Neocytolysis: When young red blood cells die first

International researchers are investigating the molecular processes involved in premature clearance from the circulation of young blood cells formed at high altitude after a descent

Two million red blood cells are removed from the circulation each second in each healthy human living at sea level. These millions of cells are replaced by the same amount of newly formed cells that leave the bone marrow as reticulocytes, which share the features of precursor cells and mature red blood cells and are vulnerable and unstable. It takes several days for reticulocytes to undergo transformations and become mature red blood cells, stable and ready to function as gas carriers for the following 100 days.

Spending days to weeks at high altitude, we initiate an adaptation sequence to help cope with low ambient oxygen levels. Urgent stimulation of production of more oxygen-carrying red blood cells is a part of this adaptation. These cells provide oxygen supply of our tissue – the more cells, the better oxygen delivery.

However, expansion of red blood cell mass is associated with the rise in blood viscosity and higher mechanical load for the heart and blood vessels. Compromised blood perfusion of extremities is a cause of freezing of fingers and toes in mountaineers.

As soon as we descend back to the sea level, the number of red blood cells in our blood sinks back to the normal level due to the selective destruction of young cells that were produced at high altitude.

Today we understand the mechanisms triggering stimulation of red blood cell production in response to low oxygen levels fairly well. They include the production of a hormone, erythropoietin (Epo), by our kidneys and its release into the bloodstream.

As soon as Epo reaches stem cells in our bone marrow, it binds to the receptors at their membrane and signals them to start dividing and to choose to become red blood cells. As a result, the number of newly formed cells (neocytes) released into the circulation may grow up to 10-fold, giving rise to more circulating red blood cells that transport more oxygen from the lungs to all the organs in our body.

Dissection of this signalling mechanism helped us to harness it, produce recombinant erythropoietin and help people that cannot produce this hormone. Athletes also made illegal use of this knowledge, applying Epo to improve performance.

The molecular processes involved in premature clearance from the circulation of neocytes formed at high altitude after descent remains much less clear.

Currently, standing hypotheses suggest that the cells formed at high altitude stem from precursors that are immature and overexposed to high levels of erythropoietin in the bone marrow, making them expose signals that render them more attractive to the macrophages and experiencing oxidative stress. These two factors make these prematurely released young cells more susceptible to fast clearance, whereas more stable mature cells survive the stress.

In order to discriminate between red blood cells produced at sea level and at the high altitude, they should be labelled and then the fate of labelled cells may be followed over time to monitor their longevity.

The labelling procedure involves using stable isotopes of carbon, or nitrogen, that incorporate into hemoglobin. Cells carrying labelled hemoglobin may be detected and their elimination monitored.

Discovering the age of a tree involves counting the growth-rings. Similar to that, the age of red blood cells may be estimated by the gradual transformation of one of the membrane proteins, historically known as protein 4.1, from native to a deamidated state, making this protein a “molecular-clock” intrinsic in all circulating red blood cells.

This “clock” may be used to assess selective removal of young cells providing direct evidence for the existence of neocytolysis.

In order to dissect the molecular mechanisms of neocytolysis, the causes of selective clearance of cells produced at high altitude have to be revealed. Tagging young cells for premature removal implies that the cells are getting unstable, possess some ‘eat me’ signals, or have lost some ‘don’t eat me’ signals. These tags are usually recognised by the macrophages that take care of damaged old cells, engulfing them and preventing the spill of free hemoglobin into plasma.

In patients suffering from pathological uncontrolled over-production of red blood cells (polycythemia), a compensatory increase in turnover rate was observed and the circulating red blood cells shared the properties common for young and old cells.

Red blood cells of polycythemic individuals are more flexible, less dense and their band 4.1 protein is less deamidated than that in a healthy test subject: a feature characteristic of young cells. At the same time, these cells are more oxidized, and oxidation is one of the key factors making red blood cells attractive for macrophages.

A decrease in oxygen levels in the atmosphere (eg at high altitude) was suggested as a cause for the weakening of antioxidative defence in stem cells giving rise to red blood cells. However, this hypothesis has never been tested for human cells. To do so, stem cells circulating in peripheral blood have to be collected before, during and after the stay of test subjects at high altitude. These cells will be then placed into the liquids resembling plasma in composition, but additionally supplemented with growth factors and hormones supporting their transformation into young red blood cells. This procedure is established in a number of research labs and is now being scaled up to produce red blood cells of rare blood groups for transfusion.

Properties of stem cells at different stages of transformation of red blood cells should be compared for these three batches of cells obtained from the same individual exposed to the changing environmental conditions. Parameters of importance will indicate any delay in maturation, impaired function of antioxidative defence proteins, membrane stability and ability to resist mechanical shear and maintain intracellular ion and water homeostasis.

This exciting programme will be fulfilled by an international research team headed by Prof. Heimo Mairbäurl (University of Heidelberg) and partners from Saarland University (both Germany), University of Zürich (Switzerland) and the University of Pavia (Italy) within a joint project funded by the German Research Foundation (DFG) and the Swiss National Science Foundation (SNSF) during the coming year.

The project will also become a training platform for the early stage researchers of the RELEVANCE Innovative Training Network consortium.

Healthy young volunteers will receive a label tagging red blood cells that were produced at sea level four months prior to the ascent to 3,450 m. One more red blood cell labelling round will be performed in one week after ascent to the Jungfraujoch research station in Switzerland, where the study participants will stay for three weeks in total.

Monitoring the disappearance of the labelled cells, along with detection of deamination state of protein 4.1, will provide information on the premature removal of neocytes. Collection of blood samples at sea level before the ascent and after the descent and at high altitude will provide red blood cells and stem cells for investigation of the impact of hypobaric hypoxia on erythropoiesis.

Why is understanding of the underlying principles of neocytolysis so important? Similar mechanisms are most likely implicated in the development of anaemia in patients on chronic erythropoietin treatment, as well as in the course of space flights.

*RELEVANCE ITN consortium receives funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 675115 — RELEVANCE — H2020-MSCA-ITN-2015/H2020-MSCA-ITN-2015. http://relevance.arivis.com/

Authors:

Bogdanova Anna, Red Blood Cell Research Group, Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.

Lars Kaestner, Theoretical Medicine and Biosciences and Experimental Physics, Saarland University, Germany.

Giampaolo Minetti, Laboratory of Biochemistry, Department of Biology and Biotechnology, “Lazzaro Spallanzani”, University of Pavia, Pavia Italy.

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Anna Bogdanova

Professor and Head of Red Blood Cell Research Group

University of Zurich

Tel: +41 (0)44 635 8811

annab@access.uzh.ch

http://relevance.arivis.com/