Printer Friendly

Neocytolysis of red blood cells following high altitude exposure.

Introduction

The discovery of erythropoietin (EPO) and of its key role in erythropoiesis allowed a comprehension of the erythropoietic response to hypoxia in molecular terms. Furthermore, studies on the activation of EPO gene paved the way for the identification of the oxygen sensing HIF (Hypoxia Transcription Factor) pathway controlling a wide range of tissue and system specific responses to hypoxia. Genomic analysis of the Tibetan highlanders has revealed a selection that has favoured variants of HIF-2 alpha associated with a haematological profile similar to that of Tibetan lowlanders (i.e.: concentration of haemoglobin and haematocrit within normal ranges). On the basis of these data, it has been suggested that the HIF-mediated increase of erythropoiesis is a misdirect response to hypobaric hypoxia that originally evolved as a response to anemia. In keeping with this conclusion is the process occurring upon transition of lowlanders from hypoxia to normoxia (for instance, mountain climbers returning to sea level after high altitude acclimatization), which consists of a decrease of EPO plasma level and a fast reduction of erythrocyte mass through neocytolysis, i.e. lysis of young erythrocytes.

Neocytolysis has been observed in healthy subjects after return from high altitude to sea level and is involved also in anemias associated to reduced production of EPO, for instance in renal diseases.

In subjects exposed to conditions triggering neocytolysis, beside the dramatic reduction of young RBCs counts, changes in neocytes membrane components have been observed contributing to a "senescent-like" phenotype and likely targeting them to macrophage phagocytosis.

Of the several cells and tissue types sensitive to hypoxia, red cells and the erythropoietic system have been one of the most extensively studied for years both in man and other mammals.

Over 100 years ago the association between hypoxia and increase of erythrocyte mass was demonstrated (1), (2). The discovery of erythropoietin (EPO) and of its key role in erythropoiesis allowed a comprehension of this process in molecular terms. Furthermore, studies on the activation of EPO gene paved the way for the identification of the molecular machinery that controls a wide range of tissue specific and systemic responses to hypoxia. Researchers focused mainly on genetic and functional features of the proteins which are components of HIF (Hypoxia Transcription Factor). This factor, an heterodimer including one of the three subunit alpha 1, 2, 3 and a beta subunit, activates the transcription of genes coding for proteins mediating the adaptive response to hypoxia, such as erythropoetin and some enzymes of the glycolytic pathway (3), (4).

Recent genomic analysis of the Tibetan highlander population, that has been living at high altitude since 25000 years, has revealed that HIF-2 alpha, the oxygen sensitive subunit involved in the regulation of erythropoiesis, and genes related to the HIF signalling pathway, have been subjected to strong and recent positive selection (5), (6). In particular, a positive selection has favoured variants of HIF-2 alpha associated with reduced blood concentration of hemoglobin and reduced haematocrit. This is associated with the haematological profile of Tibetan highlanders which is similar to the one typical of lowlanders. Indeed, an increased haemoglobin concentration and haematocrit, while can be advantageous under hypoxia, results in an elevated blood viscosity, thereby compromising tissue oxygenation and ultimately survival at high altitude.

On the basis of these data, it has been suggested that the HIF-mediated increase of erythropoiesis is a misdirect response to hypobaric hypoxia that originally evolved as a response to anemia (7).

In keeping with this conclusion is the process occurring upon transition of lowlanders from hypoxia to normoxia (for instance, mountain climbers returning to sea level after high altitude acclimatization), which consists of a decrease of EPO plasma level and a fast reduction of erythrocyte mass through neocytolysis, i.e. lysis of young erythrocytes.

Neocytolysis has been observed in healthy subjects after return from high altitude to sea level and has been shown to occur also in astronauts (8-10). In fact in microgravity, a central blood poling occurs, associated extracellular fluid release into tissues. The consequent increased diuresis and decreased plasma volume cause haematocrit augmentation, leading to EPO decrease and neocytolysis, so that red cells mass is reduced in a few days (9).

Neocytolysis is involved also in anemias associated to reduced production of EPO, for instance in renal diseases (11). It may be caused also by artificially induced EPO fluctuations, in cases of blood doping.

In subjects exposed to conditions triggering neocytolysis, beside the dramatic reduction of young red cells counts, changes in neocytes membrane components have been observed, and that contributes to a "senescent-like" phenotype and likely targets them to macrophage phagocytosis (10). The mechanism(s) leading to these membrane changes are not known, nor it is known if, and how, they could be related to decreases in the level of EPO, or other factors, in plasma. Evidence from the literature seems to support both views. After hypoxia exposure, neocytolysis could eliminate those red cells, that, after a maturation from erythroid precursors under low oxygen partial pressure condition, could have phenotypic and functional properties not appropriate to a normoxic environment. Indeed a different composition in membrane lipids (12), a higher concentration of intracellular ATP (10), and a partial fragmentation of actin (13), have been observed in a fraction of red cells populations from mountain climbers after high altitude acclimatization and return to sea level. More recently, the generation of RBCs containing fetal hemoglobin driven by hypoxia exposure has been shown (14).

Investigations about the "precarious" life of young erythrocytes, in conditions of fluctuating erythropoietin levels, as a result of physiological adaptation to extreme environments or of nephropathy, are presently conducted. The methods used for the separation of neocytes and the analysis of their biochemical and functional features have been described in several scientific reports and appropriately changed by the researcher author of this proposal. To obtain age-ranked erythrocyte populations from blood samples, red cells have been separated into three density subsets (low, middle and high density) by centrifugation on discontinuous density gradients of Percoll[R] (10). It is known that red cells density increases with age.

The neocytes separated from the whole RBCs population have been subjected to a number of analysis performed at cellular and molecular level. These studies tried to answer to the following questions:

1) Do the cellular changes, driven by hypoxia exposure, represent an adequate response to the high altitude environment making newly generated RBCs more fit to manage oxygen uptake and delivery? But are these changes still advantageous upon return to normoxia?

2) Does HIF mediated EPO's increase, in high altitude give rise to a plethora of scarcely selected, hypoxia adapted neocytes whose phenotype makes them susceptible to phagocytosis, upon return to normoxia?

3) And does EPO's decrease, occurring during deacclimatisation, the main cause triggering neocytolysis, as the abrogation of neocytolysis in vivo by EPO administration seems to suggest (8)?

Materials and methods

To deal with these issues we studied a group of four mountain climbers (age 28-43 years, two males and two females). Their hematological parameters and RBC phenotype were analysed before and after 53 days acclimatization at high altitude ([greater than or equal to] 4500 m). The RBCs populations were fractionated by density separation into age-based subsets (young, middle-aged and old), the RBCs counts of the three age classes were assessed and some phenotypical features of RBCs from these subsets were investigated by flow cytometry. In particular, the expression of CD55 and CD59 (15) that are partially lost by RBCs during in vitro and in vivo aging [16] were measured. The expression of CD47 (15) and phosphatidylserine (PS) exposure on the outer membrane were also measured. These molecules seem to be involved in the negative [17-19] and positive (20-21) regulation, respectively, of red cell phagocytosis.

Results and discussion

Upon return to sea level, this analysis showed a shift to a "senescent-like" phenotype in all RBCs subpopulations, characterised by a decrease in the level of expression of CD47 in young and middle-aged RBC subsets, exposure of PS on the membrane outer layer and a decline in the expression fluorescence intensity of CD55+ and CD59+ as measured by flow cytometry.

EPO concentration was lower in all subjects as compared with the values measured in control blood samples (mean values: 2.5[+ or -]3.3 vs. 10 [+ or -] 4.5 mIU/ml, P < 0.05).

Finally, after the 6-day descent to sea level, we observed a dramatic decrease in the number of RBCs in the low and middle density subsets and a corresponding increase (more than 3 times the control levels) of cells in the dense subset.

Further analysis of proteomes was undertaken to investigate possible changes in the membrane skeleton of these "hypoxia" adapted RBCs. By two-dimensional electrophoresis and by mass spectrometry we analysed the proteins of red cells ghosts obtained from our subjects before and after return to sea level. After the hypoxia exposure, we observed a lower expression (- 73%) and fragmentation of the same molecule (42 kDa) in 2 fragments of 27 and 28 kDa respectively [13]. This suggested an alteration in membrane skeleton structure, which was confirmed by beta-actin release in cells lysates during the ghost preparation. We observed a similar actin fragmentation and release in red cells lysates from beta-thalassaemic patients. Then, after hypoxia exposure, RBCs displayed actin modification and cytoskeleton instability.

Finally, data from a study we performed on a different group of mountain climbers who lived at high altitude (ranging from 3100 to 5600 m above sea level, Nevado Copa mountain in Peru) for 17 days, strongly support the hypothesis that, like in other instances of stress erythropoiesis such as in thalassemia or sickle cell syndromes, also in hypoxia more RBCs containing fetal hemoglobin, named fetal RBCs, are generated (in our study 5.3 times more than in normoxia). This was evaluated by comparative flow cytometry analysis of RBCs drawn before, during and after the high altitude dwelling, and the data were also supported by western blotting of partially purified hemoglobin and hemolysates of neocytes and by Q-RTPCR of m-RNA of reticulocytes enriched fractions from blood samples drawn at the three time points above mentioned (14).

The latter observation is in agreement with the notion of an hypoxia adaptation in red cells, which could shape an RBCs phenotype affecting either membrane molecules and haemoglobin The changes make the red cells function more effective even at low P[O.sub.2] targeting possibly the neocytes to phagocytosis once the mountain climbers return to sea level, i.e. to normoxia. In fact, beside the membrane changes the presence of fetal haemoglobin could imbalance the oxygen uptake/delivery by RBCs in normoxia making them more susceptible to oxidative stress and, ultimately, directing them to "senescence" and phagocytosis.

Further studies are now focused on the identification of the role of EPO or other possible factors in the survival of neocytes cultured in vitro in the presence of autologous or fetal calf serum. Survival of whole red cells populations in vitro incubated, is prolonged by autologous plasma and the protection is mediated by Bcl-X(L) (22). Studying neocytolysis in a number of physiological and pathological systems may be beneficial in the treatment of some particular blood diseases, and furthermore it may be also helpful to pave the way for the biochemical identification of hypothetical "survival factors" acting on RBCs. Moreover, studying the factor(s) that tip the balance toward survival or destruction in a relatively simple system such as the red cell can provide a model to understand maturation and survival of other cell types in other tissues. Indeed, EPO acts also on endothelial cells and on cells of the central nervous system [glia cells and neurons, ref. 23].

Conclusions

Beside the pay-load for basic science, some benefits can come from this study also for the treatment of the anaemia of uraemic patients unresponsive, or inappropriately sensitive, to EPO.

Finally, results from the proposed study may find potential application in the analysis to detect EPO abuse by athletes who want to enhance their performance. In case of EPO doping neocytolysis has not been studied, but the prediction of the theory is that, since this process is strictly related to EPO fluctuation, neocytes of athletes would be doomed to destruction after EPO withdrawal. The possibility to reveal an ongoing neocytolytic process, by analysing some property of the circulating neocytes, could be a useful complement to the commonly used analysis of urine. Although this latter method is very sensitive and reliable, it is unpractical for many laboratories and its application is limited to the detection of EPO shortly after its injection, or within a short period of time (2-3 days) since the last administration of the hormone. Neocytolysis takes 5-7 days, as shown by the group of Alfrey and Rice [8] and as reconfirmed by Risso et al. (unpublished data). Then the precise identification of neocytes, their biochemical characterisation and the definition of a "senescent" phenotype that favour cell destruction could be useful, especially when the urine analysis cannot be performed.

Declaration of interest

The authors report no conflicts of interest.

References

(1.) Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev 1992; 72: 449-89.

(2.) Porter DL, Goldberg MA. Regulation of erythropoietin production. Exp Hematol 1993; 21: 399-404.

(3.) Semenza GL. Hypoxia-inducible factor 1: master regulator of [O.sub.2] homeostasis. Curr Opin Genet Dev 1998; 8: 588-94.

(4.) Kaelin WG. Proline hydroxylation and gene expression. Annu Rev Biochem 2005; 74: 115-28.

(5.) Simonson TS, Yang Y, Huff CD, et al. Genetic evidence for high-altitude adaptation in Tibet. Science 2010; 329: 72-5.

(6.) Yi X, Liang Y, Huerta-Sanchez E, Jin X, et al., Sequencing of 50 human exomes reveals adaptation to high altitude. Science 2010; 329: 75-8.

(7.) Storz JF. Genes for High Altitudes. Science 2010; 329: 40-1.

(8.) Rice L, Ruiz W, Driscoll T, et al. Neocytolysis on descent from altitude: a newly recognized mechanism for the control of red cell mass. Ann Intern Med 2001; 134: 652-6.

(9.) Alfrey CP, Udden MM, Leach-Huntoon C, et al. Control of red blood cell mass in space-flight. J Appl Physiol 1996; 81: 98-104.

(10.) Risso A, Turello M, Biffoni F, et al., Red blood cell senescence and neocytolysis in humans after high altitude acclimatization. Blood Cells Mol Dis 2007; 38: 83-92.

(11.) Rice L, Alfrey CP, Driscoll T, et al. Neocytolysis contributes to the anemia of renal disease. Am J Kidney Dis 1999; 33: 59-62.

(12.) Gonzalez G, Celedon G, Escobar M, et al. Red cell membrane lipid changes at 3,500 m and on return to sea level. High Alt Med Biol 2005; 6: 320-6.

(13.) Risso A, Santamaria B, Pistarino E, et al. Fragmentation of Human Erythrocyte Actin following Exposure to Hypoxia. Acta Haematol 2010; 123: 6-13.

(14.) Risso A, Fabbro D, Damante G, Antonutto G. Expression of fetal hemoglobin in adult humans exposed to high altitude hypoxia, Blood Cells Mol Dis. 2012 48: 147-53.

(15.) http://www.immunologylink.com/cdantigen.html

(16.) Pascual M, Danielsson C, Steiger G, et al. Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS. Eur J Immunol 1994; 24: 702-8.

(17.) Oldenborg PA, Zheleznyak A, Fang YF, et al. Role of CD47 as a marker of self on red blood cells. Science 2000; 288: 2051-4.

(18.) Oldenborg PA, Gresham HD, Chen Y, et al. Lethal autoimmune haemolytic anemia in CD47-deficient non obese diabetic (NOD) mice. Blood 2002; 99: 3500-4.

(19.) Okazawa H, Motegi S, Ohyama N, et al. Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system. J Immunol 2005; 174: 2004-11.

(20.) Connor J, Pak CC, Schroit AJ. Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age and clearance by mononuclear cells. J Biol Chem 1994; 269: 2399-404.

(21.) Boas F E, Forman L, Beutler E. Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia. Proc Natl Acad Sci USA 1988; 95: 3077-81.

(22.) Walsh M, Lutz RJ. Cotter TG, O'Connor R. Erythrocyte survival is promoted by plasma and suppressed by a Bak-derived BH3 peptide that interacts with membrane-associated Bcl-X(L). Blood 2002; 99: 3439-48.

(23.) Jelkmann W, Wagner K. Beneficial and ominous aspects of the pleiotropic action of erythropoietin. Ann Hematol 2004; 83: 673-86.

Received: January 04, 2012

Accepted: June 04, 2012

Published: June 29, 2012

Address for correspondence:

Guglielmo Antonutto, MD

Dipartimento di Scienze Mediche e Biologiche

P.le M. Kolbe 4

I-33100 Udine (Italy)

Phone: +39-0432-494334

Fax: +39-0432-494301

email: guglielmo.antonutto@uniud.it

Guglielmo Antonutto: guglielmo.antonutto@uniud.it

Angela Risso: angela.risso@uniud.it

Authors' contribution

A - Study Design

B - Data Collection

C - Statistical Analysis

D - Data Interpretation

E - Manuscript Preparation

F - Literature Search

G - Funds Collection

(1.) Department of Medical and Biological Sciences, University of Udine, Udine, Italy (2.) Department of Agriculture and Environment Sciences, University of Udine, Udine, Italy

DOI: 10.5604/17342260.1001883
COPYRIGHT 2012 Medicina Sportiva
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2012 Gale, Cengage Learning. All rights reserved.

 
Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:ORIGINAL RESEARCH
Author:Antonutto, Guglielmo; Risso, Angela
Publication:Medicina Sportiva
Date:Jun 1, 2012
Words:2803
Previous Article:Five year experience with the upgraded dynamic flight simulator (human centrifuge) for Eurofighter/Typhoon pilot training in the German Air Force.
Next Article:Hypoxia and hearing - what do we really know?
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters