Maltooligosaccharides from JEG-3 trophoblast-like cells exhibit immunoregulatory properties.

PROBLEM: to better understand the immunoregulatory properties of trophoblasts, we have searched for small immunologically active carbohydrates derived from intact trophoblast-like cells. METHOD OF STUDY: using solid phase extraction coupled with HPLC and mass spectrometry methods, we have characterized a low molecular weight carbohydrate-rich fraction associated with JEG-3 cells. We have also tested the bioactivities of selected authentic oligosaccharides found in the oligosaccharide fraction. RESULTS: the most abundant components of the low molecular weight carbohydrate-rich fraction were maltotriose and maltotetraose, with detectable amounts of maltopentaose. When authentic maltooligosaccharides were tested using lymphocytes, IL-2 inhibition was observed. This activity was dependent upon the number of saccharide subunits, stereochemistry, and concentration. To further test maltooligosaccharide properties, maltopentose was attached to glass cover slips. Although spontaneous neutrophil motility was observed on unmodified and control surfaces, it was inhibited on maltooligosaccharide-derivatized surfaces. CONCLUSION: maltooligosaccharides are associated with the trophoblast's surface where they may exhibit immunoregulatory activities.

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcgammaRIIA or both FcgammaRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcgammaRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcgammaRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcgammaRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

2,5-Deoxyfructosazine, a D-glucosamine derivative, inhibits T-cell interleukin-2 production better than D-glucosamine.

D-Glucosamine has been widely reported to have immunosuppressive actions on neutrophils, lymphocytes, and other cells of the immune system. However, under conditions used in biological experiments (e.g., neutral pH, and phosphate buffers), we have found that D-glucosamine self-reacts to form 2,5-deoxyfructosazine [2-(D-arabino-tetrahydroxybutyl)-5-(D-erythro-2,3,4-trihydroxybutyl)pyrazine] (1) and 2,5-fructosazine [2,5-bis(D-arabino-tetrahydroxybutyl)pyrazine] (2). When tested for bioactivity at nontoxic concentrations, these D-glucosamine derivatives were more effective inhibitors of IL-2 release from PHA-activated T cells than d-glucosamine. Hence, fructosazines constitute a novel class of immunomodulators.

The hexosamine biosynthesis pathway negatively regulates IL-2 production by Jurkat T cells.

To test the hypothesis that the hexosamine biosynthesis pathway (HBP) affects cytokine production, we studied IL-2 production by Jurkat cells in response to PHA. We found that the HBP activator glucosamine (GlcN), but not glucose (Glc), dose-dependently reduced IL-2 production. Importantly, GlcN blocked trafficking of a GFP-NFAT chimeric protein to the nucleus of stimulated transfectants. Not surprisingly, changes in O-GlcNAc protein modifications were noted during cell activation with and without GlcN addition. These findings could not be explained by some non-specific change in cell metabolism because ATP concentrations did not significantly change. We speculate that HBP-active compounds may contribute to patient care in certain inflammatory and autoimmune diseases.

IFN-gamma primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking.

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-gamma. We hypothesize that synergistic cellular responses to IFN-gamma and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of approximately 20 s. IFN-gamma increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-gamma. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-gamma priming for oxidant production. MPO inhibitors blocked cell responses to IFN-gamma and CpG DNA. In the absence of IFN-gamma exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-gamma and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-gamma plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-gamma treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.

Apparent role of dynein in glucose-6-phosphate dehydrogenase trafficking in neutrophils from pregnant women.

To better understand the mechanisms of metabolic microcompartmentalization associated with neutrophil hexose monophosphate shunt activity during pregnancy, we have studied the intracellular trafficking of glucose-6-phosphate dehydrogenase (G6PDase). Microtubule motor proteins colocalize with G6PDase. Dynein inhibitors block G6PDase accumulation at the microtubule-organizing center in pregnancy cells. On this basis, we conclude that microtubule motor proteins participate in hexose monophosphate shunt enzyme transport within leukocytes.

Transaldolase is part of a supramolecular complex containing glucose-6-phosphate dehydrogenase in human neutrophils that undergoes retrograde trafficking during pregnancy.

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PDase) and 6-phosphogluconate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking in cells from pregnant women, but anterograde trafficking in cells from nonpregnant individuals. Using fluorescence resonance energy transfer techniques, we now demonstrate that transaldolase (TALase), a key regulatory enzyme in the nonoxidative branch of the hexose monophosphate shunt, is in close physical proximity with G6PDase, but not with lactate dehydrogenase, thus suggesting the formation of a TALase-G6PDase complex. Moreover, immunofluorescence microscopy demonstrated that TALase undergoes anterograde trafficking in neutrophils from nonpregnant individuals, whereas retrograde trafficking is found during pregnancy. However, pregnancy did not affect lactate dehydrogenase distribution. Colchicine treatment blocked the retrograde distribution of TALase, suggesting that microtubules are involved in TALase trafficking. We suggest that TALase is part of a supramolecular hexose monophosphate shunt complex, which likely increases the efficiency of the shunt via substrate channeling. We further suggest that TALase's retrograde motion contributes to uncoupling the shunt from its source of glucose-6-phosphate at the plasma membrane, thereby blunting nicotinamide adenine dinucleotide phosphate (reduced form) production and downstream oxidant production by neutrophils.

Molecular proximity of complement receptor type 3 (CR3) and the glycosylphosphatidylinositol-linked protein GPI-80 on neutrophils: effects of cell adherence, exogenous saccharides, and lipid raft disrupting agents.

GPI-80, a novel glycosylphosphatidylinositol (GPI)-anchored protein on polymorphonuclear leukocytes, has been reported to cooperate with CR3 in several aspects of cell function including cell activation, adhesion and migration. The present study investigates the physical proximity of CR3 and GPI-80 on living cells using resonance energy transfer (RET) techniques, which gives positive results when the separation distance is < or = 7 nm. RET from donor-labeled anti-CR3 to acceptor-labeled anti-GPI-80 was detected on adherent neutrophils, but not observed for non-adherent cells. Furthermore, RET was not observed on cells treated with cell adhesion inhibitors 4-bromophenacyl bromide (BPB), N-ethylmaleimide (NEM) or cytochalasin D, suggesting dynamic interactions between CR3 and GPI-80. CR3-to-GPI-80 proximity was blocked by N-acetyl-D-glucosamine (NADG), but not by other monosaccharides such as D-mannose, fructose, fucose, glucose, sorbitol, or galactose; molecular proximity was also disrupted by the glycolipid raft depleting agents 2-OH-propyl-betaCD and MbetaCD. Thus, lipid rafts may be important for the physical and functional cooperation of CR3 and GPI-80.

Identification of channels promoting calcium spikes and waves in HT1080 tumor cells: their apparent roles in cell motility and invasion.

Intracellular Ca(2+) signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca(2+) signaling events in HT1080 fibrosarcoma cells. We have tested the hypothesis that low voltage-activated (LVA) and nonvoltage-gated (NVG) channels of HT1080 cells participate in dynamic Ca(2+)-signaling events leading to cell migration and invasion. Immunofluorescence microscopy has shown that HT1080 cells express LVA T-type Ca(2+) channels uniformly about the cell periphery, whereas the transient receptor potential-1 (a NVG cation channel) protein appears as punctate spots about a cell's periphery. HT1080 cells exhibit periodic intracellular Ca(2+) spikes. High-speed imaging revealed that the Ca(2+) spikes were composed of a single Ca(2+) wave traveling unidirectionally about the periphery of the cytoplasm in a clockwise fashion (as viewed from basal to apical surfaces). The T-type Ca(2+) channel blocker mibefradil inhibited Ca(2+) spikes and waves on cells and, in parallel, inhibited cell motility and invasion in a dose-dependent manner. Similar changes were noted with the NVG cation channel blockers Gd(3+) and carboxyamido-triazole. The combination of LVA and NVG blockers further reduced Matrigel invasiveness. However, the Ca(2+) channel blockers nicardipine, SKF96365, diltiazem, and verapamil had no effect at appropriate doses. These results indicate that certain LVA and NVG channels regulate HT1080 cell motility. In addition to providing novel information regarding cancer cell motility, we suggest that it may be possible to design drugs that inhibit a key Ca(2+) wave, thereby enhancing the efficacy of emerging therapeutic protocols.

Hexokinase translocation during neutrophil activation, chemotaxis, and phagocytosis: disruption by cytochalasin D, dexamethasone, and indomethacin.

Neutrophils expend large amounts of energy to perform demanding cell functions. To better understand energy production and flow during cell activation, immunofluorescence microscopy was employed to determine the location of the key metabolic enzyme hexokinase during various conditions. Hexokinase is translocated from the neutrophil's cytosol to its periphery in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other activating stimuli, but not during exposure to the formyl peptide receptor antagonist N-tert-BOC-phe-leu-phe-leu-phe (Boc-PLPLP). Translocation was observed from 10(-6) to 10(-9)M fMLP. However, fMLP did not affect the intracellular distribution of lactate dehydrogenase. Hexokinase accumulated at the lamellipodium of cells exposured to an fMLP gradient whereas it localized to the phagosome after latex bead uptake. Thus, hexokinase is differentially translocated within cells depending upon the prevailing physiological conditions. Further studies noted that cytochalasin D, dexamethasone, and indomethacin blocked hexokinase translocation. Parallel regulation of reactive oxygen metabolite (ROM) production was shown. We speculate that hexokinase translocation participates in neutrophil activation.

Pregnancy alters glucose-6-phosphate dehydrogenase trafficking, cell metabolism, and oxidant release of maternal neutrophils.

Pregnancy is associated with changes in host susceptibility to infections and inflammatory disease. We hypothesize that metabolic enzyme trafficking affects maternal neutrophil activation. Specifically, immunofluorescence microscopy has shown that glucose-6-phosphate dehydrogenase (G-6-PDase), the rate-controlling step of the hexose monophosphate shunt (HMS), is located near the cell periphery in control neutrophils but is found near the microtubule-organizing centers in cells from pregnant women. Cytochemical studies confirmed that the distribution of the G-6-PDase antigen is coincident with functional G-6-PDase activity. Metabolic oscillations within activated pregnancy neutrophils are higher in amplitude, though lower in frequency, than activated control neutrophils, suggesting limited HMS activity. Analysis of radioisotope-labeled carbon flux from glucose to CO(2) indicates that the HMS is intact in leukocytes from pregnant women, but its level is not enhanced by cell stimulation. Using extracellular fluorescent markers, activated pregnancy neutrophils were found to release reactive oxygen metabolites (ROMs) at a lower rate than activated control neutrophils. However, basal levels of ROM production in polarized pregnancy neutrophils were greater than in control neutrophils. Microtubule-disrupting agents reversed the observed changes in G-6-PDase trafficking, metabolic oscillations, and ROM production by maternal neutrophils. G-6-PDase trafficking appears to be one mechanism regulating ROM production by maternal neutrophils.