ConBr, a Lectin from Canavalia brasiliensis Seeds, Protects Against Quinolinic Acid-Induced Seizures in Mice

Lectins are proteins capable of reversible binding to carbohydrates or glycoconjugates. In the central nervous system of mammals, lectins with affinity for mannose/glucose or galactose can modulate cellular communication. ConBr, a lectin isolated from the seeds of Canavalia brasiliensis, previously showed antidepressant effect in the forced swimming test in mice, with involvement of the monoaminergic system. In this study, we investigated the neuroprotective effects of ConBr against quinolinic acid (QA), a well-known NMDA agonist that produces severe neurotoxicity when administered in vivo. ConBr (10 μg/site) administered via intracerebroventricular (i.c.v.) showed a neuroprotective activity against seizures induced by QA (36.8 nmol/site; i.c.v.) when administered 15 min prior to QA, with a percentage of protection around 50%. ConBr was also able to significantly decrease the severity of the seizures but without changes in the latency of the first convulsion or the duration of the seizures. This effect was dependent on the structural integrity of the ConBr protein and its binding capacity to oligosaccharides residues. ConA, a lectin with high similarity to ConBr, did not reverse the QA-induced seizures. Moreover, ConBr was able to protect against hippocampal cell death caused by QA, which was measured by propidium iodide incorporation. QA caused activation of JNK2 and improved the phosphorylation of Ser831 and 845 on the AMPA receptor GluR1 subunit, and both of these effects were counteracted by ConBr. Our data suggest that the lectin ConBr may exert a modulatory action on NMDA receptors, which inhibits its activity in response to QA.     

Lectin from Canavalia brasiliensis (ConBr) protects hippocampal slices against glutamate neurotoxicity in a manner dependent of PI3K/Akt pathway

The excitotoxicity induced by excessive activation of the glutamatergic neurotransmission pathway is involved in several neuropathologies. In this sense, molecules that prevent the release of glutamate or the excessive activation of its receptors can be useful in preventing the neuronal cell death observed in these diseases. Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates, and some have been used in the study and purification of glutamate receptors. ConBr is a mannose/glucose-binding lectin purified from Canavalia brasiliensis seeds. In the present study, we aimed to evaluate the neuroprotective activity of ConBr against glutamate-induced excitotoxicity. Hippocampal slices were isolated from adult male mice and incubated for 6 h in Krebs saline/DMEM buffer alone (control), in the presence of glutamate or glutamate plus ConBr. The phosphorylation of Akt and mitogen activated protein kinases (MAPKs) such as ERK1/2, p38^MAPK and JNK1/2/3 was evaluated with western blotting. The results indicate that glutamate provoked a reduction in the hippocampal slice viability (−25%), diminished the phosphorylation of Akt and augmented p38^MAPK and ERK1 phosphorylation. No changes were observed in the phosphorylation of JNK1/2/3 or ERK2. Notably, ConBr, through a mechanism dependent on carbohydrate interaction, prevented the reduction of cell viability and Akt phosphorylation induced by glutamate. Furthermore, in the presence of the PI3K inhibitor LY294002, ConBr was unable to reverse glutamate neurotoxicity. Taken together, our data suggest that the neuroprotective effect of ConBr against glutamate neurotoxicity requires oligosaccharide interaction and is dependent on the PI3K/Akt pathway.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Computational model for effects of ligand/receptor binding properties on interleukin-2 trafficking dynamics and T cell proliferation response

Multisubunit cytokine receptors such as the heterotrimeric receptor for interleukin-2 (IL-2) are ubiquitous in hematopoeitic cell types of importance in biotechnology and are crucial regulators of cell proliferation and differentiation behavior. Dynamics of cytokine/receptor endocytic trafficking can significantly impact cell responses through effects of receptor down-regulation and ligand depletion, and in turn are governed by ligand/receptor binding properties. We describe here a computational model for trafficking dynamics of the IL-2 receptor (IL-2R) system, which is able to predict T cell proliferation responses to IL-2. This model comprises kinetic equations describing binding, internalization, and postendocytic sorting of IL-2 and IL-2R, including an experimentally derived dependence of cell proliferation rate on these properties. Computational results from this model predict that IL-2 depletion can be reduced by decreasing its binding affinity for the IL-2R betagamma subunit relative to the alpha subunit at endosomal pH, as a result of enhanced ligand sorting to recycling vis-à-vis degradation, and that an IL-2 analogue with such altered binding properties should exhibit increased potency for stimulating the T cell proliferation response. These results are in agreement with our recent experimental findings for the IL-2 analogue termed 2D1 [Fallon, E. M. et al. J. Biol. Chem. 2000, 275, 6790-6797]. Thus, this type of model may enable prediction of beneficial cytokine/receptor binding properties to aid development of molecular design criteria for improvements in applications such as in vivo cytokine therapies and in vitro hematopoietic cell bioreactors.     

Integrin-mediated mechanotransduction in IL-1β stimulated chondrocytes

Mechanical loading and interleukin-1β (IL-1β) influence the release of nitric oxide (^·NO) and prostaglandin E₂ (PGE₂) from articular chondrocytes via distinct signalling mechanisms. The exact nature of the interplay between the respective signalling pathways remains unclear. Recent studies have shown that integrins act as mechanoreceptors and may transduce extracellular stimuli into intracellular signals, thereby influencing cellular response. The current study demonstrates that the application of dynamic compression induced an inhibition of ^·NO and an upregulation of cell proliferation and proteoglycan synthesis in the presence and absence of IL-1β. PGE₂ release was not affected by dynamic compression in the absence of IL-1β but was inhibited in the presence of the cytokine. The integrin binding peptide, GRGDSP, abolished or reversed the compression-induced alterations in all four parameters assessed in the presence and absence of IL-1β. The non-binding control peptide, GRADSP, had no effect. These data clearly demonstrate that the metabolic response of the chondrocytes to dynamic compression in the presence and absence of IL-1β, are integrin mediated.     

Cellular heterogeneity in human transitional cell carcinoma: an anlysis of optical properties and lectin binding

Summary Flow cytometry was used to measure the binding of a panel of ten fluorescein isothiocyanate(FITC)-conjugated lectins to fifteen samples of normal and neoplastic human urothelium. Concurrent measurement of light scattering and fluorescence permitted the quantification of lectin binding to cellular subpopulations defined by their light-scattering properties. In normal urothelium, we previously demonstrated levels of lectin binding to the cellular subpopulations derived from the superficial and intermediate cell layers which were higher than levels which bound to the subpopulation derived from the basal cell layer (Wardet al., 1987). This difference was most marked withMaclura pomifera agglutinin (MPA),Ricinus communis agglutinin (RCA) andUlex europus agglutinin (UEA). We now report a similar correlation between the degree of differentiation of a cellular subpopulation and the level of lectin binding in human transitional cell carcinomas (TCCs). Morphological differentiation in human TCCs is accompanied by alterations in cell-surface carbohydrates which are similar to those which accompany cellular differentiation in the corresponding normal tissue. No systematic difference in lectin binding was observed between the corresponding subpopulations of normal and neoplastic urothelial cells.     

Metabolism of nitric oxide by Neisseria meningitidis modifies release of NO-regulated cytokines and chemokines by human macrophages

Macrophages produce nitric oxide (NO) via the inducible nitric oxide synthase as part of a successful response to infection. The gene norB of Neisseria meningitidis encodes a NO reductase which enables utilization and consumption of NO during microaerobic respiration and confers resistance to nitrosative stress-related killing by human monocyte-derived macrophages (MDM). In this study we confirmed that NO regulates cytokine and chemokine release by resting MDM: accumulation of TNF-alpha, IL-12, IL-10, CCL5 (RANTES) and CXCL8 (IL-8) in MDM supernatants was significantly modified by the NO-donor S-nitroso-N-penicillamine (SNAP). Using a protein array, infection of MDM with N. meningitidis was shown to be associated with secretion of a wide range of cytokines and chemokines. To test whether NO metabolism by N. meningitidis modifies release of NO-regulated cytokines, we infected MDM with wild-type organisms and an isogenic norB strain. Resulting expression of the cytokines TNF-alpha and IL-12, and the chemokine CXCL8 was increased and production of the cytokine IL-10 and the chemokine CCL5 was decreased in norB-infected MDM, in comparison to wild-type. Addition of SNAP to cultures infected with wild-type mimicked the effect observed in cultures infected with the norB mutant. In conclusion, NorB-catalysed removal of NO modifies cellular release of NO-regulated cytokines and chemokines.     

Amplitude and frequency modulation of metabolic signals in leukocytes: synergistic role of IFN-gamma in IL-6- and IL-2-mediated cell activation.

Many stimuli cause intracellular concentration oscillations of second messengers or metabolites, which, in turn, may encode information in their amplitudes and frequencies. We now test the hypothesis that synergistic cellular responses to dual cytokine exposure correlate with cross-talk between metabolic signaling pathways of leukocytes. Polarized RAW264.7 macrophages and human neutrophils and monocytes exhibited NAD(P)H autofluorescence oscillation periods of congruent with20 s. IFN-gamma tripled the NAD(P)H oscillatory amplitude for these cells. Although IL-6 had no effect, incubation of cells with IFN-gamma and IL-6 increased both oscillatory amplitude and frequency. Parallel changes were noted after treatment with IFN-gamma and IL-2. However, IL-1beta and TNF-alpha did not display frequency doubling with or without IFN-gamma exposure. To determine whether frequency doubling required complete IFN-gamma signaling or simply metabolic amplitude modulation, an electric field was applied to cells at NAD(P)H troughs, which has been shown to enhance NAD(P)H amplitudes. Electric field application led to frequency doubling in the presence of IL-6 or IL-2 alone, suggesting that amplitude modulation is crucial to synergism. Because NADPH participates in electron trafficking to NO, we tested NO production during cytokine exposure. Although IL-6 and IL-2 alone had no effect, IFN-gamma plus IL-6 and IFN-gamma plus IL-2 enhanced NO release in comparison to IFN-gamma treatment alone. When NO production was examined for single cells, it incrementally increased with the same phase and period as NAD(P)H. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy and entrain NO production.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.     

A Lectin from Chinese Mistletoe Increases γδ T Cell-mediated Cytotoxicity through Induction of Caspase-dependent Apoptosis

In this study, a mistletoe lectin (ML) was purified from Chinese mistletoe and the effect of this 60 kDa Chinese ML on human γδ T cell cytotoxicity, apoptosis and modulation of the cytokine network was studied. The cytotoxic properties of δ T cells was evaluated by using a ~(51)Cr release test and employed fluorescence-activated cell sorting and enzyme-linked immunosorbent assay analysis to quantify translocation of the cell membrane phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis. It was found that: (ⅰ) ML effectively stimulated γδ T cell proliferation in a dose- and time-dependent manner; (ⅱ) ML increased γδ T cell cytotoxicity; (ⅲ) ML could modulate lipopolysaccharide-induced cytokine release in a pro-inflammatory manner by increasing tumor necrosis factor (TNF)-α release and inhibiting the release of anti-inflammatory interleukin (IL)-10; (ⅳ) ML induced apoptosis in caspase-dependent and CD95-independent manner. The results indicated that ML is a potent immunomodulator to human γδ T cell cytotoxicity, apoptosis and cytokine production.     

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin-1β: role of neuropeptide Y and nitric oxide

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1β (IL-1β) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1β on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1β and NPY were also investigated. We observed that IL-1β increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1β. Moreover, IL-1β regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1β-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1β induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1β, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.     

Characterization of the Galactose-Specific Binding Activity of a Purified Soluble Entamoeba histolytica Adherence Lectin

ABSTRACT. We studied galactose (Gal)-specific binding of the soluble purified 260-kDa Entamoeba histolytica adherence protein to glycosylation deficient Chinese hamster ovary (CHO) cell mutants. Our goal was to further define the lectin's functional activity and carbohydrate receptor specificity. The adherence protein was purified by acid elution from an immunoaffnity column; however, exposure of the surface membrane lectin on viable trophozoites to identical acid pH conditions had no effect on carbohydrate binding activity. Saturable Gal-specific binding of soluble lectin to parental CHO cells was demonstrated at 4°C by radioimmunoassay; the dissociation coefficient (Kd was 2.39 × 10^?8 M^?1 with 5.97 × 10⁴ lectin receptors present per CHO cell. Gal-specific binding of lectin to Lec2 CHO cell mutants, which have increased N- and O-linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41 × 10⁵/cell) rather than a significantly reduced dissociation constant (4.93 × 10^?8 M^?1). At 4°C, there was no measurable Gal-specific binding of the adherence protein to the Lec and IdlD.Lecl CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 μg/ml) to CHO cells was equivalent at 4°C and 37°C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 μg/ml). Gal-specific binding of the lectin at 4°C was calcium independent and reduced by 81% following adsorption of only 0.2% of the lectin to CHO cells. In summary, these findings indicate that the purified E. histolytica adherence lectin demonstrates saturable Gal-specific binding to 1–6 branched-N-linked and not O-linked galactose terminal cell surface carbohydrates; however, apparently only a small percentage of purified amebic lectin molecules actually possess galactose binding activity.     

Characterization of polysaccharides from Hypnea spinella (Gigartinales) and Halopithys incurva (Ceramiales) and their effect on RAW 264.7 macrophage activity

Red algae have been reported to be an important source of polysaccharides with potential immunomodulatory properties. The objective of this study was to characterize the polysaccharides from Halopithys incurva and Hypnea spinella and to evaluate their effect on the synthesis of cytokines by murine cell line RAW 264.7 macrophages. Polysaccharides were obtained by N-cetylpyridinium bromide precipitation and characterized by Fourier transform-infrared spectroscopy. Their effect on the activity of RAW 264.7 macrophages was examined by quantification of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nitric oxide (NO) production using enzyme-linked immunosorbent assays. The activation of the cytokine IL-6 and NO increased linearly as the concentration of polysaccharides from H. incurva and Hy. spinella increased. In general, the activation of IL-6 and NO was tenfold greater when macrophages were exposed to polysaccharides from H. incurva than when exposed to polysaccharides from Hy. spinella. In contrast, TNF-α concentration did not increase when macrophages were exposed to increasing polysaccharide levels. These results indicate that polysaccharides are strong cytokine IL-6 inducers.     

Release of cytokines and apoptosis in fetal rat Type II pneumocytes exposed to hyperoxia and nitric oxide: modulatory effects of dexamethasone and pentoxifylline

The response of the fetal rat Type II pneumocyte (FTIIP), the stem cell of the alveolar epithelium, to hyperoxia would be helpful to understand the effects of oxygen-induced injury to the immature lung. In such a scenario, the presence of nitric oxide (NO) may have a protective or detrimental effect. Our goals were to evaluate the release of cytokines and apoptotic cell death in freshly isolated FTIIP (19-day) in the presence of 95% O(2) and/or NO. The effects of dexamethasone and pentoxifylline on the FTIIP cytokine response were also studied. There was no significant difference in the levels of IL-1beta and IL-10 from FTIIP, in room air, hyperoxia and/or NO at 2, 6 and 24 h. However, IL-6 release was significantly higher, when measured over time, after 2, 6 and 24 h of exposure to hyperoxia and NO. Dexamethasone in the presence of hyperoxia and/or NO increased the release of IL-10 at 24 h. There was increased apoptosis in FTIIP exposed to hyperoxia alone and in combination with NO; this was significantly attenuated in the presence of dexamethasone and pentoxifylline. We speculate that the cytoprotective effects of dexamethasone in the immature lung may, in part, be mediated via IL-10.     

Exogenous glutamine requirement is confined to late events of T cell activation

Glutamine is required for the proliferation of lymphocytes, but quantitative effects on discrete steps of activation remain unknown to date. Therefore the influence of glutamine (range: 0 mM–1 mM) on the in vitro response of human peripheral blood mononuclear cells (PBMC) to a mitogenic anti-CD3 monoclonal antibody (mAb) was investigated. Expression of surface activation markers by flow cytometry, presence of mRNA of cytokine genes by polymerase chain reaction, release of cytokines by ELISA, and entering into the cell cycle by flow cytometry were sequentially analyzed. Proliferation was measured by a ³H-thymidine incorporation assay. mRNA coding for IL-2, IL-2 receptor, IL-4, IL-5, GM-CSF, and IFN-γ was detectable independently from exogenous glutamine provision; expression of the cell surface activation marker CD69 was also glutamine independent. In contrast, later activation events including the expression of the surface activation markers CD25, CD45RO, and CD71 as well as the production of IFN-γ were found to require exogenous glutamine supply. In contrast, production of TNF-α could be observed in the absence of glutamine and was increased to a limited extent by exogenous glutamine. The overall lymphocyte response as reflected by entering into the cell cycle and proliferation was directly correlated with the glutamine concentration of the culture medium. Efficient progression through the cell cycle was found to require at least 0.5 mM glutamine and an increase in glutamine concentration from 0.1 mM to 1 mM enhanced proliferation by 50%. These results were supported by data obtained following anti-CD3 stimulation of a CD4⁺ T cell clone. Altogether, these data underline that a complete cellular immune response depends on an exogenous glutamine supply. Regarding glutamine requirements, they define early, glutamine-independent and late, glutamine-dependent lymphocyte activation stages.     

Nitric oxide regulation of human peripheral blood mononuclear cells: critical time dependence and selectivity for cytokine versus chemokine expression

NO is antiproliferative for T cells and other immune cells, but there is debate over whether it influences cytokine expression and if so whether it shows cytokine selectivity. Furthermore, the NO effect may depend on exposure time. To address these issues, we precultured human PBMC with the NO donors S-nitrosoglutathione (a natural storage form of NO) or S-nitroso-N-acetyl-D-penicillamine for up to 48 h before cell activation and then monitored proliferation and cytokine and chemokine expression. S-nitrosoglutathione or S-nitroso-N-acetyl-D-penicillamine, but not their non-NO-releasing analogues, inhibited proliferation induced by PHA or IL-2, the effect declining progressively from 48 to 0 h pre-exposure to the mitogen. This was accompanied by reduced PHA-induced IL-2 release and reduced IL-2, IFN-gamma, and IL-13 mRNA expression. In contrast, NO did not influence PHA-induced expression of mRNA for the chemokines lymphotactin, RANTES, IFN-gamma-inducible protein, macrophage-inhibitory protein-1alpha, macrophage-inhibitory protein-1beta, macrophage chemoattractant protein-1, and IL-8 or release of RANTES or IL-8. The NO effects were not toxic and were not accompanied by changes in PHA-induced CD25 expression. We conclude that exposure time to NO is critical to altered PBMC responsiveness and that NO inhibits expression of both Th1 and Th2 cytokines but not chemokines.     

Cytokine and nitric oxide production by Trypanosoma brucei infection in rats fed polyamine-deficient chow

Feeding polyamine-deficient chow (PDC) to rats decreases blood polyamines, increases the activity of ornithine decarboxylase as an index of polyamine production, and increases resistance to Trypanosoma brucei gambiense (Wellcome strain) (WS) infection. In this study, we investigated the influence on cytokine and nitric oxide (NO) production of feeding PDC to rats infected with WS. At 4 days postinfection with WS, serum concentration of interleukin (IL)-12, tumor necrosis factor-alpha, interferon-gamma, IL-10, and NO increased in PDC-fed rats; however, IL-12 concentration in normal chow (NC)-fed rats did not increase. In spleen cells cocultured with WS, levels of IL-12 and inducible NO synthase (NOS) mRNA expression were higher in PDC-fed rats than in NC-fed rats. Proliferation of WS in coculture with spleen cells from PDC-fed rats was inhibited, but inhibition of WS proliferation was not observed when an NOS inhibitor was added into the culture media. Ornithine decarboxylase (ODC) activity increased in NC-fed rats after WS infection, but decreased in PDC-fed rats. These results show that feeding WS-infected rats PDC influences the production of cytokines such as IL-12 and the regulation of NO and polyamine production, and also leads to an increase in resistance against WS.     

Zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses TNF-alpha and IL-1 beta production in monocytes by elevation of guanosine 3',5'-cyclic monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.