer (Abcam, United States). To identify the cells, the sections were stained with an aqueous solution of Aster Blue, embedded in paraffin, and visualized and photographed using a Leica DM6000 microscope (Leica, Germany). Table 1 shows the results of determination of the level of apoptosis and proliferation in the major cell populations of the thymus. The largest proportion of FLICA positive cells was accounted for the popula tion of the most immature CD4–CD8– thymocytes. The level of apoptosis in the population of mature CD4+CD8– and CD4–CD8+ cells was somewhat higher than in the population of doublepositive thy mocytes CD4+CD8+, in which the proportion of apo ptotic cells, contrary to conventional notion, is mini mal. The animals injected with hydrocortisone (2.5 mg per mouse) to induce apoptosis during the thymus atrophy served as a positive control. In addi tion to the decrease in the linear size (from 9.2 ± 0.3 to
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ulations of mature cells (CD4+CD8– and CD4–CD8+), on the contrary, were characterized by the prevalence of the levels of apoptosis over the level of proliferation, which indicated the selection processes in these cells. Figure shows the localization of caspase3con taining apoptotic cells in the thymus of mice. Panels 1 and 2 in figure confirm the data presented in the table on the low level of apoptosis in the thymus cortex, containing immature CD4–CD8– and CD4+ CD8+ cells and a higher level of apoptotic cells in the medulla, containing mature lymphocytes CD4+ and CD8+. Panel 3 in figure illustrates the increase in the level of apoptosis of immature cortical cells as early as 6 h after the administration of hydrocortisone. In the medulla, the proportion of apoptotic cells did not change significantly (panel 4 in figure), which is consistent with the concept on a high resistance of mature thymocytes to the action of glucocorticoid hormones [9]. The results obtained in this study revealed a low level of apoptosis in the immature cells CD4+CD8+ along with a significant number of proliferating cells. The results obtained by flow cytometry and immuno histochemistry indicate a high levels of apoptosis in the medullary zone of the thymus, where mature cyto toxic (CD8+) and helper (CD4+) T cells are located. This finding essentially diverges from the dominant hypothesis on the elimination of 95–99% of double positive CD4+CD8+ T cells in the course of selection. Meanwhile, the high level of apoptosis in this popula tion is one of the main bases of the modern theory of
The level of apoptosis and proliferation of thymocytes in dif ferent populations of intact mice (M ± m, n ≥ 80 in determi nation of each parameter) Percent Proportion of the total in the population, % Population number of thy FLICA+ cells PCNA+ cells mocytes CD4–CD8– 2.32 ± 0.79 CD4+CD8+ 86.49 ± 0.82 CD4+CD8– 8.69 ± 1.20 CD4–CD8+ 1.49 ± 0.20
15.34 ± 0.79 4.34 ± 0.11 6.55 ± 0.28 10.11 ± 1.02
20.21 ± 4.50 30.67 ± 2.31 1.67 ± 0.39 6.66 ± 0.98
5.0 ± 0.2 mm, p < 0.01; hereinafter, M ± m) and the weight of this organ (from 70.57 ± 8.14 to 29.97 ± 1.62 mg, p < 0.01), we observed a significant increase in the proportion of FLICA+ cells in the populations of CD4–CD8– cells (up to 48.26 ± 0.65%) and CD4+CD8+ cells (up to 66.93 ± 1.12% of the total number of cells), which was consistent with the pub lished data [9] and demonstrated the adequacy of the obtained results in the estimation of the level of apop tosis in the thymus of intact animals. Table summarizes the results of determination of the level of proliferation in the thymus. The popula tions of immature thymocytes (CD4–CD8– and CD4+CD8+) were characterized by a greater propor tion of proliferating cells compared to the proportion of cells at different stages of apoptosis, whereas the pop
1
2
3
4
Fig. 1. Localization of apoptotic cells in intact thymus and after hydrocortisone administration. Intact thymus: (1) cortex and (2) cortex and medulla; 6 h after the hydrocortisone administration: (3) cortex and (4) medulla. Staining with antibodies against caspase3. Magnification, ×40. DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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differentiation of T cells, which implies the stochastic nature of the TCR formation and the presence of pro cesses of positive and negative selection in the course of further maturation of T cells. Thus, the level of apoptosis of CD4+CD8+ thy mocytes in the thymus of adult intact mice does not exceed 5%, which contradicts the existing hypothesis about the maturation and differentiation of T cells. The percentage of apoptotic cells in the medullary zone of the thymus is much higher than in the cortex, which indirectly indicates the selection processes pri marily in the medullary zone of the thymus, contain ing mature thymocytes CD4+CD8– and CD4–CD8+.
2. Schlesinger, M., Gottesfeld, S., and Korzash, Z., Cell Immunol., 1973, vol. 6, no. 1, pp. 49–58.
ACKNOWLEDGMENTS This work was supported by the program of the Far Eastern Federal University (program no. 1326, project no. 14080625_i) and the Russian Foundation for Basic Research (project no. 150405093a).
7. Kudryavtsev, I.V., Golovkin, A.S., Zurochka, A.V., and Khaidukov, S.V., Med. Immunol., 2012, vol. 14, no. 60, pp. 461–482.
REFERENCES
9. Elmore, S.A., Toxicol. Pathol., 2006, vol. 34, no. 5, pp. 656–665.
1. Scollay, R., Kochen, M., Butcher, E., and Weissman, I., Nature, 1978, vol. 276, no. 5683, pp. 79–80.
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 462
3. Sawicka, M., Stritesky, G.L., Reynolds, J., Aboura shchi, N., Lythe, G., MolinaParis, C., and Hogquist, K.A., Front. Immunol., 2014, vol. 5. 4. Sinclair, C., Bains, I., Yates, A.J., and Seddon, B., Proc. Nat. Acad. Sci. U.S.A., 2013, vol. 110, no. 4, pp. 2905–2914. 5. Suhr, C.D. and Sprent, J., Nature, 1994, vol. 372, no. 6501, pp. 100–103. 6. Mel’nikova, V.I., Afanas’eva, M.A., Sapozhnikov, A.M., and Zakharova, L.A., Ontogenez, 2006, vol. 37, no. 4, pp. 286–291.
8. Gusel’nikova, V.V., Sinitsina, V.F., Korol’kova, E.D., Kharazova, A.D., and Polevshchikov, A.V., Mor fologiya, 2012, vol. 141, no. 2, pp. 40–45.
Translated by M. Batrukova
2015
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
Apoptosis Level in Developing T Cells in the Thymus I. S. Starskayaa, I. V. Kudryavtseva, V. V. Guselnikovab, M. K. Serebriakovaa, and A. V. Polevshchikovc Presented by Academician E.A. Korneva November 21, 2014 Received November 25, 2014
DOI: 10.1134/S1607672915030060
The thymus as the central organ of the immune system is responsible for the maturation of T cells. It is assumed that the formation of the Tcell receptor (TCR) is accompanied by the destruction of over 95% of CD4+CD8+ cells. This hypothesis is based on the results of studies obtained in 1960–1970 in vitro, which were extrapolated to the processes in vivo with out experimental confirmation [1, 2]. The mathemat ical models of the processes developed to date, which are based on the existing hypothesis, predict the death of 75% of thymocytes in the course of selection [3, 4]. Solving the problem on the level of apoptosis in maturing T cells is essential for the validation of the model of their intrathymic maturation, including pos itive and negative selection as a prerequisite. The morphological analysis of intact mammalian thymus does not reveal the whole picture of massscale apoptosis of maturing cells [5]. The use of flow cytom etry also showed that the level of apoptosis in rat thy mus reaches its maximum on prenatal day 18 (but accounts for only 25% of thymocytes), after which it drops to 5% and remains at this level in the postnatal period [6]. Due to the heterogeneity of published data and a great theoretical importance, the purpose of our work was to estimate the level of apoptosis in intact thymus by comparing the results of immunohistochemical analysis and the data obtained by flow cytometry. The study was performed with 6–10weekold out bred male mice and male CBA/JRap mice weighing 18–20 g. The animals were housed under standard vivarium conditions and received water and feed ad libitum. Work with animals were performed in accor
a
Institute of Experimental Medicine, Russian Academy of Medical Sciences, ul. akademika Pavlova 12, St. Petersburg, 197376 Russia email: [email protected] b St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg, 199034 Russia c Far Eastern Federal University, Vladivostok, Russia
dance with the rules of the Commission on Bioethics of the Research Institute of Experimental Medicine. The level of apoptosis was assessed using panspe cific caspase inhibitors FAM FLICA Poly Caspase Assay Kit conjugated with fluorochromes [7] (Immu nochemistry, United States); the results were expressed as percentage of FLICA+ cells. The level of proliferating cells in the thymus was determined using the antibodies to the nuclear protein PCNA (BioLeg end, United States); the results were expressed as per centage of PCNA+ cells. The main populations of thy mocytes were identified using the monoclonal anti bodies against mouse CD4, CD8, and CD45, which were conjugated with PE/Cy5, PE/Cy7, and APC/Cy7, respectively (BioLegend). All experiments were performed according to the protocols provided by the manufacturers. Samples were analyzed using a Navios flow cytometer (Beckman Coulter, United States). Paraffin sections of the thymus were prepared as described in [8]. To determine the localization of apo ptotic cells in the thymus, we performed immunohis tochemical staining of sections with the antibodies against caspase3 in accordance with the protocol provided by the manufacturer (Abcam, United States). To identify the cells, the sections were stained with an aqueous solution of Aster Blue, embedded in paraffin, and visualized and photographed using a Leica DM6000 microscope (Leica, Germany). Table 1 shows the results of determination of the level of apoptosis and proliferation in the major cell populations of the thymus. The largest proportion of FLICA positive cells was accounted for the popula tion of the most immature CD4–CD8– thymocytes. The level of apoptosis in the population of mature CD4+CD8– and CD4–CD8+ cells was somewhat higher than in the population of doublepositive thy mocytes CD4+CD8+, in which the proportion of apo ptotic cells, contrary to conventional notion, is mini mal. The animals injected with hydrocortisone (2.5 mg per mouse) to induce apoptosis during the thymus atrophy served as a positive control. In addi tion to the decrease in the linear size (from 9.2 ± 0.3 to
163
164
STARSKAYA et al.
ulations of mature cells (CD4+CD8– and CD4–CD8+), on the contrary, were characterized by the prevalence of the levels of apoptosis over the level of proliferation, which indicated the selection processes in these cells. Figure shows the localization of caspase3con taining apoptotic cells in the thymus of mice. Panels 1 and 2 in figure confirm the data presented in the table on the low level of apoptosis in the thymus cortex, containing immature CD4–CD8– and CD4+ CD8+ cells and a higher level of apoptotic cells in the medulla, containing mature lymphocytes CD4+ and CD8+. Panel 3 in figure illustrates the increase in the level of apoptosis of immature cortical cells as early as 6 h after the administration of hydrocortisone. In the medulla, the proportion of apoptotic cells did not change significantly (panel 4 in figure), which is consistent with the concept on a high resistance of mature thymocytes to the action of glucocorticoid hormones [9]. The results obtained in this study revealed a low level of apoptosis in the immature cells CD4+CD8+ along with a significant number of proliferating cells. The results obtained by flow cytometry and immuno histochemistry indicate a high levels of apoptosis in the medullary zone of the thymus, where mature cyto toxic (CD8+) and helper (CD4+) T cells are located. This finding essentially diverges from the dominant hypothesis on the elimination of 95–99% of double positive CD4+CD8+ T cells in the course of selection. Meanwhile, the high level of apoptosis in this popula tion is one of the main bases of the modern theory of
The level of apoptosis and proliferation of thymocytes in dif ferent populations of intact mice (M ± m, n ≥ 80 in determi nation of each parameter) Percent Proportion of the total in the population, % Population number of thy FLICA+ cells PCNA+ cells mocytes CD4–CD8– 2.32 ± 0.79 CD4+CD8+ 86.49 ± 0.82 CD4+CD8– 8.69 ± 1.20 CD4–CD8+ 1.49 ± 0.20
15.34 ± 0.79 4.34 ± 0.11 6.55 ± 0.28 10.11 ± 1.02
20.21 ± 4.50 30.67 ± 2.31 1.67 ± 0.39 6.66 ± 0.98
5.0 ± 0.2 mm, p < 0.01; hereinafter, M ± m) and the weight of this organ (from 70.57 ± 8.14 to 29.97 ± 1.62 mg, p < 0.01), we observed a significant increase in the proportion of FLICA+ cells in the populations of CD4–CD8– cells (up to 48.26 ± 0.65%) and CD4+CD8+ cells (up to 66.93 ± 1.12% of the total number of cells), which was consistent with the pub lished data [9] and demonstrated the adequacy of the obtained results in the estimation of the level of apop tosis in the thymus of intact animals. Table summarizes the results of determination of the level of proliferation in the thymus. The popula tions of immature thymocytes (CD4–CD8– and CD4+CD8+) were characterized by a greater propor tion of proliferating cells compared to the proportion of cells at different stages of apoptosis, whereas the pop
1
2
3
4
Fig. 1. Localization of apoptotic cells in intact thymus and after hydrocortisone administration. Intact thymus: (1) cortex and (2) cortex and medulla; 6 h after the hydrocortisone administration: (3) cortex and (4) medulla. Staining with antibodies against caspase3. Magnification, ×40. DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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APOPTOSIS LEVEL IN DEVELOPING T CELLS IN THE THYMUS
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differentiation of T cells, which implies the stochastic nature of the TCR formation and the presence of pro cesses of positive and negative selection in the course of further maturation of T cells. Thus, the level of apoptosis of CD4+CD8+ thy mocytes in the thymus of adult intact mice does not exceed 5%, which contradicts the existing hypothesis about the maturation and differentiation of T cells. The percentage of apoptotic cells in the medullary zone of the thymus is much higher than in the cortex, which indirectly indicates the selection processes pri marily in the medullary zone of the thymus, contain ing mature thymocytes CD4+CD8– and CD4–CD8+.
2. Schlesinger, M., Gottesfeld, S., and Korzash, Z., Cell Immunol., 1973, vol. 6, no. 1, pp. 49–58.
ACKNOWLEDGMENTS This work was supported by the program of the Far Eastern Federal University (program no. 1326, project no. 14080625_i) and the Russian Foundation for Basic Research (project no. 150405093a).
7. Kudryavtsev, I.V., Golovkin, A.S., Zurochka, A.V., and Khaidukov, S.V., Med. Immunol., 2012, vol. 14, no. 60, pp. 461–482.
REFERENCES
9. Elmore, S.A., Toxicol. Pathol., 2006, vol. 34, no. 5, pp. 656–665.
1. Scollay, R., Kochen, M., Butcher, E., and Weissman, I., Nature, 1978, vol. 276, no. 5683, pp. 79–80.
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 462
3. Sawicka, M., Stritesky, G.L., Reynolds, J., Aboura shchi, N., Lythe, G., MolinaParis, C., and Hogquist, K.A., Front. Immunol., 2014, vol. 5. 4. Sinclair, C., Bains, I., Yates, A.J., and Seddon, B., Proc. Nat. Acad. Sci. U.S.A., 2013, vol. 110, no. 4, pp. 2905–2914. 5. Suhr, C.D. and Sprent, J., Nature, 1994, vol. 372, no. 6501, pp. 100–103. 6. Mel’nikova, V.I., Afanas’eva, M.A., Sapozhnikov, A.M., and Zakharova, L.A., Ontogenez, 2006, vol. 37, no. 4, pp. 286–291.
8. Gusel’nikova, V.V., Sinitsina, V.F., Korol’kova, E.D., Kharazova, A.D., and Polevshchikov, A.V., Mor fologiya, 2012, vol. 141, no. 2, pp. 40–45.
Translated by M. Batrukova
2015
