Immunological characterization and antibacterial function of persisting granulocytes in leukemic patients receiving pulse cytosine arabinoside-consolidation chemotherapy on days 1, 3, and 5

High-dose cytosine arabinoside (HiDAC) and intermediate-dose cytosine arabinoside (IDAC) have been introduced as effective and safe consolidation chemotherapy in acute myeloid leukemia, with relatively low rates of life-threatening infections despite the high total dose of the cytostatic drug. To explore the biological background of low toxicity, we examined the numbers, immunophenotype, and functional properties of granulocytes in patients with acute myeloid leukemia receiving HiDAC or IDAC. Interestingly, the absolute numbers of neutrophils remained >500/microl until day 10 in 92 of 125 (74%) HiDAC cycles and in 106 of 113 (94%) IDAC cycles. As assessed by electron microscopy, these day-10 granulocytes surviving chemotherapy were found to be mature cells containing secondary granules and phagolysosomes. They also expressed opsonization- and phagocytosis-linked surface Ags (C3biR, CR1, C1qR, C5aR, FcgammaRI, FcgammaRII, FcgammaRIII, and G-CSF and GM-CSF receptors) like neutrophils in healthy controls. Moreover, these day-10 neutrophils exhibited oxidative burst activity and took up and digested bacteria in the same way as neutrophils in healthy controls. There was a negative correlation between absolute neutrophil counts and severe infections in HiDAC- and IDAC-treated patients with a later onset of infections in IDAC patients (median: IDAC, day 18; HiDAC, day 16). Together, functionally mature neutrophils are detectable at least until day 10 in patients treated with HiDAC or IDAC, and may explain the relatively low hematologic toxicity of these consolidation protocols. IDAC is a superior protocol in this regard and may therefore be most suitable for elderly patients and those at high risk for severe infections.     

Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils.

The lipid composition of immature myeloid cells from the bone marrow of normal persons and myeloblasts from patients with acute myeloblastic leukemia was studied and compared with the lipid composition of normal mature human neutrophils. Total cholesterol, phospholipid, and fatty acid composition was determined on each cell type. The leukemic cells showed decreased total cholesterol and cholesterol-to-phospholipid ratio, increase phosphatidylcholine and phosphatidylinositol, decreased phosphatidylethanolamine, and an increased percentage of unsaturated fatty acids when compared to normal mature neutrophils. A nearly identical pattern was seen in the normal immature myeloid precursors from normal bone marrow. We conclude that the altered lipid composition of acute myeloblastic leukemia cells is related to unexplained factors related to cell age and not to malignancy per se.     

Characterization of the human granulocyte-macrophage colony-stimulating factor receptor

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine derived from activated T cells, endothelial cells, fibroblasts, and macrophages. It stimulates myeloid and erythroid progenitors to form colonies in semisolid medium in vitro, as well as enhancing multiple differentiated functions of mature neutrophils, macrophages, and eosinophils. We have examined the binding of human GM-CSF to a variety of responsive human cells and cell lines. The most mature myelomonocytic cells, specifically human neutrophils, macrophages, and eosinophils, express the highest numbers of a single class of high affinity receptors (Kd approximately 37 pM, 293-1000 sites/cell). HL-60 and KG-1 cells exhibit an increase in specific binding at high concentrations of GM-CSF; computer analysis of the data is nonetheless consistent with a single class of high affinity binding sites with a Kd approximately 43 pM and 20-450 sites/cell. Dimethyl sulfoxide induces a 3-10-fold increase in high affinity receptors expressed in HL-60 cells, coincident with terminal neutrophilic differentiation. Finally, binding of 125I-GM-CSF to fresh peripheral blood cells from six patients with chronic myelogenous leukemia was analyzed. In three of six cases, binding was similar to the nonsaturable binding observed with HL-60 and KG-1 cells. GM-CSF binding was low, or in some cases, undetectable on myeloblasts obtained from eight patients with acute myelogenous leukemia. The observed affinities of the receptor for GM-CSF are consistent with all known biological activities. Affinity labeling of both normal neutrophils and dimethyl sulfoxide-induced HL-60 cells with unglycosylated 125I-GM-CSF yielded a band of 98 kDa, implying a molecular weight of approximately 84,000 for the human GM-CSF receptor.     

Analysis of cell-surface protein changes accompanying differentiation of HL-60 cells

The cell-surface proteins of HL-60 human promyelocytic leukemia cells have been compared to those of normal human neutrophils. Proteins of HL-60 cells surface labeled with 125I differed markedly from those of normal neutrophils, as shown by immunoprecipitation and polyacrylamide electrophoresis. Differentiation of HL-60 cells by treatment with dimethylformamide, trans-retinoic acid, or 12-O-tetradecanoylphorbol acetate did not modify the predominant surface-labeled proteins of HL-60 cells to produce a pattern similar to that of normal, mature neutrophils. However, the agents did induce greater quantities of minor cell-surface proteins immunoprecipitated by hyperimmune anti-human neutrophil serum. These immunoprecipitated proteins resembled several of the surface-labeled polypeptides of normal human neutrophils.     

Human granulocyte-specific nuclear antigen(s). II. Detection of antigens in human proliferative syndromes

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.     

Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC)

Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation (BMT) in 21 patients. Bone marrow (BM) was analyzed pre-transplant and 3–4 months post-BMT while the patients were in clinical and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR) (>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation (ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant MPFC abnormality.     

Calpain-mediated X-linked inhibitor of apoptosis degradation in neutrophil apoptosis and its impairment in chronic neutrophilic leukemia

The number of neutrophils in the blood and tissues is controlled by constitutive apoptotic programmed cell death and clearance by phagocytes such as macrophages. Here, we found that calpains cleave the X-linked inhibitor of apoptosis (XIAP) in vitro, producing fragments that are unable to inhibit caspase-3. These fragments were detected in normal neutrophils but were unstable and rapidly degraded. Calpain inhibition delayed tumor necrosis factor-alpha-induced apoptosis of normal neutrophils, consistent with a role for calpains in regulating the onset of apoptosis. Interestingly, neutrophils from three patients with chronic neutrophilic leukemia, a rare syndrome characterized by accumulation of mature neutrophils, exhibited decreased mu-calpain expression, diminished calpain activity, and impaired XIAP degradation. Neutrophils from these patients displayed a delay in spontaneous, Fas-stimulated, and tumor necrosis factor-alpha-induced apoptosis. These observations suggest that calpain-mediated XIAP degradation contributes to initiation of apoptosis in normal neutrophils and dysfunction of this regulatory pathway can lead to pathological neutrophil accumulation.     

CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.     

Inhibition of specific pathways of myeloid cell differentiation by an activated Hox-2.4 homeobox gene.

Abnormal expression of homeobox genes is one of the abnormalities associated with the development of murine and human leukemia. Myeloid leukemic cells that can be induced to differentiate to mature cells by interleukin 6 were stably transfected with an activated Hox-2.4 homeobox gene. Expression of the Hox-2.4 gene in the transfected clones inhibited specific pathways of the myeloid differentiation program induced by interleukin 6. The expression of some genes associated with differentiation was almost completely blocked, and the expression of other genes was either partially inhibited or not affected. The results support the hypothesis that abnormal expression of Hox-2.4 may contribute to the development of leukemia by interfering with the differentiation program.     

The migratory and phagocytic activity of polymorphonuclear leukocytes in rheumatoid arthritis and osteoarthritis patients

Polymorphonuclear neutrophils (PMN) play a central role in the elimination of most extracellular pathogenic microorganisms and any impairment of their functions therefore predisposes to defect immune defence. We investigated the migratory and phagocytic functions of the PMNs isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The results suggest that in RA the number and the migratory but not phagocytic capacity of synovial fluid (SF) neutrophils were enhanced, while in OA they were significantly decreased in synovial fluid cells comparatively with peripheral blood (PB). The migratory function of both PB and SF cells from RA patients was increased comparatively with that of the cells from OA patients. We found the different abnormal functions in synovial fluid neutrophils from RA and OA patients. These results may help to elucidate the underlying mechanism which leads to severe joint destruction and different susceptibility to infectious diseases in patients with rheumatic disorders.     

Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these low-density granulocytes (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I IFNs. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared with those of autologous normal-density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal-density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize neutrophils extracellular traps (NETs), which display increased externalization of bactericidal, immunostimulatory proteins, and autoantigens, including LL-37, IL-17, and dsDNA. Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by plasmacytoid dendritic cells. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and dsDNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.     

Glycosphingolipids of leukemic cells in adult T-cell leukemia-lymphoma

We analyzed lipids from leukemic cells of two patients with adult T-cell leukemia and compared them with those from T-cell lymphocytes of normal subjects. The neutral glycosphingolipids and gangliosides which were isolated were characterized by thin-layer chromatography and neuraminidase treatment. Both leukemic cells and normal lymphocytes had monoglycosylceramide and diglycosylceramide as major neutral glycosphingolipids. In one patient, diglycosylceramide was markedly increased. II3NeuAc-LacCer (GM3) and more complex gangliosides were detected in both cells. The most characteristic finding in leukemic cells was the occurrence of a disialylated ganglioside, II3(NeuAc)2-LacCer (GD3), which is not found in normal lymphocytes and neutrophils. This ganglioside may be due to the induced synthesis in association with malignant transformation.     

Chromosome Studies in Leukemia

This review discusses the significance of chromosomal abnormalities found in leukemia with the bias of belief that these have a primary role or are the mechanism of action of leukemogenic agents. The Philadelphia chromosome (Ph¹) is present in marrow cells examined without culture at any stage of most patients with chronic granulocytic leukemia (CGL). the presence of this chromosome is of diagnostic and prognostic value.Varied chromosomal abnormalities have been found in acute leukemia. Each abnormality, which may be unique, is absent in remission, found again at relapse and is seldom changed by therapy. Abnormalities may be of number of chromosomes (aneuploid) or structural rearrangements resulting in “marker” chromosomes, Ranges of abnormal numbers of chromosomes, when present, usually have related patterns which suggest origin of several cell types from one initial cell. Cells from patients with increased risk of leukemia owing to genetic factors have a high incidence of chromosome breakage and structural rearrangements suggesting a mechanism for production of clones of abnormal, possibly leukemic, cells.     

Force microscopy of nonadherent cells: a comparison of leukemia cell deformability

Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E(infinity) = 855 +/- 670 Pa for HL60 cells, E(infinity) = 48 +/- 35 Pa for Jurkat cells, E(infinity) = 156 +/- 87 for neutrophils, mean +/- SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia.     

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.     

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.     

Occurrence in human bone marrows of an antigen released from continuous cell cultures derived from human leukemia.

Fluorescent antibodies prepared against extracellular particles from a continuous culture of cells derived from a monocytic leukemia stained JIII cells but not cells infected with Rauscher leukemia virus or simian sarcoma virus. These antibodies reacted with 38% of bone marrow preparations from patients with lymphoma, 26% of preparations from patients with nonmalignant blood disorders and 6% of preparations from patients with leukemia. Bone marrow films from patients with lymphoma over the age of 50 stained less frequently than those from patients under 50. These particles released from JIII cells are not antigenically related to two of the commonly studied oncornaviruses, but may be indicative of the etiology or disease process of lymphoma in young patients.