Overview
Neocytolysis: Mechanisms and Limitations
Principal Investigator:
Clarence P. Alfrey, M.D., Ph.D.
Organization:
Baylor College of Medicine
Technical Summary
In our current studies, we are dissecting the mechanisms effecting neocytolysis, the selective destruction of young red blood cells. We have been able to demonstrate erythropoietin receptors exist on splenic endothelial cells. This is important because the spleen is the most likely site of neocytolysis. We have created an in vitro model of the process in which endothelial cells grow above macrophages. When the cells are grown in erythropoietin-containing medium and then have erythropoietin withdrawn, splenic endothelial cells become more permeable. They allow increased diffusion of large sugars and they allow increased phagocytosis of young RBCs by macrophages. Young red blood cells seem to be preferential targets in this model, as in vivo. Interestingly, endothelial cells from human aorta, umbilical veins or renal glomeruli do not respond to erythropoietin withdrawal in the manner of splenic endothelial cells. This in vitro model continues to yield insights on the mechanisms of neocytolysis.
A rodent model of neocytolysis could greatly facilitate our ability to dissect and experimentally manipulate the process. We have been very successful using AAV-viral vectors to deliver the EPO gene to mice. This establishes stable, high expression of the gene leading to marked polycythemia in the animals. We are co-delivering a tetracycline response control gene and we can successfully turn off the EPO gene expression with tetracycline in in vitro cultured cell lines. We are actively working on turning off erythropoietin with tetracycline in the polycythemic mice, which should precipitate neocytolysis and establish a model for experimental manipulation.
We have established a human model of neocytolysis by injecting volunteers with erythropoietin, increasing their red cell mass, then withdrawing the erythropoietin. We have observed a rapid fall in the number of red cells on erythropoietin withdrawal, just as predicted. One observation that has emerged is that changes in serum ferritin concentration serve as a precise inverse mirror of the changes in red cell mass. Serum ferritin reflects the amount of iron in body stores. As red cell mass increases under the influence of supplemental erythropoietin, serum ferritin falls as iron is mobilized from stores into newly synthesized hemoglobin. When red cell mass falls, as with neocytolysis, ferritin rises rapidly as iron is transferred back to stores. We have found that ferritin very precisely reflects the changes in red cell mass in space, in altitude-dwellers descending to sea level and now in this erythropoietin-driven human model. The recognition of the utility of ferritin levels in these situations should simplify our ability to study the process. Ferritin levels could also be used clinically as an early measure of the effectiveness of erythropoietin therapy in human disease.
The rate of change of ferritin concentration is much slower when erythropoietin is augmented than when the erythropoietin is decreased which matches our observations in spaceflight that deadaptation to earth's environment as manifest by decrease in red cells occurs quickly. No adverse reaction occurred in normal volunteers receiving erythropoietin for three to six weeks. The model we have established will permit determination of the minimal erythropoietin dose required to prevent deadaptation.
Our discoveries emanating from the unique environment encountered in space have yielded insights on previously unrecognized physiologic and pathophysiologic conditions on earth. We are hopeful that our studies will encourage effective countermeasures for space travelers re-entering a gravitational environment. Our observations clearly impact on such diverse situations as altitude adaptation and de-adaptation, training of elite athletes, anemia of renal disease, optimal erythropoietin dosing schedules, hemolytic anemia and polycythemias. Continuing unraveling of these phenomena will further demonstrate the unforeseen benefits that accrue when basic problems in space are scientifically addressed.