Stimulant of antibody producers isolated from the supernatants of cell cultures of normal human bone marrow and in various diseases

The biological activity of a stimulant of antibody producers (SAP) isolated from normal human bone marrow was studied and compared with that of a stimulant of antibody producers from the bone marrow of patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, lymphosarcoma, and multiple myeloma. The activity of the SAP from human bone marrow in health was similar to that of analogous transmitters from the bone marrow of other species of the mammals and birds. The activity of the SAP in patients with multiple myeloma was elevated, whereas in patients with acute myeloblastic leukemia, it was lowered.     

Expression of LSLCL, a new C-type lectin, is closely restricted, in bone marrow, to immature neutrophils.

In vitro, LSLCL is expressed by numerous myeloid, promyelocytic, and T or B lymphoblastoid cell lines. In vivo, LSLCL is strongly expressed in bone marrow and only faintly in lymphoid organs. We show here that, in bone marrow, LSLCL is detected: (i) concentrated in the cytoplasm of immature neutrophils but not in myeloblasts nor in mature neutrophils, (ii) in extracellular bone marrow fluid. Besides, numerous cDNAs, similar to LSLCL (identity of 93-99%), are found in 'expressed sequence tags' databases from various origins, mostly fetal and undifferentiated tumour tissues. Since LSLCL and various closely related cDNAs are expressed at definite stages of cellular maturation processes, we hypothesize that this class of proteins could play an important role in the control of cellular differentiation.     

Surface morphology and ultrastructure of normal and cyclic hematopoietic canine bone marrow in long-term liquid cultures

Long-term liquid cultures of normal and cyclic hematopoietic (CH) dog bone marrow produce committed granulocyte-macrophage progenitor cells (CFU-GM) and differentiated granulocytes for several weeks. Analysis of in situ fixed cultures or of cells harvested from the culture supernatants revealed that the cells had ultrastructure and surface morphology characteristic of immature and mature myeloid cells. The surface morphologies of adherent cells from both normal and CH dogs were similar. The characteristic abnormalities previously reported in neutrophils obtained from CH dogs were not observed in neutrophils obtained from long-term marrow cultures of CH dogs. These results indicate that the cellular abnormalities in the neutrophils of CH dogs may be secondary manifestations of the disease and are not inherent to the pathogenesis of the hematopoietic cells.     

Lipids of human leukocytes: relation to celltype

Significant differences in lipid composition have been found between normal human lymphocytes and polymorphonuclear leukocytes (isolated from blood by means of glass-bead columns), abnormal leukocytes from patients with acute and chronic leukemia, and leukocytes from peritoneal exudates. Lipid extracts of isolated leukocytes were analyzed for total lipid, phosphorus, cholesterol, and plasmalogens. Individual phospholipids and neutral lipids were separated by thin-layer chromatography. The major phospholipids were phosphatidyl choline, ethanolamine glycerophosphatides, sphingomyelin, phosphatidyl serine, and phosphatidyl inositol. Plasmalogen was found mainly as phosphatidal ethanolamine. The neutral lipid fractions contained free cholesterol and various amounts of triglyceride, but little esterified cholesterol. Normal lymphocytes contained about half as much total lipid per cell as normal polymorphonuclear leukocytes, with a similar cholesterol:-lipid-P ratio but relatively more lecithin and less ethanolamine glycerophosphatide. Normal mature leukocytes, compared with immature cells of the same morphological series, had a higher total lipid content per cell, more cholesterol, and a higher ratio of cholesterol to lipid-P. Little difference was found in total lipid-P per cell, but mature cells contained relatively less lecithin and more sphingomyelin. These findings may reflect differences in the relative content of various intracellular organelles as well as possible differences in the quantity and composition of the plasma membrane.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells.

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI+D+, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI+D-, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI+D- compared to the MGI+D+ clones. The MGI+D- clones produced an unusual polyunsaturated C20:5 fatty acid at 28 degrees C, whereas the MGI+D+ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI+D- clones than of the MGI+D+ clones. The cells of MGI+D+ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.     

Normal hematopoietic progenitor cells and malignant lymphohematopoietic cells show different susceptibility to direct cell-mediated MHC-non-restricted lysis by T cell receptor-/CD3-, T cell receptor gamma delta+/CD3+ and T cell receptor-alpha beta+/CD3+ lymphocytes

To evaluate the capability of NK cells and cytotoxic T lymphocytes to interact with normal hematopoietic progenitor cells (HPC), as compared to neoplastic lymphohematopoietic cells, we investigated inhibition of colony growth of these cell populations in semi-solid culture systems, after incubation with cloned cytotoxic effector cells. Three different types of cloned effector cells were investigated: TCR-/CD3- NK cells, TCR-gamma delta+/CD3+ cells, and TCR-alpha beta+/CD3+ cytotoxic T lymphocytes. Effector cells showed differential levels of tumor cell colony inhibition, but no MHC-non-restricted lysis of normal HPC was observed. Pre-stimulation of normal HPC by culturing on established stromal layers had no effect. Cell-mediated lysis of HPC only occurred by Ag-specific MHC-restricted lysis by CTL, or by antibody-dependent cellular cytotoxicity. In cell mixing experiments, irradiated tumor cells, but not normal bone marrow cells inhibited tumor cell lysis. Furthermore, cloned effector lymphocytes were able to specifically eliminate malignant cells from tumor contaminated bone marrow without damaging normal HPC. When fresh leukemic cells were used as targets, growth of acute myeloblastic leukemia colonies was inhibited after incubation with several cytotoxic effector clones, whereas chronic myeloid leukemia precursor cells showed limited sensitivity to MHC-non-restricted cytolysis. These results indicate that MHC-non-restricted cytolysis by NK cells is selectively directed against neoplastic cells and not against normal HPC.     

Monoclonal proliferation of Friend murine leukemia virus-transformed myeloblastic cells occurs early in the leukemogenic process.

Integrated Friend murine leukemia virus copies were analyzed by the Southern blotting procedure in myeloblastic cell lines obtained after in vitro infection of long-term mouse bone marrow cultures. Several steps leading to the generation of malignant myeloblastic cells after a long latency period were observed in the evolution of infected cultures. Shortly after infection, a random distribution of integrated provirus copies was observed in the DNA of normally differentiating myeloid cells. In contrast, a distinct pattern of integrated Friend murine leukemia virus copies was evident in the first non-differentiating immature myeloblastic cells appearing in cultures, suggesting a monoclonal origin of these cells. For each cell line, characteristic hybridizing fragments were conserved during the 1-year culture period necessary for the acquisition of tumorigenic properties and were also observed in tumors grafted in vivo. We can conclude that monoclonality is effective very early in the myeloid transformation process, as soon as the precursor cells are blocked in their differentiation.     

Immunocytochemical localization of lactoferrin in human neutrophils

Summary The subcellular localization of lactoferrin in human neutrophils was studied by an electron-microscopic immunoperoxidase method. This molecule was detected in small granules of blood polymorphonuclear leukocytes. A morphometrical analysis showed that there was no significant difference in the mean size between lactoferrin-positive and myeloperoxidase-negative granules. In contrast, the mean size of myeloperoxidase-positive granules was significantly larger than that of lactoferrin-positive granules. This indicates that lactoferrin is contained in the myeloperoxidase-negative, secondary, granules of human neutrophils. In immature bone marrow mononuclear neutrophils, lactoferrin was present in cytoplasmic granules of somewhat larger size than lactoferrin-positive granules of polymorphonuclear leucocytes. A morphometrical study showed that the mean size of lactoferrin-positive granules was significantly greater in immature bone marrow cells than in polymorphonuclear leucocytes. This indicates that lactoferrin-positive granules decrease in size as the cells mature. Besides cytoplasmic granules, lactoferrin was demonstrated in the Golgi complex and a part of the rough endoplasmic reticulum of immature bone marrow neutrophils, probably myelocytes and early metamyelocytes. These results show that lactoferrin is synthesized and packed into secondary granules in immature bone marrow neutrophils and therefore that the secondary granules are a type of secretory granule.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Interactions between lymphocytes and hematopoietic progenitor cells in normal and leukemic human bone marrow (phase contrast observations)

Single cell observations of normal and of leukemic human bone marrow cells demonstrated cell-cell interactions of lymphocytes with hematopoietic progenitor cells. In all cases lymphocytes and target cells were from the same individual. Lymphocyte-target cell interactions occurred more frequently with normal committed progenitor cells and leukemic blast cells from acute myeloid leukemia than with precursor cells of the proliferative cell pool, including myeloblasts, promonocytes, erythroblasts and megakaryocytes. Both induction of mitosis and degeneration of the progenitor cells occurred after cell-cell interaction with almost the same frequency. Acute myeloid leukemic blast cells degenerated after contact with lymphocytes with the same frequency as normal progenitor cells (i. e. in 16% of cell contacts), but especially during mitosis. In contrast, normal and regenerating bone marrow progenitor cells from myeloproliferative diseases demonstrated no degeneration after cell-cell interaction with lymphocytes during mitosis. Otherwise the induction of mitoses by lymphocyte-target cell interactions was more frequently observed in normal progenitor cells than in leukemic blasts.     

Up-regulation of alpha2,6 sialylation during myeloid maturation: a potential role in myeloid cell release from the bone marrow

The mechanisms by which mature myeloid cells are released from the bone marrow into the peripheral blood are not clearly understood. Glycosylation is likely to play an important role, as has been shown in the homing of lymphocytes to lymph nodes and of neutrophils to inflamed endothelia. Cell surface sialylation is an important component of many cellular adhesive interactions, both as ligand-promoting interactions, as occurs in selectin and sialoadhesin-mediated adhesion, and for reducing cell adhesion as in some cancer cells. We have studied the expression of cell surface alpha2,6-linked sialic acid in the maturation of normal bone marrow myeloid cells, the expression of alpha2,6-sialyltransferase mRNA, and the role of sialylation in the adherence of myeloid cells to bone marrow stroma. Our data show that there is a dramatic increase in cell surface alpha2,6-sialylation during the late stage of maturation. This up-regulation is restricted to specific glycoproteins including CD11b and CD18. It is associated with a relative increase in the level of alpha2,6-sialyltransferase mRNA compared with alpha2,3-sialyltransferase mRNA. The changes in mature bone marrow myeloid cells are associated with reduced cell binding to fibronectin and cultured bone marrow stroma. Our data strongly suggest that alpha2,6-sialylation may be important in the interaction between maturing myeloid cells and bone marrow stroma and may govern the release of cells from the bone marrow into the peripheral blood.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

Antibody-producing human-human hybridomas. III. Derivation and characterization of two antibodies with specificity for human myeloid cells

Peripheral blood mononuclear cells from a patient with acute myeloid leukemia (AML) and spleen cells from a patient with chronic myeloid leukemia (CML) were fused with HAT-sensitive human B lymphoma cells (RH-L4) in attempts to generate human monoclonal antibodies (Mab) against antigens with high specificity for myeloid leukemia cells. Forty-seven of 246 hybridomas secreted Ig that bound to AML cell surface constituents, as determined by FACS analysis of viable cells that were FITC-stained with the human Mab as the first-step reagent and FITC-conjugated rabbit anti-human Ig as second-step. Two of the 47 human Mab (one from each patient and designated AML-19 and CML-20, respectively) bound to both autologous and allogeneic myeloid leukemia cells. No significant binding was observed to cell surface constituents on human bone marrow cells, granulocytes, lymphocytes, erythrocytes, thymocytes, monocytes, lymphoblastic leukemia cells, fibroblasts, malignant B and T lymphocytic cell lines, and murine bone marrow cells. Both human Mab were IgG and were cytotoxic to myeloid leukemia cells in the presence of complement. About 70% of peripheral blood cell samples from 46 AML patients contained AML-19- and CML-20-positive cells, but the reactivity pattern had no correlation to the morphologic FAB classification of the samples. The promyelocytic HL60 cell line and the K562 cell line reacted with the two antibodies. Dot blot analysis of binding of AML-19 and CML-20 to cellular extracts immobilized on nitrocellulose paper showed that both human Mab in this assay also reacted with normal bone marrow cells. This was supported by microscopic immunofluorescence because both human Mab stained intracytoplasmatic structures in normal bone marrow cells, but both intracytoplasmatic and cell surface components stained in myeloid leukemia cells. Moreover, immunoblotting demonstrated that both human Mab in leukemia cells reacted with two cellular proteins with Mr approximately 14,500 and 18,000, and in normal bone marrow cells with a molecule with Mr approximately 20,000. Immunoprecipitation of cell membrane molecules with both the AML-19 and CML-20 antibody precipitated from leukemic cells only the molecule with Mr approximately 18,000 and no components from normal bone marrow cells. It is concluded that myeloid leukemogenesis may result in generation of cell surface expression of either new or abnormally processed molecules that are immunogenic in the autochthonous host. These molecules may also be useful as markers in diagnosis of myeloid leukemia.     

Alpha-smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions

A series of 217 trephine bone marrow biopsies from adult patients and specimens from 16 fetuses and 5 infants were examined for the presence of stromal myoid cells (MCs) using a monoclonal antibody recognizing alpha-smooth muscle actin. In the normal adult bone marrow, stromal cells did not contain alpha-smooth muscle actin, whereas during fetal life, many alpha-smooth muscle actin-containing MCs were connected with vascular sinusoids in the primitive bone marrow. This cell type reappeared in various characteristic distribution patterns in adult bone marrow during different neoplastic and non-neoplastic conditions including metastatic carcinoma, Hodgkin's disease, multiple myeloma, hairy cell leukemia, acute myeloid leukemia (FAB M4, 5, 7) and chronic myelo-proliferative diseases. In general, the appearance of MCs was associated with a slight to pronounced increase in the deposition of reticulin and collagen fibers. We propose that bone marrow MCs represent a distinct subpopulation of fiber-associated or adventitial reticular cells undergoing cytoskeletal remodeling in response to various stimuli.     

The cellular oncogenes c-myc, c-myb and c-erb are transcribed in defined types of avian hematopoietic cells

The possible role of normal chicken cellular sequences c-erb, c-myb and c-myc, together referred to as c-onc genes and related to the oncogenes of defective avian acute leukemia retroviruses (DLVs), was investigated by determining the accumulation of c-onc RNA in different avian cells an cell lines. Levels of c-myc and in some instances c-myb RNA are elevated in immature hematopoietic cells or cell lines from various lineages but more mature hematopoietic cells, as well as non-hematopoietic cells, contain only low levels. In contrast, the level of c-erb RNA is generally low, but high in a small number of normal bone marrow cells. The results indicate that the cellular homologues of the viral oncogenes are differentially expressed during hematopoiesis. They also indicate that the hypothesis that DLV target cells express their homologous c-onc genes might hold for c-erb, but is not valid in its simple form for c-myc and c-myb.     

α-Smooth muscle actin is expressed in a subset of bone marrow stromal cells in normal and pathological conditions

A series of 217 trephine bone marrow biopsies from adult patients and specimens from 16 fetuses and 5 infants were examined for the presence of stromal myoid cells (MCs) using a monoclonal antibody recognizing α-smooth muscle actin. In the normal adult bone marrow, stromal cells did not contain α-smooth muscle actin, whereas during fetal life, many α-smooth muscle actin-containing MCs were connected with vascular sinusoids in the primitive bone marrow. This cell type reappeared in various characteristic distribution patterns in adult bone marrow during different neoplastic and non-neoplastic conditions including metastatic carcinoma, Hodgkin’s disease, multiple myeloma, hairy cell leukemia, acute myeloid leukemia (FAB M4, 5, 7) and chronic myeloproliferative diseases. In general, the appearance of MCs was associated with a slight to pronounced increase in the deposition of reticulin and collagen fibers. We propose that bone marrow MCs represent a distinct subpopulation of fiber-associated or adventitial reticular cells undergoing cytoskeletal remodeling in response to various stimuli.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.     

Action of inhibitor released from neutrophils and leukemic blast cells.

The concept that polymorphonuclear leukocytes, or neutrophils, play a role in feedback control of granulopoiesis has been supported by the finding in bone marrow culture studies that mature neutrophils inhibited formation of granulocytic colonies. The study described in this paper was done to investigate the mechanisms involved. With the use of a modified assay it was found that mature neutrophils released factors that reduced the proliferation of colony-forming cells in cultures stimulated by cell-free colony-stimulating factor. In myeloproliferative and myelodysplastic disorders the amount of inhibitor released by the neutrophils varied greatly. Leukemic blast cells also released inhibitor, and in some cases the amount released per cell was greater than the amount released from normal mature neutrophils. The inhibitory factors released from the neutrophils differed from those previously described in the literature in terms of mode of action and apparent molecular size.