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T G Cotter

Publications and source records attributed to T G Cotter.

At least 109 records · Page 6Linked to original sources
PubMed1990

HL-60 cells induced to differentiate towards neutrophils subsequently die via apoptosis.

The human promyelocytic HL-60 cell line can be induced to differentiate to neutrophil-like cells in response to a variety of chemical stimuli. We have found that retinoic acid-treatment of HL-60 cells over a period of 6-8 days resulted in a progressive increase in the proportion of cells with mature neutrophil morphologies and was closely followed by an increase in the proportion of cells exhibiting the morphological characteristics of apoptosis, the non-pathological mode of cell death. Using Percoll step-density gradients we have demonstrated a marked increase in the buoyant density of these cells and have used this density difference to obtain enriched fractions of cells for more detailed study. Degradation of the nuclear DNA of these cells into integer multiples of about 200 base pairs, indicative of endogenous endonuclease activation a major characteristic of programmed cell death, was also demonstrated. From these observations we conclude that the mode of cell death in cultures of terminally differentiated HL-60 cells is that of apoptosis. These results parallel those of a recent report which has shown apoptosis to be the mode of cell death of ageing peripheral blood neutrophils. Because of this, we believe that our observations further validate the use of the HL-60 cell line as a model system for the study of human granulopoiesis in vitro and further, that this model system may be useful for gaining insight into the underlying mechanisms involved in apoptosis.

Cell Differentiation↗
PubMed1989

Identification of a leukocyte alloantigen with a high-frequency expression in leukemia patients.

In this report we describe the production and characterization of a monoclonal antibody to the human promyelocytic leukemia cell line HL-60. The antibody, NC-2, is of the IgG1 subclass and precipitates a 50-Kd protein from 125I-labeled HL-60 cells. The antigen is insensitive to treatment with trypsin, papain, or neuraminidase. NC-2 did not react with a number of established human cell lines, including Daudi, Molt-4, K562, U937, KG-1, CEM, Raji, and Gash-P. Neutrophils and monocytelike cells derived from HL-60 cells that were induced to differentiate continued to express the antigen. NC-2 reacted with all peripheral-blood cells except erythrocytes from eight (5%) of 150 normal individuals tested. Bone marrow samples from patients with myelogenous leukemias were more frequently reactive with NC-2 than were those from normal individuals (12/33 v 1/10). Family studies indicated that the antigen was inherited in an autosomal-dominant manner. These findings suggest that the expression of the above alloantigen is associated with an increased incidence of leukemia.

Animals↗
PubMed1988

The degranulation response in differentiated HL-60 cells.

HL-60 cells differentiate into mature granulocytes in response to treatment with a variety of chemical agents. Such HL-60 cell derived granulocytes display many of the properties associated with their peripheral blood counterpart. In this study we have investigated the development of the degranulation response in dimethylsulfoxide (DMSO) or retinoic acid differentiated HL-60 cells over a six day period. The release of a number of enzymes in response to stimulation by a variety of agents was examined. Soluble aggregated IgG (SAIgG) stimulated the release primarily of elastase from HL-60 derived granulocytes with little or no release of other granule enzymes, in particular myeloperoxidase. This contrasted to what was seen when peripheral blood granulocytes were used. The lack of myeloperoxidase release was not due to the parallel release of enzyme inhibitors or failure of the stimulus to bind to the cells. Neither was it due to variations in the kinetics of enzyme release or the presence of myeloperoxidase and elastase in discrete sub-populations of HL-60 cells. When other stimuli such as fMet-Leu-Phe, A23187, or phorbol myristate acetate (PMA) were used a relatively normal degranulation response was seen. Thus, the degranulation response in granulocytes derived from HL-60 cells appears relatively normal when a range of commonly used stimuli are used but is impaired when aggregates of IgG are used.

Calcimycin↗
PubMed1987

Production and characterization of a monoclonal antibody to a myeloid specific differentiation antigen.

A monoclonal antibody termed NC-1 was produced which binds to an antigen present on the human promyelocytic leukemia cell line HL-60 and peripheral blood neutrophils. The antibody bound to the majority of promyelocytes in the HL-60 culture, but not to myeloblasts. No antibody reactivity was detected to a range of other cell lines or to a limited number of normal human tissues. Peripheral blood lymphocytes, monocytes, basophils, eosinophils, erythrocytes and platelets did not react with the antibody. Bone marrow smears exhibited binding of NC-1 to myeloid cells at the promyelocyte and later stages of differentiation along the granulocyte lineage. The KG-1 myeloblastoid and U937 myelomonocytic lines could be induced to express the antigen, when exposed to neuraminidase which removes terminal sialic acid from carbohydrate residues. Similarly myeloblasts in bone marrow samples bound NC-1 when they were treated with neuraminidase. HL-60 cells induced to differentiate to granulocytes by treatment with retinoic acid continued to express the antigen. A similar result was observed when HL-60 cells were induced to differentiate to monocytes, even though blood monocytes failed to bind the antibody. These data indicate that the antibody NC-1 reacts with an antigen expressed on myeloid cells beyond the promyelocyte stage of differentiation.

Antibodies, Monoclonal↗
PubMed1986

Age-related differences in granulocyte chemotaxis and degranulation.

In the present study we have demonstrated that human peripheral blood granulocytes from elderly subjects exhibit reduced chemotaxis and degranulation in response to stimulation with fMet-Leu-Phe. Cyclic AMP levels in non-stimulated cells were not significantly different (mean +/- SEM): 4.8 +/- 0.6 and 3.9 +/- 0.4 pmol/10(7) cells for young and elderly respectively. Stimulation with fMet-Leu-Phe (5 X 10(-7) mol/l) for 30s induced the production of cyclic AMP: 8.4 +/- 0.5 and 6.7 +/- 0.3 pmol/10(7) cells for young and elderly. These findings, together with those previously published by us, suggest that cyclic AMP production and cellular functions which this molecule participates in are reduced in cells from elderly individuals.

Adolescent↗
PubMed1984

Purification and characterization of an antigen involved in neutrophil chemotaxis and degranulation using a monoclonal antibody.

In a previous study we described an anti-neutrophil monoclonal antibody, which inhibited human neutrophil chemotaxis and degranulation without any detectable effect on phagocytosis or oxidative metabolism (Cotter, T. G., Spears, P. and Henson, P. M., J. Immunol. 1981. 127: 1355). This antibody was termed NCD 1. In this study we determined the number of NCD 1-binding sites per neutrophil. Approximately 25 000 NCD 1 IgG-binding sites per cell were found with an equilibrium dissociation constant (Kd) of 6.5 microM for antibody binding. NCD 1 Fab bound to approximately 39 000 sites per cell with a Kd of 16.5 microM. Affinity chromatography columns prepared by coupling NCD 1 to Sepharose 4B beads were used to purify the antigen which bound this antibody. The antigen was a 110-kDa glycoprotein which was not susceptible to reduction by 2-mercaptoethanol. The antigen was not internalized following phagocytosis of opsonized sheep erythrocytes by neutrophils.

Antibodies, Monoclonal↗
PubMed1983

Decreased neutrophil cyclic AMP response to isoprenaline stimulation in the elderly.

Neutrophils from drug-free elderly subjects produced approximately 50% less cyclic AMP in response to isoprenaline than did neutrophils from young subjects. A significant difference in basal cyclic AMP levels was also evident (elderly 2.8 +/- 0.37; young 4.9 +/- 0.36 pmol of cAMP/10(7) cells; P less than 0.05). With a range of anti-neutrophil monoclonal antibodies no evidence of age-related neutrophil population heterogeneity was found. These findings indicate that the age-related decline in beta-adrenoceptor responsiveness is not due to changes in the neutrophil population. The present results support the hypothesis that there is a generalized decline in beta-adrenoceptor-mediated responsiveness in the elderly.

Adult↗
PubMed1983

A cell surface differentiation antigen involved in human neutrophil chemotaxis and degranulation.

In this study we have demonstrated that the human promyelocytic leukaemia cell line (HL-60 cells) completely lack the biological functions of chemotaxis and degranulation. In addition, they were also unable to bind the anti-neutrophil monoclonal antibody NCD 1 which has been shown to inhibit these functions in the peripheral blood neutrophil. When HL-60 cells were induced to differentiate by culturing for 6 days at 37 degrees C in the presence of either 1.25% dimethylsulphoxide (DMSO) or 0.5% dimethylformamide (DMF), up to 35% bound NCD 1. Differentiated HL-60 cells are capable of chemotaxis, degranulation and these newly acquired functions were inhibited by NCD 1 in a manner similar to that seen for the peripheral blood neutrophil. The results correlate the appearance of an antigen on the cell surface of DMSO- or DMF-induced HL-60 cells with the acquisition of two specific cellular functions.

Antibodies, Monoclonal↗
PubMed1981

A monoclonal antibody inhibiting human neutrophil chemotaxis and degranulation.

The hybridoma technique of Kohler and Milstein was used to raise a monoclonal antibody against human neutrophils it has been found to inhibit neutrophil chemotaxis and degranulation. The antibody has been termed NCD 1. Incubation of 12 micrograms of NCD 1 with neutrophils (2 X 10(6)), followed by incubation with cytochalasin B and stimulation with either C5a, fMet-Leu-Phe, or soluble aggregated immunoglobulin, resulted in the inhibition of lysosomal enzyme release. The inhibition was not cytochalasin B dependent, since lysosomal enzyme release induced by opsonized zymosan or opsonized sheep erythrocytes was also inhibited, even in the absence of cytochalasin B. NCD 1 had no affect on enzyme release induced by the calcium ionophore A23138 or by phorbol myristate acetate. The antibody also inhibited neutrophil chemotaxis to zymosan-activated plasma and fMet-Leu-Phe. By contrast, superoxide production stimulated by phorbol myristate acetate, opsonized zymosan, or fMet-Leu-Phe was not affected by NCD 1. Similarly, uptake of 51Cr-labeled opsonized sheep erythrocytes into neutrophils was resistant to the effects of the antibody. NCD 1 Fab fragments did not inhibit lysosomal enzyme release or chemotaxis, althugh binding to the neutrophil surface was demonstrated by immunofluorescence. NCD 1 F(ab')2 fragments behaved analogously to the whole antibody. Binding of both fMet-Leu-[3H]Phe or 125I-soluble aggregated immunoglobulin to the cells was not inhibited by the antibody, which implied that NCD 1 was inhibiting some post-ligand binding event.

Animals↗
PubMed1981

A monoclonal antibody-inhibiting FMLP-induced chemotaxis of human neutrophils.

A monoclonal IgG1 antibody, termed NCD3, was raised against human neutrophils and has been shown to inhibit neutrophil chemotaxis. NCD3 displayed a considerable degree of stimulus specificity in that it inhibited N-formyl-methionyl-leucyl-phenylalanine- (FMLP) induced chemotaxis up to 80%, C5a and zymosan-activated plasma induced chemotaxis by only 20% and had no effect on leukotriene B4- (LTB4) or casein-mediated chemotaxis. NCD3 did not inhibit granule enzyme release from neutrophils in response to stimulation by various secretagogues, including FMLP, in the presence or absence of cytochalasin B (CB). Neutrophil phagocytosis of 51Cr-labeled opsonized sheep erythrocytes (51Cr-EAC) and superoxide anion (O2-) production in response to FMLP or phorbol myristate acetate (PMA) stimulation were not affected by pretreating cells with NCD3. Divalency of NCD3 was necessary for inhibition of chemotaxis, suggesting a requirement for cross-linking of the antigenic determinants on the neutrophil membrane surface.

Anaphylatoxins↗
PubMed1980

Purification and characterisation of an 'elastase-like' enzyme from rabbit polymorphonuclear leucocytes.

An enzyme with proteolytic activity has been isolated from the subcellular granules of rabbit polymorphonuclear leucocytes. Purification of the enzyme involved extraction of the granule membranes with 0.01 M sodium phosphate buffer, pH 7.4, containing 1 M NaCl and 0.1% Triton X-100, followed by gel exclusion chromatography on Sephadex G-75. The enzyme hydrolysed p-nitrophenol-N-tert-butyloxylcarbonyl-L-alaninate, a synthetic substrate for elastase, but failed to hydrolyse elastin. The enzyme also hydrolysed azo-albumin with a pH optimum between 7.5 and 8.5. Inhibition studies indicated that the enzyme was a serine proteinase (EC 3.4.21.-) and it was found to have an apparent molecular weight of 25 000 by polyacrylamide gel electrophoresis. The purified enzyme behaved as a single on SDS-polyacrylamide gel electrophoresis, had a single isolectric point at pH 5.9, yet showed multiple components on centrifugation.

Animals↗
PubMed1979

Studies on the filtration properties of isolated renal basement membranes.

1. The filtration properties of films of renal basement membrane were studied in vitro using pressure filtration chambers. 2. Retention of cytochrome c by the films was found to be dependent upon the filtration pressure indicating that it was transferred across the films by convective as well as diffusive flow. In contrast, serum albumin was transferred by diffusive movement only. 3. When solutions containing both cytochrome c and IgG were filtered it was found that increasing the filtration pressure reduced the flux of cytochrome c across the films. A similar phenomenon occurred when serum was filtered, less protein passed through the films at high filtration pressures. These phenomena are explained by concentration-polarisation effects. 4. The flux of cytochrome c through the films was found to decrease in a non-linear manner as the films thickness was increased. With thin films, the flux of cytochrome c increased in a non-linear manner as the concentration of the protein in the overstanding solution was increased. With thicker films the flux was linearly dependent on concentration. These findings are interpreted as supporting the view that movement of cytochrome c occurs, at least in part, by convective flow.

Animals↗
PubMed1978

Effects of concentration-polarization on the filtration of proteins through filters constructed from isolated renal basement membrane.

1. Filters comprising multiple layers of rabbit renal tubular basement membrane were constructed with conventional pressure filtration chambers. The effects of concentration-polarization on the behaviour of these filters was assessed by studying the filtration of proteins and of serum under turbulent (stirred) and unstirred conditions. 2. With stirring bovine serum albumin was effectively rejected by the filter barriers (sigma = 0.95) but rejection was diminished (sigma = 0.18) without stirring. The hydraulic permeability of the filters also fell without stirring. 3. In the presence of horse immunoglobulin G, a wholly rejected protein, the rejection of cytochrome c was increased and hydraulic flux was reduced. 4. Filtration studies of serum showed that serum protein was effectively rejected with stirring (sigma greater than 0.999) but rejection diminished when stirring ceased (sigma = 0.98). Albumin was the only protein detected in the filtrate with stirring but alpha- and beta- globulins appeared when stirring ceased. 5. These results show that concentration-polarization markedly affects the behaviour of these basement membrane filters in vitro, since without stirring a polarization layer of rejected protein is formed, which reduces hydraulic permeability and results in increased protein permeation through the filter.

Animals↗
PubMedDate not supplied

The induction of apoptosis by chemotherapeutic agents occurs in all phases of the cell cycle.

A1.1 T-cell hybridoma cells exposed to either actinomycin D (1 microgram/ml), camptothecin (200 ng/ml) or aphidicolin (10 micrograms/ml) for 16 hrs at 37 degrees C die via apoptosis. The cell death was independent of RNA synthesis, in contrast to previous data reported for other forms of apoptosis in murine lymphocyte cells and their derived lines. Each of the three agents described appeared to induce death in all phases of the cell cycle in asynchronously proliferating cells. G1 cells appeared to be more susceptible to the effects of camptothecin and contrasts with other reports which detail its selectivity for S and G2 phase cells. This might indicate that cells are progressing into S phase before dying or, alternatively, cells may indeed be dying in G1. When elutriated synchronised cells were exposed to each of the three cytotoxic agents cell death occurred in all phases of the cell cycle. In view of the fact that G1 and S phase cells did not cycle to any appreciable extent during drug exposure, it was likely that ensuing death, occurred specifically from these phases. G2/M cells, however, moved rapidly into G1 in the presence of each drug, thus making it difficult to determine whether G2/M cells were capable of undergoing drug-induced apoptosis. To overcome this problem, nocodazole (50 ng/ml) was used to block asynchronous cells in M phase. When these cells were exposed to actinomycin D, aphidicolin or camptothecin, cell death ensued via apoptosis.

Animals↗