P. A. T. Tetteroo

Megakaryoblastic differentiation of proerythroblastic K562 cell-line cells.

The human proerythroblastic leukemia cell-line K562 was induced to differentiate into megakaryocytic cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Megakaryocytic differentiation was detected when lineage-specific monoclonal antibodies were used to monitor the effect of TPA on K562 cells. A monoclonal anti-platelet antibody (C17) directed against an epitope present on GP IIIa appeared to react with K562 cells after induction. This was observed together with the disappearance of glycophorin A, the erythrocyte-specific lineage antigen. The induced megakaryocytic cells were also detected by ultrastructural platelet peroxidase (PPO). Immunoprecipitation, after ectolabeling of the cells with the C17 antibody and SDS-polyacrylamide gel electrophoresis, proved that TPA-induced K562 expressed both GP IIIa and GP IIb. However, the monoclonal antibody C15 directed against another epitope of platelet GP IIIa reacted only partially, or not at all, indicating that GP IIIa expressed on TPA-induced K562 differs structurally from that on normal platelets. K562 clones, expressing glycophorin A in all cells, were obtained by limiting dilution and culture. When these clones were treated with TPA, again megakaryocytic cells were obtained. These findings are discussed in relation to normal megakaryocytopoiesis.

Immunological study of in vitro maturation of human megakaryocytes

Summary. Human megakaryocyte colonies were grown from the bone marrow in plasma clot or methyl cellulose cultures. Maturation of the megakaryocytic cells was sequentially studied from day 5 to day 16 of culture by fluorescent labelling with a panel of monoclonal and polyclonal antibodies against different platelet glycopro‐teins (Gp), PI AI antigen, factor VIII RAg platelet factor 4 (PF 4), fibrinogen and platelet‐derived growth factor (PDGF). Expression of Gp Ib was also studied by immunogold technique at electron microscopy. The first cells identifiable by these antibodies were found at day 5 of culture. They had the size of a lymphocyte. These small megakaryocyte precursors already expressed all the platelet antigens, HLA‐DR and transferrin receptors and were devoid of erythroid or myeloid markers. Among the platelet antigens, Gp IIIa was the most sensitive marker for the identification of these precursors. However, double‐fluorescent labelling demonstrated that the different platelet markers were coexpressed in a large majority of cells. Interestingly, cytoplasmic markers demonstrated that these small megakaryocyte precursors were themselves heterogenous by morphological criteria. During maturation, expression of Gps, particularly of Gp Ib, increased while the labelling pattern of anti factor VIII RAg and anti PF 4 antibodies switched from diffuse to granular staining. PDGF could also be detected in the megakaryocytes grown in culture.

Monoclonal antibodies against human platelet glycoprotein IIIa

Summary. Two murine monoclonal antibodies specific for human platelets were prepared and characterized by immunofluorescence, immunoprecipitation and by studying their effect on platelet function.

A murine monoclonal IgM antibody specific for blood group P antigen (globoside)

A murine monoclonal IgM erythrocyte antibody appeared to have anti‐P (anti‐globoside) specificity. The antibody was a relatively weak cold agglutinin, but a strong haemolysin and its reactivity with red cells was markedly enhanced by enzyme treatment. This antibody was used to study the cell and tissue distribution of globoside. Globoside was not only detectable on red cells and erythroblasts, but also on endothelial cells and on subsets of platelets, megakaryocytes and fibroblasts. It was not detectable on granulocytes, monocytes and most peripheral blood lymphocytes. Neither was it present on erythroblast precursors (CFU‐E, BFU‐E), pro‐erythroblasts or on the cells of the pro‐erythroblastic cell lines K562 and HEL. However, K562 cells expressed globoside when induced to mature into erythroblasts by sodium butyrate. Cells of patients with various leukaemias were also tested. A significant number of positively reacting cells was frequently (six out of 18) seen in cases with a CML blast crisis (CML‐BC) and rarely in AML (four out of 37 cases). In CML‐BC the P‐positive cells were probably erythroblasts and/or megakaryoblasts. Thus, globoside appeared to be an interesting marker in CML‐BC of the erythroblastic or mixed erythroblastic‐megakaryoblastic type.

Characterization of myeloid leukemia by monoclonal antibodies, with an emphasis on antibodies against myeloperoxidase

Since the last workshop on human leukocyte differentiation antigens, there are 14 well defined cluster-designated (CD) antigens which characterize myelomonocytic cells. Of these, 5 are potentially useful for myeloid leukemia typing (i.e. CD13, CD14, CD15, CD33, CD36) because they are cell lineage-specific and also expressed on immature cells. However, the reactivity of monoclonal anti-CD antibodies, directed against these antigens, with myeloblastic leukemia cells was found to be quite low. We produced monoclonal antibodies against myeloperoxidase. These antibodies react also with promyeloperoxidase, synthesized in HL-60 cell line cells. Monoclonal antimyeloperoxidase was found to be the most sensitive reagent to diagnose acute myeloid leukemia, even more sensitive than cytochemical stains (Sudan black, myeloperoxidase).

Localization of the Human NA1 Alloantigen on Neutrophil Fc-γ-Receptors

The neutrophil-specific-NA antigen system is a biallelic system. It comprises the antigens NA1 and NA2, which in Caucasian populations show a phenotype frequency of 46% and 88%, respectively (1). It is clinically an important system because it may be involved in diseases such as neonatal alloimmune neutropenia and autoimmune neutropenia as well as in blood transfusion reactions such as rigors, fever, and respiratory distress (1,2).

Serological and Biochemical Characterization of Human Myeloid-Associated Antigens and Their Expression in Human-Mouse Cell Hybrids

Recently many monoclonal antibodies (MoAb) which define new granulocyte- and/or monocyte-specific antigens have been described. These antibodies have been applied to the study of myeloid and monocyte differentiation [8, 9, 11], as well as to the typing of the cells of patients with leukemia [5, 10]. In this report we describe three new monoclonal antibodies, two reacting with granulocytes and one reacting with granulocytes, monocytes, and a population of non-T lymphocytes. The molecular identity of the antigens recognized by the antibodies and the expression of the antigens in hybrids derived from fusion of human and mouse myeloid cells was studied. They were also compared with monoclonal antibodies against granulocytes from other laboratories.

In-vitro differentiation of cells of patients with acute undifferentiated leukaemia

Summary The diagnosis of acute undifferentiated leukaemia (AUL) is made when the cells of patients with acute leukaemias cannot be classified as myeloid or lymphoid by means of morphological, cytochemical and immunological criteria. The mononuclear cells of eight different AUL patients were cultured in suspension for 3 d with or without TPA. After culture, especially in the presence of TPA, the cells of all patients expressed at least one myeloid membrane antigen. It was shown that this antigen expression was dependent on de novo protein synthesis and not influenced by inhibition of proliferation.

Colony-forming cells in chronic granulocytic leukemia--II. Analysis of membrane markers.

Membrane markers and functional properties in vitro of blast cells from the peripheral blood of 2 patients with chronic granulocytic leukemia were studied. Buffy-coat cells were enriched for colony-forming cells by density centrifugation (d less than or equal to 1.062 g cm-3). Upon culture, a large proportion of the (cryopreserved) low-density cells from both patients formed hemopoietic colonies that were heterogeneous with respect to size and cellular composition. Expression of membrane markers on the cells, which had the morphology of undifferentiated blasts, was studied using flow cytometry with a panel of monoclonal antibodies. A striking heterogeneity was observed in that variable numbers of cells were found to express myelomonocytic, megakaryocytic and erythroid membrane markers. Antigenic properties of colony-forming cells were studied by sorting of cells with a fluorescence activated cell sorter. Low numbers of cells (10, 4 and 1, respectively) were sorted directly into the wells of Terasaki microtest plates. With this system, it was shown that myeloid colony-forming cells from patient 1 were exclusively present in HLA-DR-positive cell fractions. Colony formation from the level of a single sorted cell was documented. Sorting of cells labeled with anti-blood-group-H antibody showed that small erythroid colony-forming cells from patient 2 were blood-group-H antigen-positive. These cells did not express HLA-DR. The other colony-forming cells from this patient and essentially all colony-forming cells from patient 1 were HLA-DR-positive and blood-group-H-negative. Although only 2 patients were tested, our studies clearly demonstrate that low-density cell fractions from the blood of patients with CGL provide distinct advantages for the study of membrane properties of hemopoietic cells and of hemopoietic differentiation in general.

Expression of CD15 (FAL) on myeloid cells and chromosomal localization of the gene

SummaryDifferent CD15 murine monoclonal antibodies were studied. These antibodies appeared to react specifically with the human myeloid-lineage-derived cell types in both peripheral blood and bone marrow.The antigens recognized by these antibodies were immunoprecipitated from lysates of125I-labelled neutrophilic PMNs of healthy donors and subsequently analysed by electrophoresis on SDS-polyacrylamide gel and autoradiography. All antibodies precipitated the same membrane polypeptides from the membrane-iodinated PMN lysates: 105 and 150-kDa as most prominent, together with 260-, 230-, 67- and 52-kDa polypeptides.Absorption studies were performed with synthesized carbohydrate molecules. Antibody B4.3 appears to be directed against 3-α-fucosyl-N-acetyl-lactosamine (FAL). Competition experiments with125I-labelled B4.3 demonstrated complete inhibition of binding by B4.3 and three other CD15 antibodies (VIM D5, UJ308, MI/N1), and partial inhibition by three additional antibodies (FMC10, FMC12, FMC13), indicating binding to the same antigenic structure. None of the antibodies reacted with monocytes using the immunofluorescence technique, but after neuraminidase digestion of these cells, positive reactions were obtained with all antibodies. Immunoprecipitation with lysates of both native and neuraminidase-digested monocytes showed no polypeptide bands. Monocytic differentiation of the myeloid cell line HL60 by 12-O-tetradecanoylphorbol-13-acetate (TPA) was accompanied by a decrease in reactivity with the antibodies, which could be reversed by neuraminidase digestion. This indicates that 3-α-fucosyl-N-acetyl-lactosamine is masked for the detection with antibodies upon monocytic differentiation by sialylation.Human x mouse myeloid cell hybrids were obtained after fusion of human myeloid cells and the HPRT-deficient murine myeloid cell line WEHI-TG. These hybrids were tested for reactivity with the anti-CD15 antibodies.The CD15 panel exhibited very similar reactivity patterns with the hybrid clones. Chromosomal analysis of hybrid cell metaphases revealed that the gene(s) involved in the expression of FAL must be located on human chromosome 11 in the region q12-qter.