L. Morra

T lymphocyte subsets in chronic uremic patients treated with maintenance hemodialysis.

Blood T lymphocyte subsets have been studied using monoclonal antibodies in 10 chronic uremic patients treated with maintenance hemodialysis. Both total T lymphocytes identified by the antibody OKT3, and the helper-inducer T lymphocyte subset identified by the antibody OKT4 were found to be significantly lower than normal. The cytotoxic-suppressor T cell subset was only moderately, even if significantly reduced, so that the T4/T8 ratio in uremic patients was significantly lower than normal. These data provide an additional contribution to the interpretation of immunological and hematological deficiencies observed in chronic uremia.

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.

Defective in vitro growth of BFU-E of elderly subjects revealed by hydrocortisone

The effect of aging on hematopoiesis and bone marrow exhaustion have long been debated. Unexplained anemia and impaired in vitro proliferation of erythroid precursors is frequently observed in the elderly. As hydrocortisone is known to increase the BFU-E in vitro growth, we have studied the response of BFU-E to hydrocortisone in a group of nonanemic elderly subjects. The BFU-E growth in methylcellulose from blood mononuclear cells (MNC), stimulated by lymphocyte-conditioned medium (LCM), either with or without hydrocortisone, of 10 subjects aged 76-91 years was compared with the BFU-E growth from MNC of a group of ten young subjects. While LCM induced a significant increase of BFU-E growth, both in young and old subjects, hydrocortisone induced a significant increase of BFU-E growth only in young subjects. This study shows that in the elderly, there is a latent defect of erythropoiesis, possibly consisting of a defective ability to modulate the receptors for erythropoietin on BFU-E, and that hydrocortisone offers a useful tool to identify it.

In vitro growth of erythroid progenitor cells (BFU-E) and production of burst-promoting activity (BPA) by T lymphocytes in patients with myelodysplastic syndromes

The in vitro growth of circulating erythroid progenitors (BFU-E) populations and the production of burst-promoting activity (BPA) by T lymphocytes have been studied in 17 patients with myelodysplastic syndromes. Based on the in vitro growth patterns of BFU-E, four groups of patients have been identified: i) normal BFU-E growth; ii) low spontaneous BFU-E growth, but normal response to LCM; iii) impaired BFU-E response to LCM; iv) no BFU-E growth. The pattern of BFU-E growth seems to be related to the clinical stage of the disease rather than to the FAB subgroup to which the patients belong. The ability of T lymphocytes to stimulate BFU-E growth was significantly reduced in all patients. The possible mechanisms inducing the impaired production of BPA by T lymphocytes are discussed. The in vitro evaluation of circulating erythroid precursors can supply useful prognostic information and possibly indications concerning the responsiveness of erythropoietic stem cells to recombinant human erythropoietin in vivo.

Effect of cimetidine on burst-promoting activity of normal T lymphocytes.

SummaryThe effect of cimetidine, an inhibitor of suppressor T lymphocytes, on the burst-promiting activity (BPA) of normal T lymphocytes has been studied. Cimetidine has been shown to increase the BPA of normal T lymphocytes, both when added to the culture and when T lymphocytes were preincubated for 1 h with it. Cimetidine had no direct effect on the in vitro growth of burstforming units (BFU-E). Our results show that CD 8 suppressor T lymphocytes inhibit the in vitro growth of BFU-E in normal conditions, either directly or through inhibition of BPA of CD 4 helper T lymphocytes. Cimetidine has proved to be a useful tool for investigating the hematological role of T-lymphocyte subsets in normal and pathological conditions.

Effect of corticosteroid treatment on hemopoiesis in vivo and in vitro in a patient with Felty's syndrome

We have studied the CFU-GM and BFU-E in vitro growth in a neutropenic and anemic patient with Feltys syndrome, either before or one and three months after steroid therapy when neutrophils and erythrocytes returned to normal. Both CFU-GM growth and CSA production were found to be low before therapy, and prednisone was shown to raise them to normal levels. The in vitro growth of BFU-E and the production of BPA by T lymphocytes of the patient were significantly lower than normal when studied before therapy. However, the T lymphocytes incubated in vitro with hydrocortisone regained their ability to stimulate the BFU-E growth. After prednisone therapy both BFU-E growth and BPA production by T lymphocytes returned to normal. Possible pathogenetic mechanisms of impaired granulo- and erythropoiesis in Feltys syndrome are discussed. The in vitro study with hydrocortisone can help to identify steroid-sensitive patients.

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.