A. Ponassi

Normal range of blood colony-forming cells (CFU-C) in humans: Influence of experimental conditions, age, sex, and diurnal variations

ZusammenfassungKoloniebildende Zellen (CFU-C) aus dem Blut und Koloniestimulierte Aktivität aus dem „feeder layer“ peripherer Blutleukozyten (Leukozyten-CSA) wurden in einem halbflüssigen Agarkultur-System bei 69 Normalpersonen untersucht. Gesunde Freiwillige wurden nach Geschlecht und Alter (20–45 Jahre und älter als 60 Jahre) in Gruppen unterteilt. Der Anteil der zirkulierenden CFU-C war bei jungen Frauen (20–45 Jahre) signifikant niedriger als in der Gruppe älterer Männer (älter als 60 Jahre), jedoch waren die Unterschiede zwischen den übrigen Gruppen nicht signifikant. Leukozytäre CSA unterschied sich in den verschiedenen Gruppen nicht signifikant. Bei 5 jungen Männern wurden zirkulierende CFU-C am selben Tag morgens um 8.00 und nachmittags um 16.00 Uhr untersucht: Es ergaben sich keine unterschiedlichen Werte. Bei 18 Personen wurden die Untersuchungen nach längeren Zeitintervallen wiederholt: Die Anzahl der gebildeten Kolonien variierte maximal um das Fünffache. Plasma und segmentkernige Granulozyten, wie sie im angewandten Kultursystem üblich waren, inhibierten das Wachstum der Kolonien nicht. In den meisten Fällen ließen sich in doppelschichtigen Kultursystemen höhere Koloniezahlen erreichen als in einschichtigen, jedoch schienen die „feeder layer“ einiger normaler Personen das Koloniewachstum zu inhibieren.SummaryBlood colony-forming cells (CFU-C) and colony-stimulating activity obtained from feeder layers of peripheral blood leucocytes (leucocyte CSA) have been studied in 69 normal subjects by means of semisolid agar culture system. Groups of normal volunteers were selected according to sex and age (20 to 45 and older than 60 years) and the results compared. The mean number of circulating CFU-C was significantly lower in young women (20–45 years old) than in males over 60 years of age, but no differences were found among the other age and sex groups. Leucocyte CSA did not significantly differ among these groups. In 5 young males the blood CFU-C did not show significant variations at 8 AM and at 4 PM of the same day. When the study was repeated in 18 subjects at longer time intervals, the number of colonies showed a maximum fivefold variation. The amount of plasma and polymorphonuclear granulocytes present in our culture system did not inhibit the colony growth. In most cases, double layer cultures grow a higher number of colonies than single layer, but feeder layers of some normal subject seem to inhibit the colony growth.

Inadequate Ability of T-Lymphocytes from Chronic Uremic Subjects to Stimulate the in vitro Growth of Committed Erythroid Progenitors (BFU-E)

The growth of normal burst-forming units (BFU-E) is known to depend on a burst-promoting activity (BPA) produced by T-lymphocytes. Few BFU-E colonies have been observed in cultures of blood mononuclear cells (MNC) of uremic patients. The aim of the present study was to examine the concentration of BFU-E in the blood of uremic patients and to evaluate the ability of uremic T-lymphocytes to produce BPA. We have studied 6 chronic uremic patients treated with maintenance hemodialysis. When 5 X 10(5) blood MNC depleted of T-lymphocytes of uremic subjects were stimulated by 1 X 10(6) normal T-lymphocytes in a methylcellulose culture system we observed the growth of a number of BFU-E colonies that did not differ significantly from normal (29 +/- 11.8 colonies). On the contrary, when 5 X 10(5) blood MNC depleted of T-lymphocytes of normal subjects were stimulated by 1 X 10(6) T-lymphocytes of uremic patients, the number of BFU-E colonies obtained was significantly lower than normal (1.9 +/- 3.1 colonies). These data show that the decreased number of BFU-E colonies obtained from blood MNC of uremic patients is due to a defect of uremic T-lymphocytes. The impairment of T-lymphocytes can be due to inhibitors of T-lymphocyte function or to variations in T cell subsets, leading to a decrease in the OKT4/OKT8 cell ratio. In any case it is a significant pathogenetic mechanism contributing to anemia in chronic uremia.

Effect of Hydrocortisone on BFU-E Growth and on Burst-Promoting Activity of T Lymphocytes in Man

Glucocorticosteroid hormones have been reported either to stimulate or to inhibit human erythropoiesis. We have studied the in vitro effect of hydrocortisone, 10(-6) mol/l, on human BFU-E when stimulated by preconstituted burst-promoting activity (BPA) in a medium conditioned by T lymphocytes. Hydrocortisone was found to stimulate BFU-E growth, even at largely suboptimal concentrations of BPA, through hormone receptors, as the effect was blocked by preincubation of BFU-E with equimolar progesterone. The possibility that glucocorticosteroids may increase the number and/or affinity of erythropoietin receptors on BFU-E is discussed. We have also studied the effect of hydrocortisone on the production of BPA by human T lymphocytes stimulated by phytohemagglutinin. Preincubation of T lymphocytes for 1 h with hydrocortisone, 10(-6) mol/l, significantly reduced the BPA of lymphocyte-conditioned medium. Again the inhibition of BPA production was reversed by incubation of lymphocytes with equimolar doses of progesterone. The conflicting results previously reported on the effect of glucocorticosteroids on erythropoiesis may be due in part to the opposing effects of the hormones on BFU-E growth and BPA production. The role hydrocortisone plays in the physiological regulation of human erythropoiesis is at present largely unknown.

Alterations of Granulopoiesis in Chronic Uremic Patients Treated with Intermittent Hemodialysis

Granulocyte-macrophage progenitor cells (CFU-GM), leukocyte colony-stimulating activity (CSA), granuloblast differentiation and proliferation and the effect of uremic serum on the in vitro growth of normal CFU-GM have been studied in 8 chronic uremic patients treated with intermittent hemodialysis three times a week. The studies were performed in the postabsorptive state twice in each patient, that is at the longest and shortest dialytic interval. CFU-GM growth in agar and leukocyte CSA did not differ significantly from the normal level in uremic subjects. The granulocytic and macrophagic differentiation in a liquid culture system was significantly reduced in uremic patients, notwithstanding the appearance of high numbers of undifferentiated blastic cells. The serum of uremic patients had no effect on normal CFU-GM and leukocyte CSA.

Inhibition of BFU-E in vitro Growth and of Burst-Promoting Activity of T Lymphocytes by Serum of Chronic Uremic Patients Receiving Hemodialysis

The effects of uremic serum on the in vitro growth of normal BFU-E and on the burst-promoting activity by normal T-lymphocytes were evaluated separately. The effect of hemodialysis on the removal of possible serum inhibitor(s) was also tested. Sera of 12 uremic patients were shown to provoke a 60% inhibition of the in vitro growth of normal BFU-E and almost complete abolition of burst-promoting activity by T lymphocytes. While hemodialysis significantly removed the inhibition of uremic sera on BFU-E growth, it was rather ineffective in removing the inhibitor(s) of T lymphocytes. The different effects of uremic serum on BFU-E and on T lymphocytes are discussed.

Blood granulocyte-macrophage progenitor cell concentrations and differentiation in vitro in patients with hepatic cirrhosis.

Blood granulocyte-macrophage progenitor cells (CFU-GM) and myeloid differentiation were studied in 16 patients with hepatic cirrhosis by culturing blood mononuclear cells in semisolid and in liquid medium. The numbers of CFU-GM from normal and cirrhotic subjects were not significantly different, even when increased numbers of monocyte-macrophagic colonies and decreased numbers of granulocytic colonies were observed in cirrhotic patients. Significantly reduced granulocytic growth and increased monocyte-macrophagic cell growth were found in liquid culture of cirrhotic patients. These data seem to indicate that in hepatic cirrhosis, besides granulocyte sequestration within the spleen, there is a disorder of granulocytic versus monocyte-macrophagic differentiation.

Influence of the spleen on the blood distribution of the colony-forming cells (CFU-C) in man

The incidence of the blood committed granulocyte progenitor cells (CFU-C) before and after epinephrine administration has been studied in 10 normal, 16 splenomegalic and 8 splenectomized subjects through a double-layer agar culture system. A significant increase of the mean values of CFU-C per milliliter of blood has been observed after epinephrine administration in normal and in splenomegalic subjects. In splenectomized patients the baseline mean values of CFU-C per milliliter of blood were higher than those observed in the other groups of subjects, but they did not increase after epinephrine infusion. The concentration of CFU-C per 10(6) total blood leukocytes was the same in all three groups of subjects and it was not modified by epinephrine administration. Our results seem to indicate that the CFU-C are distributed in two blood compartments, the spleen representing an important section of the marginal compartment of the blood CFU-C.

Influence of the spleen on the blood distribution of the leukocytes producing colony-stimulating activity (CSA) in man

SummaryThe colony-stimulating activity (CSA) produced by the blood leukocytes has been studied before and after epinephrine administration in ten normal, 15 splenomegalic, and seven splenectomized subjects through a double layer agar culture system.A significant increase of mean values of the CSA per milliliter produced by blood monocytes has been observed after epinephrine administration in the groups of normal and of splenomegalic subjects. In the group of splenectomized subjects the baseline mean value of CSA per milliliter of blood was higher than those observed in the other groups, but it did not show any increase after epinephrine infusion.The CSA produced by 106 blood leukocytes was similar in all three groups of subjects, and it was not similarly modified by epinephrine administration.Our results seem to indicate that the leukocytes producing CSA are distributed within two rapidly exchangeable blood compartments, the spleen representing an important section of the marginal compartment of blood monocytes.