Geoffrey Keighley

TWO HEREDITARY MOUSE ANEMIAS (Sl/Sld and W/Wv) DEFICIENT IN RESPONSE TO ERYTHROPOIETIN

INTRODUCTION Two different anemia-producing mutants in the mouse are of special interest to experimental hematologists concerned with control of erythropoiesis because of their particular responses to anoxia, erythropoietin and tissue transplant therapy. These mutations, although strikingly similar in their phenotypic manifestations, occurred spontaneously at loci in two different genetic linkage groups and we have reason to believe that members of the two allelic series act on two different parts of the hematopoietic system. Semidominant alleles at the steel (S l ) locus in linkage group IV, and alleles at the W locus in linkage group XVII, show very similar pleiotropic effects on pigmentation, fertility, and blood-formation. Animals bearing double doses, either of mutant S1 alleles or of mutant W alleles, are all black-eyed, white sterile mice afflicted with severe macrocytic anemia. Of these anemias, the W series mutants have been the most thoroughly described, while steel series mutants have as yet been inadequately reported in the literature. We will neither examine the genetic features of either type of anemia in this paper nor present a comprehensive treatise on their pathology, physiology, histology, biochemistry, longevity, or breeding performance; for these, the reader is referred to review articles (Bernstein, 1963; Russell & Bernstein, 1966; Russell & Meier, 1966). Rather, we will present only that information which is directly applicable to our analysis of the hematopoietic responses of W/Wv and S1/Sld anemic mice. A tabulation of selected characteristics of the mutants and their normal littermates is presented in TABLES 1 and 2 as a reminder to those who have examined previous literature on the subject, and as a starting point for those who have a different orientation. The tabulation indicates that untreated W/ Wv anemics and S1 /S1 anemics are phenotypically indistinguishable, except that the S1/Sld anemia tends to be more severe, with lower mean hematocrit levels and higher relative and absolute numbers of reticulocytes. Even though the experienced investigator is unable to tell them apart, they still differ in some interesting and significant ways in their reactions to experimental manipulation. It is our intention, therefore 1 ) to describe several types of physiological experiments which we employed in our search for the steps in hematopoiesis that are deranged by the action of these genetic determinants; 2) to present information obtained thereby; and 3 ) to consider briefly the possible significance of these results.

Response of Normal and Genetically Anaemic Mice to Erythropoietic Stimuli

results in a lifelong macrocytic anaemia (Russell, 195.5, 1962). Gene action leading to thc liacmatopoietic defect of WWv animals is known to occur in the haernatopoietic cells themselves, rather than being imposed from another part of the body. This has been demonstrated repeatedly by successful implantation of haematopoietic cells froin normal tvzv foctal liver into adult and juvenile WWv anaemic mice (Russell, Smith and Lawson, 1956; Bernstein and Russell, 1959). The implanted cells function autonomously according to their own wzv gcnotypc, and thc blood picture of the host changcs gradually but pcrmanently to tha t of a normal mouse. In the present experiments four types of adult mice were used: sevcrcly anaemic WWv mice ; haematologically nornial ww and Ww mice ; very slightly aiiaeniic WUzv inice ; and chimaeric individuals whose original genotype was WWv but which had acquired a lloriiial wtu blood picture following implantation of isologous ww hacmatopoictic cells. An erythropoictic hormone, erythropoictin (EP), niay be obtained from sevcrely aiiacniic animals, for instance froin the plasma or rabbits or sheep made anaemic by blccdiiig or by phenylhydrazinc-induced hacmolysis. It is also found in the urine of some anaeniic aninials

The Relation between Erythropoiesis and Plasma Erythropoietin Levels in Normal and Genetically Anaemic Mice during Prolonged Hypoxia or after Whole‐Body Irradiation

When normal mice (B6D2F1, WBB6F1‐ +/+ and WCB6F1‐ +/+) are exposed to constant hypoxia, they respond with a steady increase of PCV for about 3 weeks. Plasma erythropoietin increases sharply for the first 1 or 2 days only, then falls to control levels, with occasional temporary increases through the next 30–40 days. Genetically anaemic mice (WBB6F1‐W/Wv and WCB6F1‐Sl/Sld) respond differently. The W/W mice show an increase of PCV, and Sl/Sld mice do not, but both respond with large increases of plasma erythropoietin levels which persist, except for some temporary falls, through 16–40 days. The response to hypoxia of chimeric W/W mice, their anaemia cured by implantation of +/+ marrow cells, resembles that of normal +/+ mice much more than that of anaemic W/W mice. Although normal WBB6F1‐ +/+ mice recover after 200 R whole‐body irradiation at rates similar to those of other normal mice, anaemic WBB6F1‐W/W mice show delayed onset of haemopoietic recovery in spite of significant production of erythropoietin. Exogenous erythropoietin injected into mice disappears more slowly from the plasma of anaemic W/W mice than from the plasma of normal +/+ mice, and erythropoietin which has been passed through WBB6F1‐ +/+ normal mice is not thereby made as effective in anaemic W/W mice as it is in normal mice. These findings are consistent with the view that although both W/W and Sl/Sld anaemic mice can produce large amounts of erythropoietin over prolonged periods, W/W mice remain anaemic because of a genetic defect in RBC precursors, and Sl/Sld mice remain anaemic because of a genetic defect in some part of the internal environment in which the RBC must develop.

Stimulation by Serotonin of Erythropoietin‐dependent Erythropoiesis in Mice

Summary. Serotonin stimulates erythropoiesis in normal mice but not in the presence of anti‐erythropoietin serum. It also stimulates erythropoiesis in adrenalectomized or hypophysectomized mice. It increases erythropoietin titres in the plasma of normal but not of nephrectomized mice. The serotonin precursor l‐5‐hydroxytryptophan stimulates, but d‐5‐hydroxytryptophan and the serotonin catabolite 5‐hydroxyindoleacetic acid do not.

Inactivation of erythropoietin by Koshland's tryptophan reagent and by membrane filtration

Abstract 1. 1. Reaction with 2-hydroxy-5-nitrobenzylbromide ( Koshland s reagent) inactivates human and rabbit erythropoietin. Fifteen amino acids and two hexosamines are found unchanged but most of the tryptophan is missing in the reacted erythropoietin. It is probable that one or more intact tryptophan moieties are obligatory for the biological activity. 2. 2. There are great losses when mg quantities of erythropoietin are filtered through Millipore membranes. Protein relatively rich in tryptophan along with the activity appears to be selectively held on the membrane. Selas Flotronics membranes (made of silver) cause almost no loss of activity and are therefore preferable for sterilization of small amounts of erythropoietin, as in cell culture experiments. 3. 3. Human urinary erythropoietin reacts with anti-human albumin serum but differs from albumin by carbohydrate components (hexoses, hexosamines and sialic acid) linked to the protein. Since the tryptophan content of the erythropoietin is higher than that of albumin the immunological reaction cannot be due to albumin which by linkage with carbohydrates has acquired erythropoietin character.

Question of Purity of Erythropoietic Factor Concentrates.

Summary The bulk of erythropoietic activity of anemic plasma can be concentrated in a fraction which represents less than 0.5% of plasma proteins. Yet from the similarity in yield and electrophoretic behavior of the corresponding inactive fraction from normal plasma, it appears likely that the erythropoietic factor constitutes only a small portion of the present concentrates.