Survival and senescence of human young red cells in vitro.
Angela Risso1, Annarita Ciana2, Cesare Achilli2, Giampaolo Minetti2
1Department of Agriculture and Environment Sciences, University of Udine, Udine, Italy
2Laboratories of Biochemistry, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
BACKGROUND: A number of experimental investigations in vivo suggest that in humans a decrease of circulating erythrocyte number ensues whenever erythropoietin (EPO) plasma level decreases. Since the process seems to selectively eliminate young red cells (neocytes), it has been named neocytolysis. The experimental models in vivo have revealed and documented multiple forms of neocytolysis but have not fully elucidated the specificity of the target red cells and the relation with EPO level changes. In an attempt to better characterize the neocytolytic process, we have undertaken an in vitro investigation on age-ranked human red cells.
METHODS: By centrifugation on Percoll density gradient we separated the red cells population into three subsets, neocytes, middle-aged and old. Then we comparatively investigated the kinetics of survival of the subsets cultured under different conditions: with medium alone, with 10% autologous plasma, with EPO, alone or in combination with autologous monocytes.
RESULTS: Neocytes showed a viability and a survival rate lower than the other red cells when cultured in medium or with 10% plasma. EPO at physiological doses increased their survival rate, but not that of the other subsets. This effect was enhanced by co-culture with monocytes.
CONCLUSION: Likely neocytes are more sensitive than the other RBCs subsets to presence or absence of survival signals, such as EPO or plasma or monocytes derived factors. These observations could provide an insight into the link between the decrease in EPO plasma level and the reduction of circulating red cells mass and account for the specificity of neocytes clearance.
The control of red blood cell (RBC) mass is related to RBC lifespan, which, in humans, is normally of about 120 days, but can be shortened in pathological conditions such as in sickle cell disease or thalassemia. In these cases, excessive destruction of pathological RBCs can lead to a decrease in red cell mass, to anaemia and to stress erythropoiesis (where massive red cell production occurs). Human RBC lifespan can also be shortened as a physiological adaptive response to environmental cues that require a decrease of the circulating red cells mass. In this latter case the number of circulating RBCs decreases not by clearance of altered or defective cells, but by a selective lysis of early generated RBCs, or neocytes (young cells). The process, that has been named neocytolysis (1), is associated with a decline in erythropoietin (EPO) plasma levels, and it has been described in subjects exposed to a large panel of different environmental signals (2): for instance, in hypoxia-adapted subjects upon their return to normoxia, or in astronauts during the first days of space flight (3-5). In the latter, a central blood pooling occurs due to microgravity, which is associated to plasma release into tissues. The consequent increased diuresis and decreased plasma volume cause pseudopolycythemia. Hence, EPO decreases and neocytolysis ensues, so that red cells mass is reduced in a few days since the beginning of the space flight (5).
Neocytolysis seems to be associated also to anaemia, such as when, as in renal failure, or in chronic infection or inflammation, EPO plasma level decreases (2,6). Finally, artificially-induced fluctuations in EPO levels, as in blood-doping, seem to trigger neocytolysis (7). In all the instances where environmental, pathological or artificial cues trigger lysis of neocytes, two features appear to be shared by all the different types of neocytolysis, i.e. the decline in EPO plasma levels and the elimination of young red cells.
To understand the paradoxical selectivity of neocyte elimination and its association with EPO plasma decrease, investigations have been undertaken both in murine in vivo and in human in vitro model systems. The murine system, focussed on neocytolysis from post-hypoxia exposure, points to the role of hypoxia in erythropoiesis. In hypoxia, hypoxic inducible factors not only increase EPO synthesis, and hence the number of circulating RBCs, but also cause molecular changes (decrease of microRNA 21, hence reduction of catalase expression) in erythroid precursors, that make red cells born under hypoxia more sensitive to Reactive Oxygen Species upon return to normoxia. This could be enhanced by the augmented mitochondrial mass of reticulocytes (8).
The hypothesis that neocytes can be particularly sensitive to changes in EPO levels, was investigated by comparatively testing the survival of human young, middle-aged and old red cells that were separately incubated in vitro in the presence of different concentrations of recombinant human EPO (9).
Although the experimental conditions were far from those of circulating red cells, the following observations suggested a role of EPO on neocyte survival:
- Young red cells incubated at 37°C were more sensitive than the other RBC subsets to the culture conditions, both without and with autologous plasma added to the culture medium (RPMI, 8 mM HEPES, 5 mM glucose).
- EPO added to the cultures at physiological concentration (10 mIU/ml) was able to prolong survival of neocytes, especially in the presence of 10% plasma, and the effect was reinforced by the presence of adherent cells. This pro-survival effect was not detected on the other RBC age classes.
- Finally, EPO at higher than normal doses not only failed to have a pro-survival effect but proved to be toxic in a dose dependent manner, as shown by the rate of survival that decreased linearly and at a rate dependent of EPO concentration (Fig 1).
Although the expression of the EPO receptor by red cells, in particular by young red cells, is still debated, there is strong evidence in favour of this hypothesis, (10).
Then, EPO seems to be active on neocytes cultured in vitro in a paradoxical way. The effects of EPO on young red cells [which survive at physiological concentrations and are driven to senescence and death at high concentrations of the hormone (see Fig. 1)] are reminiscent of the effects of IL-2 on the homeostasis of T lymphocytes cultured in vitro (11). While studying the control of population density of murine T lymphocytes, Hart et al. showed that in response to IL-2, activated T cells, when cultured at different densities, reached a dual homeostatic state. The cultures starting at low density reached a low stable density, those cultured at higher density reached homeostasis at higher density, but below the maximum allowed by the available resources. This was related to the ability of IL-2 to both trigger proliferation rate cooperatively and cell death linearly. After elaborating a mathematical model suited to describe the cell and cytokine dynamics, they suggested that the paradoxical, dual signalling property of IL-2 provides cell populations with dynamical features that “are resistant to environmental perturbations” (11).
Although we do not know whether the effects exerted by EPO on erythroid precursors, in some steps of their development, are similar to those mediated by IL-2 on T lymphocytes (proliferation vs. death), it is tempting to hypothesize that the dual role of EPO on young red cells, i.e. pro-survival and death-inducing, could be inserted into the framework provided by Hart et al. for IL-2, and partially account for the ability of the erythropoietic system to cope efficiently with external perturbations.
Importance of the study. Neocytolysis involves topics related to basic science and also to clinical and practical applications, as in the treatment of the anaemia associated with different pathologies, and the elaboration of new strategies to detect blood doping. Study of this process could be beneficial to improve the management of some forms of anaemia and to better understand the role of EPO in the control of red cell mass.
Acknowledgements. This work was supported by the “Ministero dell’Università e della Ricerca”, Italy, with PRIN2008 funds to G.M. and A.R.
Dr Giampaolo Minetti, University of Pavia, Department of Biology and Biotechnology “Lazzaro Spallanzani”, Laboratories of Biochimica, via Bassi, 21, 27100 Pavia, Italy. Tel.:+39 0382 987891; FAX: +39 0382 987240; email: firstname.lastname@example.org.
Dr Angela Risso, University of Udine, Dept of Agricultural and Environmental Sciences, Via delle Scienze 91, 33100 Udine, Italy. Tel. +39 0432 558789; FAX +39 0432 558784; email: email@example.com.
- Alfrey CP, Rice L, Udden MM, Driscoll TB. Neocytolysis: physiological down-regulator of red-cell mass. Lancet. 1997;349:1389-1390. doi: 10.1016/S0140-6736(96)09208-2.
- Risso A, Ciana A, Achilli C, Antonutto G, Minetti G. Review. Neocytolysis: none, one or many? A reappraisal and future perspectives. Front Physiol. 2014;5:54. doi: 10.3389/fphys.2014.00054.
- Rice L, Ruiz W, Driscoll T, Whitley CE, Tapia R, Hachey DL, Gonzales GF, Alfrey CP. Neocytolysis on descent from altitude: a newly recognized mechanism for the control of red cell mass. Ann Intern Med. 2001;134:652-656. doi:10.7326/0003-4819-134-8-200104170-00010.
- Risso A, Turello M, Biffoni F, Antonutto G. Red blood cell senescence and neocytolysis in humans after high altitude acclimatization. Blood Cells Mol Dis. 2007;38:83-92. doi: 10.1016/j.bcmd.2006.10.161.
- Alfrey CP, Udden MM, Leach-Huntoon C, Driscoll T, Pickett MH. Control of red blood cell mass in spaceflight. J Appl Physiol. 1996;81:98-104.
- Alfrey CP, Fishbane S. Implications of neocytolysis for optimal management of anaemia in chronic kidney disease. Nephron Clin Pract. 2007;106:c149-c156. doi:10.1159/000104425.
- Chang CC, Chen Y, Modi K, Awar O, Alfrey C, Rice L. Changes of red blood cell surface markers in a blood doping model of neocytolysis. J Investig Med. 2009;57:650-654. doi: 10.231/JIM.0b013e3181aa0978.
- Song J, Yoon D, Christensen RD, Horvathova M, Thiagarajan P, Prchal JT. HIF-mediated increased ROS from reduced mitophagy and decreased catalase causes neocytolysis. J Mol Med (Berl). 2015;93:857-866. doi: 10.1007/s00109-015-1294-y.
- Risso A, Ciana A, Achilli C, Minetti G. Survival and senescence of human young red cells in vitro. Cell Physiol Biochem. 2014;34:1038-1049. doi: 10.1159/000366319.
- Mihov D, Vogel J, Gassmann M, Bogdanova A. Erythropoietin activates nitric oxide synthase in murine erythrocytes. Am J Physiol Cell Physiol. 2009;297:C378-C388. doi: 10.1152/ajpcell.00543.2008.
- Hart Y, Reich-Zeliger S, Antebi YE, Zaretsky I, Mayo AE, Alon U, Friedman N. Paradoxical signaling by a secreted molecule leads to homeostasis of cell levels. Cell. 2014;158:1022-1032. doi: 10.1016/j.cell.2014.07.033.