Antonio Camagna

Interleukin-2 bolus therapy induces immediate and selective disappearance from peripheral blood of all lymphocyte subpopulations displaying natural killer activity: role of cell adhesion to endothelium.

As early as 10-15 min after the start of a 30 min interleukin-2 (IL-2) infusion, a rapid, virtually complete disappearance of all natural killer (NK) lymphocyte subpopulations (including both CD3- CD56+ and CD3+ CD56+ cells with either alpha/beta or gamma/delta T-cell receptor) was observed from peripheral blood. In contrast, the number of T lymphocytes (CD3+ CD56-) was unmodified for at least 2 h after IL-2 injection. The IL-2-induced, rapid disappearance from peripheral blood of NK and NK-like lymphocytes may be related to their massive adherence to the activated endothelium. In this regard, IL-2 infusion caused a very rapid rise of tumour necrosis factor-alpha (TNF-alpha) plasma concentration, whereas other cytokines, such as interferon-gamma (IFN-gamma), were induced only at later times. In vitro experiments indicated that IL-2, either alone or better combined with TNF-alpha, exerts a rapid and selective stimulatory effect on NK adhesion to endothelial cells. On the basis of these findings, we suggest that the activation of NK lymphocytes induced by IL-2, alone or combined with TNF-alpha, plays a key role in mediating the massive and selective adherence of NK and NK-like cells following IL-2 bolus infusion.

Red Blood Cell Age, Pyruvate Kinase Activity, and Insulin Receptors: Evidence that Monocytes and RBCs May Behave Differently

Data emerging from insulin receptor studies performed on red blood cells (RBCs) and monocytes from the same subject are not always in agreement; dichotomy might occur since variations in mean RBC age are not taken into account or because insulin receptors on the two cell types behave differently. In the present investigation RBCs from normal male subjects were separated into five populations of different mean age by means of centrifugation of RBCs on a discontinuous gradient of buffered Percoli for 10 min at 1000 x g. Insulin binding varied significantly depending upon the RBC population tested and was closely correlated to the activity of pyruvate kinase (r2 = 0.86), a well-known marker of RBC age. These data suggested that pyruvate kinase assay might be helpful in studies of RBCs. To confirm this hypothesis, RBCs from 10 normal male subjects and 13 male patients with hemolytic anemia were studied; insulin binding was correlated to pyruvate kinase activity. By adjusting insulin binding to 2 x 109 RBCs/ml the range of data was abnormally high, but it became acceptable after adjusting insulin binding to pyruvate kinase activity (0.75 U/2 x 109 RBCs). The overalr2 = 0.82) but only slightly to reticulocyte number (r2 = 0.56) since not only reticulocytes but also erythrocytes lose receptors during maturation. Pyruvate kinase activity was measured in RBCs from normal men and from normally menstruating women at the seventh and twenty-fourth days of the cycle; results demonstrated that adjustment of data, according to mean RBC age, broadens dichotomy of monocyte and RBC data. In conclusion, this paper demonstrates that (1) insulin binding experiments may be carried out on RBCs separated into populations of different mean age; (2) by assaying the pyruvate kinase activity, it is possible to study RBC samples of different mean age and to reveal insulin binding variations provoked by changes in mean RBC age; (3) RBCs lose insulin receptors physiologically and continuously with age; (4) RBC insulin receptors of patients with hemolytic anemia are normal; and (5) monocyte and RBC insulin receptors may behave differently.

Pure Human Hematopoietic Progenitors: Direct Inhibitory Effect of Transforming Growth Factors-β1 and -β2

TGF-beta 1 and TGF-beta 2 are effective inhibitors of hematopoiesis. We report that colony formation by pure peripheral blood CD34+CD33- BFU-E and CFU-GM (100 cells/dish) is effectively inhibited by both molecules, although TGF-beta 1 is up to 10-fold more potent than TGF-beta 2. Therefore, the effect of these molecules is apparently direct, rather than mediated by accessory cells. The maximal inhibitory activity of TGF-beta is exerted essentially at the early progenitor level, whereas BFU-E/CFU-GM primed for 48 h and IL-3, GM-CSF, and erythropoietin become insensitive to its action. In addition, [3H]TdR suicide experiments indicate that TGF-beta 2 blocks the IL-3-induced progression of early progenitors into the S phase of the cell cycle, whereas IL-6 and bFGF potentiate their entry into the mitotic process. Altogether, these results are compatible with the hypothesis that TGF-beta plays a relevant regulatory role in the homeostasis of early hematopoietic proliferation/differentiation.

Polyclonal expansion of CD3+/CD4+/CD56+ large granular lymphocytes and autoimmunity associated with dysregulation of Fas/FasL apoptotic pathway

Evidence is accumulating regarding CD95/CD95 ligand (Fas/FasL) pathway dysregulation in clonal diseases of the lymphohaemopoietic lineages. According to these observations, it has been proposed that this defect may represent one of the mechanisms of tumour progression. In large granular lymphocyte (LGL) leukaemia, dysregulated apoptosis may represent a key event in the development of malignancy and autoimmunity. This case report describes dysregulation of the Fas/FasL pathway in a chronic polyclonal expansion of CD3+ LGLs associated with numerous serological immune abnormalities.

Expression of transferrin receptors: differential regulatory mechanisms in monocytes-macrophages versus other hemopoietic cells.

Interaction of a cell membrane receptor with its ligand induces either activation of a specific biological process or cellular uptake of an essential nutrient. On this basis, receptors have been classified in two categories:’ class I receptors transmit a specific piece of information (e.g., epidermal and platelet-derived growth factor receptors modulate the growth of a variety of cell types); class I1 receptors interact with and internalize glycoproteins carrying essential metabolic factors (e.g., low-densitylipoproteins (LDL) and transferrin (Trf) receptors allow respectively the uptake of cholesterol and iron). Upon exposure to ligand, class I receptors are rapidly downregulated. In contrast, binding of ligand with class I1 receptors does not usually lead to modulation of their number. A paradigmatic class I1 receptor is the human receptor for LDL.’ Incubation of cells with cholesterol-saturated LDL results in a decrease in the number of receptors. Conversely, incubation in the absence of LDL causes a rise in their number. Both phenomena are mediated by modulation of receptor synthesis, which is in turn controlled by the intracellular concentration of free cholesterol.2 Recent studies suggest that the Trf receptor may similarly represent a typical class I1 receptor: the level of intracellular iron apparently modulates the rate of Trf receptor synthesis,’ thus resembling the regulation of LDL receptors via cholesterol. All types of cells require iron to sustain essential metabolic pathways. Additionally, actively dividing cells need iron for their growth, presumably because the metal is

Erythrocyte Uroporphyrinogen DecarboxylaseActivity: Diagnostic Value and RelationshipWith Clinical Features in Hereditary PorphyriaCutanea T arda

ABSTRACT A marked discrepancy betweenmild and late clinical features and a nearly completeabsence of erythrocyte uroporphyrinogendecarboxylase activity (Ery-UROD activity) wasobserved in a case of inherited porphyria cutaneatarda. The entity and time of appearanceof clinical features, the onset of clinical symptomsafter exposure to contributing factors, theeffectiveness of phlebotomies and heterozygosityof the mother alone for uroporphyrinogendecarboxylase (UROD) deficiency were typicalfor familial porphyria cutanea tarda (F-PCT),whereas the extremely low UROD activity waspeculiar to hepatoerythropoietic porphyria(HEP). These observations indicate that: 1) EryURODactivity may not always be useful to discriminatebetween F-PCT and HEP; 2) EryURODactivity does not always correlate withclinical symptoms; 3) in inherited UROD deficiency,the genetic defect may be heterogeneous.Finally, the observed discrepancy may provideadditional evidence for the existence of tissuespecificisozymes.

The synergistic effect of simultaneous addition of retinoic acid and vitamin D3 on the in-vitro differentiation of human promyelocytic leukemia cell lines could be efficiently transposed in vivo

Both human cell lines HL-60 and AML-193 exhibit a myeloblastic and promyelocytic morphology, respectively, but may be regarded as bipotent leukemic precursors. They can be triggered to differentiate to either granulocytes or monocytes upon retinoic acid (RA) or 1,25-dihydroxyvitamin D (D3) addition, respectively. We have investigated the effect of combined addition of these chemical inducers on the in-vitro differentiation of both cell lines. RA and D3 added together exert synergistic effects on the in-vitro maturation of these myeloid cell lines. Interestingly, the additive effects were lost if the cells were incubated with the inducers added at sequential times. The synergistic effect could be transposed in vivo and could be clinically significant in the treatment of the promyelocytic leukemia. This clinical strategy may help to prevent retinoic acid resistance or to overcome it in patients relapsed after RA therapy and usually unresponsive to a reinduction therapy with RA alone.

Characterization of differences in insulin receptors from young and old red blood cells.

It has been demonstrated that young RBCs (reticulocytes and early mature erythrocytes) possess more insulin receptors than old RBCs (late mature erythrocytes) but it is not yet known whether insulin receptors on young and old RBCs are regulated similarly. In the present investigation insulin receptors on young and old RBCs have, therefore, been studied in five normal male subjects before and after 2 days dexamethasone ingestion (0.5 mg tablet every 6 h) and, in the same subjects, before and 5 h after ingestion of 75 g glucose. The results obtained clearly demonstrate that dexamethasone increases insulin receptor concentration while glucose ingestion increases both insulin receptor affinity and concentration on young RBCs. By contrast, neither stimuli modify insulin receptors on old RBCs. Studies on RBCs are usually performed on the whole RBC population not taking into account this differential responsiveness of receptors on young versus old RBCs; consequently, this phenomenon might be responsible of the fact that some data reported on RBCs are not in agreement with those reported on monocytes or adipocytes and it should be taken into consideration when using RBCs to evaluate insulin receptor regulation.

IL-2 induces the release of secondary cytokines which stimulate the cytotoxic activity of either NK or CD8(+) lymphocytes.

The therapeutic potential of interleukin-2 (IL-2) for the treatment of human cancer has been extensively investigated (cfr. 1). In cell cultures IL-2 activates not only the lymphocytes which mediate MHC-restricted recognition of specific target cells, but also the natural killer (NK) lymphocytes which lyse in vitro particular tumor targets, The lymphokine-activated killer (lAK) cells, obtained by culturing peripheral blood lymphocytes (PBL) with IL-2 (2) exhibit the capacity of lysing a wide range of fresh and cultured malignant cells and virally-transformed normal tissues with minimal destruction of normal cells (2). Clinical trials have shown that significant anti-tumor responses are observed in a minority of patients with disseminated malignancies following infusion of large dosages of 11-2, combined or not with IAK cells (3). However severe toxicity is associated with high-dose IL-2 therapy (3). The administration of IL-2 results in a series of typical modifications of circulating blood cells. Thus IL-2 has a pronounced effects on the number of circulating lymphocytes: lymphopenia develops early after initiation of bolus IL-2 infusion and persists during the entire period of IL-2 administration. Within 24-48 h after discontinuance of IL-2, a marked rebound lymphocytosis develops (4); the level of rebound is doseand schedule-related (4). Furthermore, IL-2 infusion also elicits a series of secondary effects on the hematopoietic system: i) a progressive anemia may develop, which often requires blood transfusion; ii) thrombocytopenia is occasionally monitored; iii) eosinophilia often occurs, particularly after prolonged IL-2 administration (5). Prelimirary studies suggested that these hematological changes were associated with a decline in circulating erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitors (5). In the present study we have investigated the mechanisms responsible for IL-2-induced hematological modifications and the possible role of secondary IL-2-induced cytokines in the activation of cytotoxic lymphocytes.