Increase In Hexose Monophosphate Shunt Activity Research Articles

IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils.

IL-4 is a T-helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL-4 on neutrophilic maturation using the cell line HL-60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL-60 cells with recombinant hu IL-4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL-60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL-4 increased surface expression of the neutrophil-lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL-4 treated HL-60 cells demonstrated enhanced Fc- and complement-mediated phagocytic capacity and increased hexose-monophosphate shunt activity. In addition, IL-4 was capable of sustaining the neutrophil maturation of HL-60 cells that had been pre-treated for 24 h with DMSO. To investigate the effect of IL-4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL-4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti-human IL-4 abolished phagocytic stimulation. Finally, IL-4 treatment also stimulated resting neutrophils to migrate toward zymosan-activated serum (ZAS) and human IL-5. The results demonstrate that IL-4 is a potent maturation factor for myelocytes to become neutrophils and that IL-4 can stimulate resting mature neutrophils.

Nitric oxide attenuates cellular hexose monophosphate shunt response to oxidants in articular chondrocytes and acts to promote oxidant injury.

Nitric oxide (NO) has been implicated in both cartilage degradation and cell survival. Importantly, NO has been shown, in a cell-type-dependent manner, to directly cause cell death or indirectly promote cell death by compromising the ability of cells to detoxify intra- or extracellular oxidants. In this study we examined the role of NO in the survival of bovine chondrocytes exposed to catabolic cytokines (interleukin-1 (IL-1); tumor necrosis factor [TNF]) with or without the addition of an exogenous oxidant stress (e.g., H2O2, HOOCl, etc.). The exposure of chondrocytes to a mixture of IL-1 and TNF (IL-1/TNF) results in the release of NO but did not alter cell viability. However, there was evidence of NO-dependent oxidative responses in the IL-1/TNF group, as we observed an increased level of intracellular oxidants as well as the appearance of a 55 kD nitrated protein which reflects the formation of peroxynitrite. We next analyzed viability with H2O2. The LD50 for IL-1/TNF-treated cells was 0.1 mM (vs. 1 mM for control). The enhanced sensitivity was completely reversed when cells were incubated with the NO synthase inhibitor 1-n5-1-iminoethylornithine (NIO). To test whether cell death was caused by compromising the ability of cells to detoxify extracellular oxidants, we examined the hexose monophosphate shunt (HMPS) response in cells given H2O2. Treatment of control cells with H2O2 resulted in a fourfold increase in HMPS activity. In contrast, IL-1/TNF cells exhibited no increase in HMPS activity. The attenuation of stimulated HMPS activity was reversed by the coaddition of NIO. Thus, these data indicate that 1) endogenous NO mediates cytokine-dependent susceptibility to oxidant injury and 2) this effect is in part due to impaired activation of the HMPS. In inflamed joints replete with cytokines and oxidants, NO may contribute to chondrocyte death and progressive joint destruction.

Potentiation of retinoic acid-induced U-937 differentiation into respiratory burst-competent cells by nitric oxide donors

All- trans retinoic acid (tretinoin) is a known inducer of differentiation of the human monoblastic cell line, U-937. We now report that the ability of retinoic acid (RA) to induce differentiation of U-937 cells into cells possessing respiratory burst activity is enhanced by the known nitric oxide-donating drugs glyceryl trinitrate, molsidomine and CAS 936, and by tetranitromethane in combination with cysteine. RA alone was a strong inducer of U-937 differentiation as indicated by the following responses to 12- O-tetradecanoylphorbol-13-acetate (TPA) stimulation: (1) increase in the percentage of cells staining with nitroblue tetrazolium (NBT); (2) increase in the total amount of formazan (the product of NBT reduction by O 2 −) as determined spectrophotometrically; (3) increase in hexose monophosphate shunt (HMPS) activity as assessed by [ 14C]CO 2 released from d-[1- 14C]glucose. RA was also able to increase mRNA levels for two respiratory burst-related genes and for glucose-6-phosphate dehydrogenase (G6PD), an HMPS enzyme. Other indications of differentiation were reduced cell proliferation, increased adherence and altered nuclear morphology. The observed increase in formazan production and HMPS activity and the reduction of cell proliferation due to RA were augmented by co-treatment with either glyceryl trinitrate, molsidomine, CAS 936 or tetranitromethane plus cysteine. Glyceryl trinitrate alone increased HMPS activity and G6PD mRNA levels and also reduced cell proliferation. Glyceryl trinitrate, molsidomine and CAS 936 are presumed to release nitric oxide and increase intracellular cGMP levels by stimulation of soluble guanylate cyclase. The mechanism of action of tetranitromethane is less certain, although it may also generate reactive nitrogen intermediates. These data suggest that a NO·/ cGMP pathway may augment a retinoic acid-mediated pathway to enhance maturation of U-937 cells with respect to the respiratory burst. Glyceryl trinitrate may act additionally by another pathway.

Inhibition of human neutrophil function by 6-aminonicotinamide: The role of the hexose monophosphate shunt in cell activation

Stimulation of polymorphonuclear leukocytes with phorbol myristate acetate (PMA) or chemotactic factors such as f-Met-Leu-Phe (fMLP) activates a membrane oxidase which results in the generation of the superoxide anion (O2−) and the oxidation of NADPH to NADP+. The subsequent reduction of NADP+ to NADPH is believed to be directly dependent upon activation of the hexose monophosphate shunt (HMPS). To further understand the role of the HMPS in the oxidative burst, we examined the kinetics of HMPS activation by fMLP and PMA. Both of these agents stimulate an increase in HMPS activity that parallels their production of O2−. To examine the role of the HMPS in cell activation, we treated polymorphonuclear leukocytes with the specific HMPS inhibitor, 6-aminonicotinamide. This pretreatment inhibited fMLP- and PMA-stimulated HMPS activity and O2− release by 80% and 60% respectively with a 50% inhibitory dose (ID50) of 5 × 10−7 M. Measurement of reduced NADPH using 350 nm ultraviolet light-stimulated fluorescence and flow cytometry indicated that 6-aminonicotinamide had no effect on resting levels of NADPH fluorescence but significantly inhibited the fluorescence recovery following stimulation with fMLP or PMA. In contrast, PMA- and fMLP-stimulated membrane depolarization measured with the carbocyanine dye 3,3′-dihexyloxacarbocyanine iodide and chemotaxis to fMLP were unaffected by 6-aminonicotinamide treatment. On the contrary, fMLP- or PMA-stimulated myeloperoxidase release by fMLP or PMA was enhanced by 30% and 150%, respectively, following treatment with 6-aminonicotinamide, suggesting a decreased oxidative inactivation of myeloperoxidase. These findings show that 6-aminonicotinamide can be used to preferentially inhibit the oxidative burst in neutrophils by inhibiting the HMPS and suggest that the HMPS functions primarily to reduce NADP+ for the production of O2− and has minimal involvement in neutrophil chemotaxis and enzyme release.

Human Monocyte to Macrophage Differentiation In Vitro: Characterization and Mechanisms of the Increased Antibody-Dependent Cytotoxicity Associated With Differentiation

Human monocyte-to-macrophage differentiation in vitro is associated with marked enhancement in the capacity to bind and lyse antibody-opsonized red blood cells (rbc). We previously demonstrated that this was due in part to an increased number of Fc receptors. The current study further characterized the mechanism of this enhanced macrophage as antibody-dependent cellular cytotoxicity (ADCC). We observed that 1) macrophages but not monocytes lysed "innocent bystander" rbc as well as opsonized rbc; 2) macrophages exhibited an increased hexose monophosphate shunt activity compared to monocytes; 3) macrophage produced H2O2 but not OH.; and 4) macrophage lyses of opsonized rbc were 80% O2 dependent. We conclude that human monocyte-to-macrophage maturation in vitro is associated with an enhanced O2-dependent cytotoxic mechanism normally present in monocytes. The enhanced H2O2 production noted in macrophages may reflect an increased generation of several other reactive oxygen species in these cells. One of these oxygen radicals may be the mediator of the enhanced macrophage cytotoxicity and of the "innocent bystander" phenomenon observed.

H 2O 2 production, modification of the glutathione status and methemoglobin formation in red blood cells exposed to diethyldithiocarbamate in vitro

Human red blood cells treated with the CuZn superoxide dismutase inhibitor diethyldithio-carbamate (DDC) undergo metabolic modifications in addition to the superoxide dismutase inhibition: oxidation of the reduced glutathione (GSH) to oxidized glutathione (GSSG), methemoglobin formation, and increased hexose monophosphate shunt activity were observed. The magnitudes of these changes are dependent on the DDC concentration. Under nitrogen, only superoxide dismutase inhibition occurs. After removal of the GSH with N-ethylmaleimide, production of H 2O 2 can be detected by measuring the red cell catalase inhibition in the presence of 3-amino-1,2,4-triazole. H 2O 2 production is not altered by conversion of oxyhemoglobin to methemoglobin by sodium nitrite prior to incubation. GSH oxidation and methemoglobin formation are stopped when DDC is eliminated from the incubation medium after completion of the superoxide dismutase inhibition. These data indicate that methemoglobin formation and modification of the GSH status in red cells treated by DDC are not a direct consequence of the CuZn Superoxide dismutase inhibition but are due rather to a DDC-dependent production of H 2O 2.

Ascorbate-mediated stimulation of neutrophil motility and lymphocyte transformation by inhibition of the peroxidase/H2O2/halide system in vitro and in vivo

Neutrophil migration, postphagocytic hexose-monophosphate shunt activity and myeloperoxidase-mediated iodination of ingested Candida albicans and lymphocyte mitogen-induced transformation were assessed in six normal volunteers before and 1 h after a single intravenous injection of 1 g ascorbate. Increased neutrophil motility was observed which was associated with decreased myeloperoxidase-mediated iodination of C. albicans and a slight increase in hexose-monophosphate shunt activity. Lymphocyte transformation was also increased. Alterations in these activities were related to serum ascorbate levels. To investigate the relationship of ascorbate-mediated increased neutrophil motility and lymphocyte transformation to decreased peroxidase activity neutrophils and lymphocytes from normal individuals were exposed to the horseradish peroxidase/H2O2/sodium iodide system in the presence and absence of ascorbate and tested for migratory and proliferative responses respectively. Exposure to the horseradish peroxidase/H2O2/halide system caused inhibition of neutrophil motility and lymphocyte responsiveness to mitogens. However, inclusion of ascorbate protected both the neutrophils and lymphocytes from the inhibitory effects of the horseradish peroxidase/H2O2/halide system.

Transport and utilization of amino acids and glucose in human monocytes: activation of glucose metabolism by insulin.

The influence of insulin on transport and utilization of amino acids and glucose in purified human peripheral blood monocytes has been studied. Insulin had an immediate stimulating effect on the uptake of 3-O-methylglucose and 2-deoxyglucose; the maximal effects were 55% and 47% increases, respectively, during the first 2 min, in which energy-dependent hexose uptake dominates. Later, with advancing free diffusion, values declined to 16% and 25%. After a lag of 30 min, the rise in glucose uptake was followed by a small rise in glucose oxidation, documented by an 18% increase of 14CO2 production from [1-14C]glucose in the presence of hormone. No effect of insulin on sodium dependent alpha-aminoisobutyric acid or sodium-independent leucine uptake in monocytes could be found. The incorporation of amino acids into monocyte protein remained unchanged as well. Our results prove that the well documented binding of insulin to human monocytes initiates specific cellular reactions. The increased hexose monophosphate shunt activity may result in increased immune reactivity of the monocyte.

Alterations in hexose monophosphate shunt during lymphoblastic transformation

This study characterized the alteration in hexose monophosphate shunt (HMPS) activity occurring during lymphoblastic transformation to nonspecific mitogens. Average HMPS activity was threefold higher in phytohemagglutinin (PHA)-stimulated cultures incubated for 68 hr when compared to unstimulated cultures. Serial measurement of HMPS activity indicated that this increased activity was delayed for several hours after the addition of the mitogen and reached a peak of fivefold higher activity during the second day of culture. Glycolysis increased before HMPS activity and was increased during the entire culture period although it was also maximal during the second day of culture. Pokeweed mitogen also resulted in increased glucose metabolism. The increase was of lesser magnitude than PHA and probably reflected the lower degree of lymphoblastic transformation of pokeweed cultures. These studies demonstrate that lymphocytes undergoing blastic transformation have increased HMPS activity which follows a predictable temporal pattern as does glucose consumption. Also, this study demonstrates that the ionization chamber electrometer technique for the direct measurement of 14CO 2 can be applied to the study of long-term tissue cultures.

Uptake and reduction of oxidized and reduced ascorbate by human leukocytes.

Incubation of human leukocytes with dehydroascorbate (DHA) results in an increase in their reduced ascorbate (AA) content and hexose monophosphate shunt (HMS) activity, independent of oxygen supply. Incubation with AA induces these changes only in the presence of oxygen. The increase in HMS activity observed as cell AA increases by 1 micromol is the same during incubation with either DHA or AA. We propose that human leukocytes take up ascorbate as DHA (AA after oxidation to DHA) and reduce it promptly to AA, and that HMS stimulation upon incubation with either AA or DHA is a result of DHA reduction.

Effects of various antisera on the morphology, phagocytic ability, and metabolism of guinea-pig peritoneal macrophages.

ability to adhere to glass at low concentrations (0.2%-2%) and vacuolization with cell death at high concentrations of antisera (10%). The stages of attachment and ingestion were prolonged by exposure to 1%-2% antiserum, and it became possible to distinguish between them. Metabolic studies revealed that low concentrations of antiserum stimulated consumption of oxygen and hexose-monophosphate-shunt (HMS) activity without significantly affecting production of lactate; these metabolic changes were similar to those during phagocytosis. High concentrations of antiserum inhibited metabolic activity. When macrophages were exposed to 1%-2% AMS, increased HMS activity during phagocytosis appeared to occur during attachment rather than ingestion. Absorption of antisera with macrophages, lymphocytes, or renal tissue abolished antimacrophage effects. Antilymphocyte effects of ALS were not absorbed by exposure to macrophages. Immunosuppression produced by ALS may result from antimacrophage as well as antilymphocyte effects.

The role of hydrogen peroxide and glutathione in glucose oxidation by the thyroid

Catalase depressed the 14CO 2 yield from [I- 14C]glucose in vitro in both TSH-stimulated and unstimulated thyroid slices. This observation was specific for the enzyme and seemed related to its action on H 2O 2, since H 2O 2 in the presence of GSH induced a significant stimulation of the hexose monophosphate shunt in thyroid homogenates. This effect of H 2O 2 and glutathione was specifically linked to the increased reoxidation of NADPH to NADP +, as measured by the spectrophotometric disappearance of the pyridine nucleotide at 340 mμ. These results offered proof for glutathione peroxidation in thyroid and suggested a role of glutathione in regulating thyroid hexose monophosphate shunt activity. It is postulated that the increased hexose monophosphate shunt activity induced by TSH could be at least partially related to H 2O 2 production in the course of colloid droplet or phagosome formation.

Increase in hexosemonophosphate shunt activity during tissue repair

Increase in hexosemonophosphate shunt activity during tissue repair