Decay-accelerating factor is present on paroxysmal nocturnal hemoglobinuria erythroid progenitors and lost during erythropoiesis in vitro.

A glycoprotein that regulates the deposition of C3b on the erythrocyte surface, called decay-accelerating factor or DAF, is absent from the red blood cells (RBC) of patients with paroxysmal nocturnal hemoglobinuria (PNH), explaining in part their abnormal sensitivity to complement. We used a specific antiserum to DAF, flow microfluorometry, and clonogenic assays for erythroid progenitor cells to study PNH erythropoiesis in vitro. By fluorescence-activated cell sorter analysis, all RBC from normal individuals are DAF+. In contrast, the RBC of six patients with PNH showed discrete populations of DAF- cells (10-44%; x +/- SEM = 31 +/- 6%). The DAF- RBC population was partly eliminated by prior acidified serum lysis. To determine whether erythropoietic progenitors expressed DAF, bone marrow cells were sorted by flow microfluorometry and the separated DAF+ and DAF- populations then cultured in vitro. In two normal individuals, but also in six patients with PNH, erythroid colonies formed only from cells in the DAF+ fraction. However, a variable proportion of the normoblast progeny of these DAF+ progenitor cells from patients with PNH was DAF-. Individual bursts removed from cultures of PNH bone marrow showed two discrete populations by fluorescence; the majority of normoblasts were DAF-, only 3 of 27 individual bursts had greater than 50% DAF+ cells, and in three patients, DAF- normoblasts averaged 79%. In contrast, the progeny of individual bursts from normal individuals comprised a unimodal DAF+ population. In each PNH patient, one normal burst (greater than 80% DAF+ normoblasts) was detected, possibly reflecting a normal residual population of erythroid progenitors. By the criterion of DAF expression, there was no evidence of separate populations of normal and PNH type progenitor cells. The phenotypically normal erythroid progenitors of PNH bone marrow acquire the PNH characteristics during differentiation in vitro.

Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule.

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.

Expression of transforming growth factor-beta 1 in specific cells and tissues of adult and neonatal mice.

We have used immunohistochemical techniques to detect transforming growth factor-beta 1 (TGF-beta 1) in many tissues of adult and neonatal mice. Each of two antibodies raised to the amino-terminal 30 amino acids of TGF-beta 1 selectively stained this molecule in either intracellular or extracellular locations. Strong intracellular staining was found in adrenal cortex, megakaryocytes and other cells of the bone marrow, cardiac myocytes, chondrocytes, renal distal tubules, ovarian glandular cells, and chorionic cells of the placenta. Marked staining of extracellular matrix was found in cartilage, heart, pancreas, placenta, skin, and uterus. Staining was often particularly intense in specialized cells of a given tissue, suggesting unique roles for TGF-beta within that tissue. Levels of expression of mRNA for TGF-beta 1 and its histochemical staining did not necessarily correlate in a given tissue, as in the spleen. The present data lend further support to the concept that TGF-beta has an important role in controlling interactions between epithelia and surrounding mesenchyme.

Determination of the hemoglobin F program in human progenitor-derived erythroid cells.

The absolute adult and fetal hemoglobin (HbF) contents of the erythroid cells derived from the differentiation of normal human and simian erythroid progenitors and of the peripheral blood erythroid burst-forming units (BFU-E) of patients with nondeletion hemoglobinopathies have been measured with a sensitive radioligand immunoassay. The HbF content varied between 0.13 and 2.96 pg/cell, representing between 0.7% and 19.6% of the total hemoglobin with a mean value of 7.0%. The absolute content of HbF was indistinguishable in the well-hemoglobinized progeny of marrow erythroid colony-forming units, marrow or blood BFU-E, or of mixed colony-forming units. The term HbF program refers to this inherent capacity to produce fetal hemoglobin (HbF) in the erythroid cells derived from these progenitors in vitro. The HbF content of marrow erythroblasts as determined by the same radioligand immunoassay was similar to that found in the peripheral blood, suggesting that the switch off of gamma-chain production occurs after the erythroid colony-forming unit stage of maturation. Increasing concentrations of a crude erythropoietin-containing preparation induced higher numbers of erythroid colonies, which were larger in size, but the HbF program was unaffected. In contrast to the hemoglobin accumulation in human progenitor-derived colonies, simian progenitor-derived colonies produced considerably more HbF, and the amount of HbF was strongly influenced by progenitor maturity. Assays of the HbF content of erythroblasts derived from culture of the peripheral blood BFU-E of patients with nondeletion hemoglobinopathies and their parents showed that the HbF program in the progenitors of such patients is highly variable. Some produce only a slight excess of HbF in progenitor-derived erythroblasts, whereas others have extraordinarily high HbF programs. The molecular basis of this variability is presently unknown.

Nuclear and cytoplasmic distribution of cellular myb protein in human haematopoietic cells evidenced by monoclonal antibody.

A monoclonal antibody was used for analysing the expression of the cellular myb (c-myb) protein in a variety of established human tumor cell lines and its decrease after induction of differentiation. Differentiated resting human T-cells and B-cells do not express detectable amounts of c-myb protein. However, upon mitogenic stimulation in vitro T-cells exhibit strong expression of the c-myb protein as demonstrated by immunocytochemical staining and indirect immunoprecipitation. In contrast to the transformed T-lymphoblastic cell line Molt-4, where c-myb protein is a nuclear antigen, it was found in proliferating normal T-cells almost exclusively distributed in the cytoplasm. Screening of a total of 70 fresh human malignant lymphomas by immunohistochemical staining indicates the presence of the c-myb protein primarily in non-Hodgkin's lymphomas with a large growth fraction, i.e. precursor cell-derived lymphoblastic lymphomas of B-cell type and T-cell type (9/10, 3/4, respectively) and anaplastic large cell Ki-1 lymphomas (5/9), which originate from activated lymphoid cells. The c-myb protein was located predominantly in the nucleus and in some cases additionally in the cytoplasm. The different subcellular locations suggest a dual functional role. While nuclear localisation is exhibited by transformed haematopoietic cells, cytoplasmic localisation appears to be characteristic for proliferating normal T-cells and points to a second property of the c-myb protein other than interaction with DNA.

Isolation and structural analysis of murine c-fes cDNA clones.

The murine c-fes protein is encoded by an mRNA of 2.8 kb which is expressed predominantly in haemopoietic cell lines of the myeloid lineage. We have isolated over-lapping cDNA clones encompassing the entire coding region of murine c-fes. The predicted translation product of the c-fes mRNA is an 820 amino acid protein with 89% overall similarity to feline and human c-fes sequences, and 66% similarity to the chicken c-fps sequence. The murine c-fes proto-oncogene is related to other members of the protein tyrosine kinase family of oncogenes, although it is structurally distinct from both the receptor type, and the src family of tyrosine kinases. A comparison of the predicted protein sequence of murine, feline, human and chicken c-fps/fes proteins highlights several domains of possible functional significance in the molecule.

TdT+/nonlymphoid antigen+ acute leukemias: immunologic and karyotypic monitoring during therapy and at relapse suggests the transformation of a bipotential stem cell.

Immunophenotype and karyotype were monitored in 19 adult acute leukemia patients with blast cell populations expressing terminal transferase (TdT) and nonlymphoid antigens either at presentation or at relapse. Three patterns of immunophenotypic course were observed when following the patients through at least one, sometimes two (six patients), or three relapses (one patient). Induction chemotherapy induced predominantly TdT+ leukemias with a minor monoblastic component to become TdT-negative, purely monoblastic without clinical response or change in karyotype in five patients (group 1). In group 2, relapse was associated with the disappearance (four patients) or the appearance of TdT+/nonlymphoid antigen+ features (four patients). In two instances, new nonrandom cytogenetic abnormalities, in one case, evolution of an initial abnormal cytogenetic clone, were found at relapse. Six patients (group 3) presented and relapsed with identical TdT+ myeloblastic, promyeloblastic, monoblastic immunophenotype and karyotype. In general, FAB classification did not reflect expression of TdT in nonlymphocytic leukemias or the presence of nonlymphoid blast features in lymphocytic leukemias. Lymphoid-specific antigens in addition to TdT were not detected in any of the cases at the time of nonlymphoid antigen expression. In 11 of the 19 patients, simultaneous expression of TdT and myeloid or monocytic antigens could be demonstrated at the single cell level using double-fluorescence staining. These follow-up data are best consistent with a drug-induced maturation drive of a TdT+/monocytic (majority of cases) or TdT+/myelocytic leukemic stem cell with its differentiation commitment being influenced by chemotherapy or by other as yet undefined conditions predisposing to proliferation of the leukemic cell at relapse.

A monoclonal antibody against migration inhibitory factor (MIF) obtained by immunization with MIF from the human lymphoblast cell line Mo.

A monoclonal antibody-secreting hybrid cell line, E7, was constructed from myeloma cells and spleen cells from BALB/c mice immunized with partially purified human MIF from culture fluid of the human T-lymphoblast cell line Mo. The hybrid cell line E7 was selected by screening hybridoma cultures for their capacity to adsorb added MIF activity when assayed together with rabbit anti-mouse IgG-coupled to protein A-Sepharose. The monoclonal antibody produced by the cloned hybridoma E7 also directly neutralized MIF activity from Mo cells and two species of MIF from the culture fluid of human peripheral blood lymphocytes, but did not neutralize IFN-gamma from Mo cells and from human peripheral blood lymphocytes. This antibody reacted also with a component in phytohemagglutinin preparations with an apparent molecular weight of 60,000; however, it did not react with the active tetramer of phytohemagglutinin.

Detection of messenger RNAs within single hemopoietic cells by in situ hybridization on small slide areas.

In situ hybridization provides a powerful tool to detect specific mRNA sequences at the cellular level. We have applied a modified in situ hybridization technique using specifically prepared regular glass microscope slides to evaluate mRNA levels in cells of small samples. Cells were derived from in vitro colonies or isolated by fluorescence-activated cell sorting and deposited on the slides. These slides were coated with polysiloxane, sparing small circular areas where adherent cells attach and can be grown directly; after preincubation of the collection areas with fibronectin, the slides can also be used to deposit and to grow nonadherent cells. In situ hybridization was performed with 35S-labeled probes. Acetylation of the slides and the cells prior to hybridization, the addition of vanadyl-ribonucleoside complexes, and a prehybridization step were found to be necessary to optimize signal-to-noise ratios, as shown by evaluation of c-myc-specific mRNA in phytohemagglutinin-stimulated T4-lymphocytes. This technique might be very useful to study mRNA expression in small samples of hemopoietic cells.

Cyclic changes in fluorescence polarization of a membrane probe during the cell cycle of an erythroid precursor cell line.

The membrane lipid bilayer of the K562 cell line undergoes marked changes during cell cycling. These changes can be detected by measuring the fluorescence polarization of the rod-like hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene. Single-cell measurements were performed by flow microfluorometry on synchronized K562 cells. The fluorescence polarization of the probe increased after cell division and was maximal during the S phase. Concomitant with the transition from S to G2 and M phase was a decrease in fluorescence polarization. The data are interpreted as reflecting a minimal membrane lipid fluidity during the S phase and a maximal fluidity during the G2/M-phase of the above erythroid precursor cell line.

Detection of membrane lectins on the surface of hemopoietic progenitor cells and their changing pattern during differentiation.

To probe for the presence of membrane lectins on hemopoietic progenitors capable of reacting with specific sugars, we synthesized a group of neoglycoprotein reagents by covalently binding biologically relevant monosaccharides to bovine serum albumin (BSA). These reagents were incubated with marrow cells that were subsequently agglutinated on a layer of BSA. The concentrations of various progenitor cells were determined in agglutinated and nonagglutinated fractions using standard clonal assays. Spleen colony-forming units, mixed lineage colony-forming units, and colony-forming units in culture were preferentially agglutinated by galactosyl- and mannosyl-BSA, but not by fucosyl-BSA, indicating the presence of membrane lectins on the surface of these cells with galactosyl and mannosyl specificities. Differentiation in the erythroid direction was associated first with the loss of galactosyl-recognizing system so that erythroid burst-forming units demonstrated only mannosyl-recognizing system. Further differentiation into erythroid colony-forming units was associated with the loss of mannosyl-reacting system as well but the development of a new fucosyl-reacting system. In every case, the agglutination was reversible and preventable in the presence of competing sugars, indicating the specificity of the reaction. Further characterization of these membrane lectins may shed light on their function in cellular interactions in hemopoiesis.

Evaluation of a mouse Y chromosome probe for assessing marrow transplantation.

The multicopy mouse Y chromosome DNA probe 80Y/B (1) has been used to probe genomic DNA isolated from male and female bone marrow cells, mixed together in known ratios. It was found that in a mixture with a ratio of 1 male:200 female marrow cells the male cells could readily be detected by this method. The potential use of this technique for assessing repopulation kinetics of marrow transplantation is discussed.

Benzidine positive multicentric eosinophilic colonies in human blood mimicking erythroid bursts.

When normal human mononuclear blood cells are cultured in plasma clot or methylcellulose in the presence of erythropoietin, two types of benzidine positive multicentric colonies can be recognized in the light microscope at high magnification: one containing diffusely stained cells with the morphology of erythroblasts (erythroid bursts), and another containing diffusely and/or granular stained benzidine positive cells with a morphology of neutrophils/macrophages. Based on their cytochemical properties, lack of inhibition of the benzidine reaction by potassium cyanide, and strong granular staining with luxol fast blue and alkaline eosin, the cells from the latter are identified as eosinophils.

Analysis of human hemopoietic progenitor cells for the expression of glycoprotein IIIa.

Human hemopoietic progenitor cells were examined for the expression of glycoprotein IIIa (GPIIIa). This protein, which forms the beta-subunit of the GPIIb/IIIa receptor for cytoadhesive proteins as well as the beta-subunit of the vitronectin receptor, represents the most sensitive cell surface marker so far identified for the megakaryocytic lineage. Bone marrow cells were fractionated by a discontinuous Percoll gradient to separate cells that form megakaryocytic colonies in culture (1.05 greater than rho less than 1.077 g/ml). Density centrifugation was followed by indirect immunopanning to select for an enriched population of progenitor cells depleted of most of the mature cells of the myeloid, lymphoid, and erythroid lineages. This cell suspension was labeled with antibodies directed against determinants of GPIIIa and analyzed using a fluorescence-activated cell sorter (FACS). Fractions of cells were sorted and analyzed for the ability to form hemopoietic colonies in culture. Our study demonstrated that megakaryocytic progenitor cells (CFU-M) as well as granulocyte-macrophage colony-forming units (CFU-C), erythroid colony-forming units (BFU-E), and mixed lineage colony-forming units (CFU-GEMM) express HLA-DR antigens but lack GPIIIa. Therefore GPIIIa represents a marker that is not present on hemopoietic progenitor cells, but is expressed on the progenies of CFU-M. In view of the importance of GPIIIa as a component of receptors for cytoadhesive proteins, this finding may help to elucidate the adhesive interactions between early hemopoietic cells and bone marrow interstitium.

Binding of iodinated erythropoietin to rat bone marrow cells under normal and anemic conditions.

Specific binding sites for erythropoietin (Epo) were shown in normal and anemic rat bone marrow cells using [125I]labeled human recombinant Epo. When rats were treated once or several times with phenylhydrazine or malotilate, or by phlebotomy, the serum Epo level determined by RIA began to increase rapidly. Thereafter, both the number of erythroid colony-forming unit (CFU-E)-derived colonies and the Epo binding capacity of bone marrow cells increased almost simultaneously in response to induced anemic states, suggesting that the amount of Epo binding in bone marrow cells may reflect in vivo erythropoiesis. Scatchard analysis of the binding data from normal rats revealed the presence of a single class of binding sites (Kd = 0.18 +/- 0.04 nM, 38 +/- 5 sites/cell). In anemic states, the apparent average receptor number per cell increased (52-62 sites/cell) without changing in binding affinity toward Epo. Furthermore, [125I]Epo was cross-linked to the cell surface molecule of approximately 165 kd in nonreducing conditions and 75 kd in reducing conditions. Autoradiographic analysis indicated that Epo receptors were distributed on immature erythroid cells. Proerythroblasts were the most heavily labeled, whereas orthochromatic erythroblasts and cells of myeloid and lymphoid lineages were not labeled. Calculations based on Scatchard and autoradiographic analysis showed that proerythroblasts have 390 receptor sites per cell, twice as many as basophilic or polychromatophilic erythroblasts have. These results are consistent with the stage-specific action of Epo in physiological differentiation of erythroid cells.

A monoclonal antibody identifies a 215 000-dalton nuclear envelope protein restricted to certain cell types.

The monoclonal antibody, AGF2.3, was isolated from mice immunised with the human promyeloid cell line HL60. By immunofluorescence and immunoelectron microscopy the antibody was shown to bind to the nuclear envelope in uninduced HL60 cells. Immunofluorescent staining was reduced to very low levels in HL60 cells induced to mature to monocytes or neutrophils by addition of 12-0-tetradecanoylphorbol-13-acetate or dimethyl sulfoxide respectively. Blood neutrophils did not express the antigen. Weak immunofluorescent staining of cell nuclei was observed in peripheral blood lymphocytes and in sections of normal human kidney, tonsil and skin epithelium. The AGF2.3 antigen was strongly expressed on the nuclei of 21/21 haemopoietic cell lines and 21/25 permanent non-haemopoietic cell lines representing various cell types. In contrast, the antigen was not expressed by any of six primary (untransformed) cell cultures. These included fibroblasts, endothelial cells and keratinocytes. The antigen was expressed in the Q10 SV-40 transformed cell line derived from a non-expressing primary fibroblast culture. AGF2.3 antibody precipitated a protein with an apparent subunit molecular weight of approximately 215 kDa from Triton X-100 extracts of HL60 and HeLa cells labelled with 35S-methionine. This protein was not detectable in extracts of primary skin fibroblasts prepared in parallel. We conclude that AGF2.3 antibody recognises a previously undescribed protein associated with the nuclear envelope which is expressed at high levels in most transformed cell lines but which is weakly expressed or absent in normal tissues and primary cell cultures.

Prosomes, small cytoplasmic RNP particles, contain glycoproteins.

Prosomes, ubiquitous small ribonucleoprotein complexes, were isolated from the cytoplasm of erythropoietic mouse cells induced by Friend leucemia virus. We present evidence that some of the prosomal proteins are glycosylated. Specific reactions with the biotinylated lectins concanavalin agglutinin (Con A), Solanum tuberosum agglutinin (STA) and Limulus polyphemus agglutinin (LPA) indicate that the carbohydrate moieties contain N-acetylneuraminic acid, N-acetylglucosamine and mannosyl- or glucosyl-residues. Glycosylation of prosomal proteins could explain the resistance of prosomes to proteinase K digestion.

Structure and function of transferrin receptors and their relationship to cell growth.

The transferrin receptor binds the major serum iron-transport protein, transferrin, and mediates cellular iron uptake. The receptor is a major immunodominant cell surface glycoprotein of cultured cells and its expression on the cell surface is co-ordinately regulated with cell growth. Recent structural and functional studies of the transferrin receptor are reviewed. The properties of monoclonal antibodies against the transferrin receptor that inhibit transferrin-mediated iron uptake are described. Studies with these antibodies establish that the transferrin receptor plays an important role in cell growth and suggest that monoclonal antibodies that interfere with the function of growth-related receptors may be useful in regulating tumour cell growth.

Detection of RNA transcripts in normal lymphoid and myeloid colonies.

A procedure is described for the routine detection of RNA transcripts in small numbers of hematopoietic cells growing in semi-solid agar. It is suggested that hybridization depends upon RNA expression and that as few as 2500 mRNA molecules per colony are easily detected. Applications of this technique are described in three diverse experimental systems; immunoglobulin gene expression in B cell colonies; neo expression in normal and transformed B cell clones derived from multipotent stem cells infected with a neo-containing retrovirus; and c-myc expression in factor-dependent myeloid colonies.

A radioimmunoassay that sandwiches human interleukin-2 between radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line.

Two monoclonal antibodies were raised against human interleukin-2 (IL-2) produced by E. coli harboring recombinant complemental DNA. Both antibodies did not neutralize its activity, nor did they inhibit the binding of IL-2 to the receptor on target cells. Taking advantage of the ability of monoclonal antibodies to detect IL-2 that had bound to the receptor, a radioimmunoassay was developed that sandwiched IL-2 between the radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line infected with human T cell leukemia virus Type I. The assay had the advantage of detecting only IL-2 with the ability to bind to the receptor, and displayed a linear dose-response relationship over concentrations ranging from 5 to 100 ng/ml.