Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.

Autocrine and paracrine effects of an ES-cell derived, BCR/ABL-transformed hematopoietic cell line that induces leukemia in mice.

During differentiation in vitro, Embryonic Stem (ES) cells generate both primitive erythroid and definitive myeloid lineages in a process that mimics hematopoiesis in the mammalian yolk sac. To investigate leukemic transformation of these embryonic hematopoietic progenitors, we infected differentiating cultures of ES cells with the Chronic Myeloid Leukemia-specific BCR/ABL oncoprotein. Following a period of liquid culture, we isolated two transformed subclones, EB57 and EB67, that retained characteristics of embryonic hematopoietic progenitors and induced a fatal leukemia in mice characterized by massive splenomegaly and granulocytosis. Histopathology of the spleen revealed an abundance of undifferentiated blast-like cells. Investigation of the clonal origins of the granulocytes in the peripheral blood demonstrated that the injected donor cells contributed modestly to the granulocyte population while the majority were host-derived. EB57 secretes IL-3 and unidentified cytokines that can stimulate autocrine and paracrine cell proliferation, presumably accounting for the reactive granulocytosis in diseased mice. These BCR/ABL transformed hematopoietic derivatives of ES cells recapitulate the relationship of BCR/ABL expression to IL-3 production that has been described for primitive hematopoietic progenitors from human CML patients, and illustrates the potential for autocrine and paracrine effects of BCR/ABL-infected cells in murine models.

Hydroxyurea promotes the reduction of spontaneous BFU-e to normal levels in SS and S/beta thalassemic patients.

We have studied the effects of hydroxyurea on growth and differentiation of early erythroid progenitor cells (BFU-e) from peripheral blood of sickle cell disease patients (five SS and two Hb S/beta-thalassemia) in the presence or absence of exogenous stimulating factors. When the mononuclear cells from the sickle cell disease patients were cultured at diagnosis (before hydroxyurea treatment), there was an increased number of BFU-e in relation to controls (p < 0.05, Wilcoxon test) when cells were grown in the presence or absence of 5637 conditioned medium and erythropoietin. Colonies that developed in the absence of added growth factors were considered "spontaneous". A significant difference was observed after hydroxyurea treatment in the number of BFU-e obtained in the presence and absence of stimulus, with a higher reduction in the spontaneous BFU-e number. As expected, there was an increased Hb F level in these patients when compared with their pretreatment levels. There was no correlation between spontaneous BFU-e and hemoglobin levels in all patients studied.

Spontaneous erythroid colony formation in Brazilian patients with sickle cell disease.

The ability of circulating progenitor cells to develop erythroid colonies was studied in vitro in the presence or absence of growth factors (5637-CM and erythropoietin) in 63 patients with sickle cell disease (SCD) (36 homozygotes for hemoglobin [Hb] S, 13 double heterozygotes for Hb S and beta thalassemia, and 14 SC patients) in Southeast Brazil. In the presence of growth factors, SCD patients (all genotypes) presented significantly higher numbers of circulating burst-forming unit-erythroid (BFU-E/5 x 10(5) MNC), when compared with control subjects. However, when the progenitor cells were cultured in the absence of added stimulus, high numbers of BFU-E were observed only in the genotypes SS and S/beta thalassemia. SC patients presented a similar response to the control subjects. Moreover, there was an inverse correlation between spontaneous (without stimulus) BFU-E and Hb levels in SCD patients. These results suggest that the formation of spontaneous BFU-E observed in SCD may be due to an expanded erythropoiesis secondary to hemolysis.

Immunoglobulin levels in workers exposed to hexachlorobenzene.

The serum immunoglobulin (IgG, IgM and IgA) concentrations of 52 chlorinated-exposed workers were examined and compared with those of non-exposed, age- and sex-matched individuals. At the time of testing, the exposed population had mean hexachlorobenzene (HCB) blood levels of 3.84 micrograms/dl with a range of 0.1 to 16 micrograms/dl. Increased IgG and IgM levels were found in the HCB-exposed workers (P < 0.05 and P < 0.01, respectively). Hepatic function was evaluated by serum aspartate (AST) and alanine aminotransferase (ALT) activities, as well as by bilirubin levels. IgM concentrations were positively correlated with three of the studied parameters, namely, length of exposure (r = 0.367) and the activities of both AST (r = 0.367) and ALT (r = 0.507).

Early circulating erythroid progenitor cells and expression of erythropoietin receptors in sickle cell disease.

The ability of circulating progenitor cells from 22 patients with sickle cell disease (SCD) to develop erythroid colonies was studied in vitro in the presence or absence of growth factors (5637-CM and erythropoietin). In both conditions, SCD patients presented significantly higher numbers of circulating burst-forming unit-erythroid (BFU-E/5x10[5]MNC) when compared to control subjects. The study of the expression of erythropoietin receptors revealed an increased level in SCD patients. Moreover, there was a correlation between both stimulated and autocrine (without stimulus) BFU-E and the expression of erythropoietin receptors. These results are of particular interest since they indicate that the phenomenon of spontaneous BFU-E-derived colonies observed in SCD patients may be due to an increased expression of erythropoietin receptors.

Defective neutrophil function in workers occupationally exposed to hexachlorobenzene.

In this work we have studied the respiratory burst and chemotaxis of polymorphonuclear leukocytes from 51 workers exposed to chlorinated compounds, which were compared with those of non-exposed, age- and sex-matched individuals. These two neutrophil functions were significantly reduced as compared to controls. No correlation was observed between the length of exposure, hexachlorobenzene (HCB) blood concentrations and neutrophil chemotaxis or the extent of nitroblue tetrazolium reduction.

Measurement of the respiratory burst and chemotaxis in polymorphonuclear leukocytes from mercury-exposed workers.

The chemotactic and nitroblue tetrazolium reducing activities of neutrophils from 48 mercury-exposed workers were examined and compared with those of non-exposed, age- and sex-matched individuals. At the time of testing, the exposed population had a mean (+/- s.d.) urinary mercury concentration of 24.0 +/- 20.1 micrograms g-1 creatinine and in 44 of these workers urinary mercury levels were below the accepted threshold level (TLV) of 50 micrograms g-1 creatinine. The two neutrophil functions were significantly reduced in the mercury-exposed workers compared with the controls. In 28 of these workers, chemotaxis was re-evaluated 6 months later. During the intervening 6 months, the level of hygiene was improved throughout the plant and urinary mercury concentrations were determined monthly in each worker. Despite a significant reduction in urinary mercury concentrations, neutrophil migration did not return to within the normal range. These results suggest that 'safe' level mercury exposure may lead to impairment of neutrophil function.

Polymorphonuclear phagocytosis and killing in workers exposed to inorganic mercury.

The ability of neutrophils to phagocytose and kill Candida species as well as the splenic phagocytic function were investigated in workers from a mercury-producing plant. In the neutrophil phagocytosis study, two species of Candida were used since in individuals with myeloperoxidase deficiency neutrophils are unable to kill Candida albicans, while Candida pseudotropicalis can be effectively lysed. Phagocytosis of both antigens and splenic phagocytic function were normal in all the workers studied. However, following ingestion of the organisms there was considerable reduction in the ability of neutrophils from exposed workers to kill both species of Candida, and this was not explained by a mild impairment of phagocytosis. After improvement in the hygiene conditions in the factory, a new evaluation was performed, 6 months later, in the same workers and urinary mercury concentrations were determined monthly in each worker. Despite a significant reduction in urinary mercury concentrations, a greater impairment in the ability of neutrophils to kill C. albicans was observed. The killing of C. pseudotropicalis presented no further impairment when compared to the previous evaluation. These results suggest that impairment of the lytic activity of neutrophils from workers with urinary mercury concentrations within the safe level for exposed population is due, at least in part, to some interference with myeloperoxidase activity. In addition, the mercury-NADPH complex, once formed, could limit the utilization of reduced pyridine nucleotides by NADPH-dependent enzymes such as NADPH oxidase, thereby inhibiting the PMN respiratory burst.

Engulfment and killing capabilities of neutrophils and phagocytic splenic function in persons occupationally exposed to lead.

Phagocytosis and intracellular killing of Candida albicans and Candida pseudotropicalis by neutrophils as well as phagocytic splenic function from lead-exposed workers were studied. Two species of Candida were used since in individuals with myeloperoxidase deficiency neutrophils are unable to kill C. albicans, whereas C. pseudotropicalis can be effectively lysed. Phagocytosis with both antigens and phagocytic splenic function were normal in all the workers studied. However, lytic activity towards C. albicans, but not C. pseudotropicalis was impaired. This defect was observed in lead-exposed workers with blood lead levels and urinary delta-aminolevulinic acid (ALA-U) concentrations in the "safe" (below 60 micrograms/dl and 6 mg/l, respectively) and toxic ranges. An impaired ability to kill C. albicans suggests that lead exposure may lead to a myeloperoxidase deficiency. With the exception of blood lead levels and ALA-U concentrations, there was no correlation between any of the other parameters examined.

Immunoglobulin levels in workers exposed to inorganic mercury.

The serum immunoglobulin (IgG, IgM and IgA) concentrations of 44 mercury-exposed workers were examined and compared with those of non-exposed, age- and sex-matched individuals. At the time of testing, the exposed population had a mean (+/- S.D.) mercury urinary concentration of 24.7 +/- 19.1 and in 40 of them urinary mercury levels were below the currently accepted limit of 50 micrograms/g creatinine. Increased IgG, IgA and IgM levels were found in the mercury-exposed individuals and in 16, a second evaluation was performed six months later. During the intervening six months, the level of hygiene was improved throughout the plant, and urinary mercury concentrations were determined monthly in each worker. Despite a significant reduction in mercury urinary concentrations, serum immunoglobulin levels did not return to the normal range. There was no correlation between the length or level of exposure and the immunoglobulin levels. Liver protein synthesis, as studied by factor V, prothrombin time, prealbumin and transaminase activity, was normal and liver injury, as evaluated by serum aspartate and alanine aminotransferase activities (AST and ALT, respectively), was not observed. No haematological abnormalities were noted. These results indicate that "safe" levels of mercury exposure may lead to humoral immunological stimulation.

Immunoglobulin levels and cellular immune function in lead exposed workers.

The immunological status of lead acid battery workers with blood lead levels and urinary delta-aminolevulinic acid (ALA-U) concentrations ranging from safe to toxic levels has been examined and compared with those of non-exposed, age and sex matched controls. No differences in the serum concentrations of IgG, IgA and IgM between the populations were observed and there existed no correlation between blood lead level or ALA-U concentrations and serum immunoglobulin levels. In addition assessment was made of the capacity of peripheral blood mononuclear cells to respond to the mitogen phytohaemagglutinin (PHA), a correlate of T cell function. As before, there was no difference between exposed and control populations and no correlation between reactivity and blood lead concentration. Our data suggest that chronic exposure to lead fail to compromise lymphocyte function in man.