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The nitric oxide donors, azide, and hydroxylamine, inhibit the programmed cell death of cytokine-deprived human eosinophils
Abstract
Azide and hydroxylamine release nitric oxide (NO) enzymatically in biological conditions. We observed that both compounds were able to inhibit in vitro the programmed cell death of human eosinophils from peripheral blood. This protective effect could be mimicked by permeable cGMP analogs and by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Moreover, the soluble guanylate cyclase inhibitor LY-83583 inhibited in a dose-response manner the effects of the NO donors. Consequently, via the increase of eosinophil survival, NO could contribute to the amplification of inflammatory and allergic processes. This effect appears to be mediated, at least in part, by the soluble guanylate pathway.
... Nasal breathing has been reported to reduce pulmonary vascular resistance (PVR) and improve arterial oxygenation compared with oral breathing in subjects without lung disease (Lundberg, I996;Lundberg et al, I996c;Settergren et al, 1998) Th2 cells which putatively produce a cytokine profile that has been associated with exacerbation of asthma (Taylor-Robinson et al, 1993;Bames & Liew, 1995). NO has also been shown to promote chemotaxis (Ferreira et al, 1996) and increase survival o f eosinophils (Beauvais et al, 1995). On the other hand, NO has been found to be mainly secreted in the nose and paranasal sinuses (Lundberg et al, 1994b;Lundberg et al, 1995a), with diseases involving them modulating nasal NO levels (Kharitonov et al, 1997b;Lindberg et al, 1997b). ...
... Furthermore, some studies in murine cells have suggested that NO may amplify inflammation by altering the balance between T-helper (Th) 1 and Th2 cell types, leading to the proliferation o f Th2 cells which putatively produce a cytokine profile that has been associated with exacerbation o f asthma (Barnes & Liew, 1995). NO has also been shown to promote chemotaxis (Ferreira et al, 1996) and increase survival o f eosinophils (Beauvais et al, 1995). These observations, however, have not yet been extended to humans. ...
Chronic rhinosinusitis (CRS) is a prevalent disease that has marked effects on the society. Its definition, pathophysiology, microbiology and treatment are still a source of debate. Likewise, there is still considerable confusion in the literature concerning the relationship and the effect of therapy of CRS upon the concomitant asthma. The aims of the thesis were: To conduct the first prospective randomised controlled trial, evaluating and comparing the medical and surgical treatment of CRS and its subgroups; to study whether the presence of nasal polyps serves as a poor prognostic factor for the efficacy of CRS therapy; to study whether asthma serves as a poor prognostic factor for the efficacy of CRS therapy; to elucidate the relationship between upper and lower airway diseases, considering specifically the relationship between CRS and asthma; to conduct the first prospective randomised study evaluating and comparing the effect of the medical and surgical treatment of CRS and its subgroups upon asthma, applying a range of subjective and objective parameters, and including exhaled NO as an easy and sensitive detector of inflammation; to study whether the presence of nasal polyps serves as a poor prognostic factor tor asthma control in CRS patients; to study the effect of medical and surgical therapy of CRS upon nasal NO levels; to investigate the value of nasal NO as an objective indicator of the effect of therapy on CRS. 2. Subjects were 90 patients with CRS, of whom 43 were asthmatics. Patients were randomised either to medical or surgical therapy of CRS. All patients underwent pre and post-treatment assessments of visual analogue score (VAS), chest score, overall asthma control score, use of anti-asthma medication, hospitalisation tor asthma, the Sinonasal Outcome Test-20, the Short Form 36 Health Survey (SF-36), nitric oxide (NO), acoustic rhinometry, saccharine clearance time, spirometry, and nasal examination including anterior rhinoscopy and endoscopy. 3. Both the medical and surgical treatment of CRS significantly improved almost all the subjective and objective parameters of CRS (P<0.01), with no significant difference being found between the medical and surgical groups (P>0.05) except for total nasal volume in CRS (P<0.01) and CRS without polyposis (P<0.01) groups in which the surgical treatment demonstrated greater changes.. 4. Both the medical and surgical treatment significantly improved almost all the subjective and objective parameters of CRS in asthmatic patients (P<0.01 in total groups and <0.05 in subgroups), with no significant difference being found between the surgical and medical groups. 5. The asthmatic surgical groups showed a general trend for improvement in the subjective and objective lower airway measurements. However, this did not reach a statistical significance except for use of bronchodilator inhalers (P<0.05), use of oral corticosteroids (P<0.05), hospitalisation (P<0.05), overall asthma control score (P<0.05), exhaled NO (P<0.05) and FEV1% (P<0.05) in the total surgical group of CRS. The medical groups showed higher significance values than the corresponding surgical groups. However, the difference between the medical and surgical groups did not reach a statistical significance except for exhaled NO (P<0.05) and FEVl% (P<0.05) measurements in CRS with polyposis. On the other hand, no significant difference was found between the surgical groups of CRS without and with polyposis, although CRS without polyposis tended to show higher improvement percentage values in the subjective and objective lower airway measurements. 6. Nasal NO increased significantly with medical (P<0.01) and surgical (P<0.01) treatment of CRS. The surgical groups experienced higher levels of improvement, although not significant (P>0.05), than the medical groups. Nasal NO correlated inversely with VAS (P<0.001), SCT (P<0.001), endoscopic score (P<0.001), polyp grade (P<0.01) and CT score (P<0.001). 7. Chronic rhinosinusitis should be targeted with maximal medical therapy in the first instance, with surgical treatment being reserved for cases refractory to medical therapy. Neither presence of nasal polyps nor asthma serves as a poor prognostic factor for the efficacy of CRS therapy. The evidence of the link between CRS and asthma is too striking to be denied. Both medical and surgical therapy of CRS improve the clinical course of asthma, with the medical treatment being superior to the surgical, especially in CRS with polyposis. On the other hand, it seems that sinus surgery can trigger or aggravate asthma in a subgroup of CRS patients. Finally, nasal NO is a valuable objective measurement in monitoring medical and surgical therapy for CRS.
... IL-10, transforming growth factor beta 1 (TGF-β1), interferon (IFN)-γ, and Fas antigens, Fas ligand are involved in the regulation of the EOS apoptosis. These cytokines can also promote EOS apoptosis [16][17][18]. ...
Background
Eosinophils (EOS) is one of the most important cells involved in the pathogenesis of chronic airway inflammation in asthma, and its apoptosis is part of the mechanisms of asthma. Therefore, this study aimed to observe the effect of Chinese medicine Wentong decoction (WTD) in EOS apoptosis in asthmatic rats. This work also explored the mechanism of WTD regulation in EOS apoptosis and provided a new target for clinical treatment of asthma.
Methods
Asthmatic rats induced by ovalbumin were treated with WTD. Lung function of rats in each group was detected, and lung tissue pathology, EOS counts in blood and bronchoalveolar lavage fluid were observed. The degree of the EOS apoptosis in rats was detected. The expression content of interleukin (IL)-5, IL-10, chemokine (C–C motif) ligand 5 (CCL5), granulocyte–macrophage colony-stimulating factor (GM-CSF), transforming growth factor beta 1 (TGF-β1), interferon (IFN)-γ, and other cytokines in rat serum and the genes of Eotaxin mRNA, Fas mRNA, FasL mRNA, Fas/FasL and Bcl-2 mRNA in the lung tissues were determined.
Results
WTD can reduced airway resistance in rat models and improved airway compliance. The pathological changes of lung tissue in WTD group were significantly alleviated, at the same time, WTD could reduce the EOS count in the blood and BALF smears of the asthmatic model rats. Compared with the model group, the apoptosis degree of EOS significantly increased in rats in the WTD group. The expression of IL-5, CCL5, and GM-CSF in the serum and the expression of Eotaxin mRNA, Bcl-2 mRNA in the lung tissues in rats in the WTD group rats decreased. Moreover, the expression of IL-10, TGF-β1, and IFN-γ in the serum and the expression of Fas mRNA, FasL mRNA in the lung tissues in rats in the WTD group rats increased compared with that in rats in the model group.
Conclusions
Wentong decoction may accelerate EOS apoptosis, reduce asthma inflammation, and alleviate the disease through regulating and controlling the factors related to the anti-apoptosis and pro-apoptosis.
Electronic supplementary material
The online version of this article (10.1186/s13020-018-0180-2) contains supplementary material, which is available to authorized users.
... However, a deficiency in inducible nitric oxide synthase (iNOS) suppresses ozone-induced airway tissue injury and LPS-induced acute airway inflammation in mice [30,31]. NO inhibitors block eosinophil recruitment in the lungs [32]. NO is also a potent vasodilator in the bronchial circulation and may play a major role in airway circulation [33]. ...
Background
Retinoid X receptors (RXRs) are members of the nuclear receptor (NR) superfamily that mediate signaling by 9-cis retinoic acid, a vitamin A (retinol) derivative. RXRs play key roles not only as homodimers but also as heterodimeric partners—e.g., retinoic acid receptors (RARs), vitamin D receptors (VDRs), liver X receptors (LXRs), and peroxisome proliferator-activated receptors (PPARs). The NR family was recently associated with allergic diseases, but the role of RXRs in allergen-induced airway responses is not well defined.The goal of this study is to elucidate the role of RXRs in asthma pathogenesis and the potency of RXR partial agonist in the treatment of allergic airway inflammation and airway hyperresponsiveness using a murine model of asthma. Methods
We investigated the effect of a novel RXR partial agonist (NEt-4IB) on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in a murine model of asthma. Balb/c mice were sensitized (days 0 and 14) and challenged (days 28–30) with ovalbumin (OVA), and airway inflammation and airway responses were monitored 48 h after the last OVA challenge. NEt-4IB was administered orally on days 25 to 32. ResultsOral administration of NEt-4IB significantly suppressed AHR and inflammatory cell accumulation in the airways and attenuated the levels of TNF-α in the lung and IL-5, IL-13 and NO levels in bronchoalveolar lavage (BAL) fluid and the number of periodic acid Schiff (PAS)-positive goblet cells in lung tissue. Treatment with NEt-4IB also significantly suppressed NF-κB expression. Conclusion
These data suggest that RXRs may be of crucial importance in the mechanism of allergic asthma and that the novel RXR partial agonist NEt-4IB may be a promising candidate for the treatment of allergic airway inflammation and airway hyperresponsiveness in a model of allergic asthma.
... The consequences of these nitrogen species are diverse, ranging from inhibition of virus protein and RNA synthesis to potentiation of inflammation and injury to the respiratory tract (39). Nitric oxide also regulates many other physiologic processes, including eosinophil migration (40) and survival (41,42), airway epithelial cell ciliary motility (43), and airway caliber through airway smooth muscle relaxation (44). Eosinophil-derived nitric oxide may affect multiple functions in the airway beyond what our experiments were designed to assess. ...
Respiratory viruses cause asthma exacerbations. Since eosinophils are the prominent leukocytes in the airways of 60-70% of patients with asthma, we evaluated the effects of eosinophils on a common respiratory virus, parainfluenza 1, in the lung. Eosinophils recruited to the airways of wild-type mice following ovalbumin sensitization and challenge significantly decreased parainfluenza virus RNA in the lungs 4 days after infection compared to non-sensitized animals. This antiviral effect was also seen in IL-5 transgenic mice with an abundance of airway eosinophils (NJ.1726), but was lost in transgenic eosinophil-deficient mice (PHIL) and in IL-5 transgenic mice crossed with eosinophil-deficient mice (NJ.1726-PHIL). Loss of the eosinophil granule protein, eosinophil peroxidase, using eosinophil peroxidase-deficient transgenic mice (EPX-/-) did not reduce eosinophils' antiviral effect. Eosinophil antiviral mechanisms were also explored in vitro. Isolated human eosinophils significantly reduced parainfluenza virus titers. This effect did not involve degradation of viral RNA by eosinophil granule RNases. However, eosinophils treated with a nitric oxide synthase inhibitor lost their antiviral activity, suggesting eosinophils attenuate viral infectivity through production of nitric oxide. Consequently, eosinophil nitric oxide production was measured with an intracellular fluorescent probe, CuFL. Eosinophils produced nitric oxide in response to virus and to a synthetic agonist of the virus-sensing innate immune receptor Toll-like receptor 7. Interferon-γ increased expression of eosinophil Toll-like receptor 7 and potentiated Toll-like receptor 7-induced nitric oxide production. These results suggest eosinophils promote viral clearance in the lung and contribute to innate immune responses against respiratory virus infections in humans.
Background
MRL/MpJ -Tnfrsf6 lpr (MRL/ lpr) mice, a murine model of systemic lupus erythematosus (SLE), have defective expression of Fas, substantially reducing signaling for apoptosis via this mechanism. However, it is known that MRL//pr mice have increased spontaneous apoptosis of leukocytes. These conflicting observations have stimulated interest in apoptosis in this SLE model. MRL/ lpr mice overproduce nitric oxide (NO) as autoimmune disease progresses. In vitro administration of NO may induce or decrease apoptosis depending on the cell type. Therefore, we hypothesized that NO induces MRL/ lpr spleen lymphocyte apoptosis independent of Fas receptor engagement.
Methods
Percentages of apoptotic spleen lymphocytes from MRL/ lpr and BALB/cJ mice were determined ex vivo after in vivo treatment with N G -monomethyl-l-arginine (NMMA), a nitric oxide synthase (NOS) inhibitor. After culture in varying concentrations of a slow-acting NO donor, the following were determined in spleen lymphocytes: (1) levels of apoptosis, (2) the effect of phorbol myristate acid (PMA) on levels of NO-induced apoptosis, and (3) protein kinase C (PKC) activity.
Results
Spleen lymphocytes from MRL/ lpr mice with active disease had increased levels of ex vivo apoptosis when compared with BALB/cJ controls. This increase was reduced by pharmacologic inhibition of NOS in MRL/ lpr but not in BALB/cJ mice. Exogenous administration of NO in vitro reduced PKC activity and induced apoptosis in MRL/ lpr spleen lymphocytes, an effect that could be reduced via coadministration of PMA in vitro.
Conclusion
These results suggest that NO plays a role in spleen lymphocyte apoptosis in MRL/ lpr mice, possibly via inhibition of PKC, despite a Fas defect.
Prior to the 1980s, nitric oxide (NO) was best known as a toxic reactive free radical found in atmospheric pollutants and carcinogens [1], a by-product of microbial nitrogen metabolism [2,3] and a potent activator of the mammalian enzyme heme-containing soluble guanylate cyclase [4]. As early as 1916, evidence for a mammalian nitrogen metabolic pathway was reported with the finding that several types of animals, including humans, excreted more urinary nitrate than could be accounted for by dietary intake [5]. Further evidence was reported by Tannenbaum and co-workers [6,7] who showed that in vivo mammalian nitrate formation was substantially enhanced by administration of the immunostimulant lipopolysaccharide (LPS) [8]. Stuehr and Marletta [9] first demonstrated that murine macrophages stimulated in vitro with LPS expressed nitrogen oxide synthetic activity and produced nitrite (NO2) and nitrate (NO3-). Simultaneously, other investigators were trying to elucidate the identity of a short-lived, diffusable endothelium-derived relaxing factor (EDRF) produced by acetylcholine-treated endothelial cells that was responsible for mediating smooth muscle cell relaxation [10]. Based on the similarities in the pharmacological properties of EDRF and NO generated from acidified nitrite, Furchgott suggested that EDRF may be NO in 1986 [11]. At the same time, Ignarro et al. also proposed that EDRF may be NO or a closely related species [12]. The following year, two independent groups of investigators [13,14] demonstrated EDRF was indeed nitric oxide (NO).
Endogenous exhaled nitric oxide (NO) is increased in asthma patients, especially in the exacerbated state. To evaluate the role of NO in airmay mucosa of toluene diisocyanate (TDI)- induced asthina in relation to clinical and immunologic findings, 20 TDI-induced asthma patients were enrolled and classified into two groups: 9 newly diagnosed patients (group I) and 11 patients having persistent asthma symptoms for >5 years despite work withdrawal (group II). Immunohistocytochemistry was applied to compare the expression of endothelial constitutive NOS (c-NOS) and induced NOS (i-NOS) in airway mucosa of both groups. They were observed in four areas: epithelium (EP), smooth muscle (SM), vascular endothelium (VE), and mucous gland (MG). The intensity of expression was graded on a scale of one to four and mean values were presented front blind readings observed by two experts. Serum-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibodies to TDI-human serum albumin conjugate were measured by enzyme-linked immunosorbent assay. e-NOS was found most frequently in VE (516 [83.31] versus 7/10 [70%]) followed by MG, EP, and SM in both groups. i-NOS was found most frequently in EP (7/9 177.8%] versus 7/11 [63.6%]), followed by MG, VE, and SM in both groups. The intensities of i-NOS and c-NOS of MG were significantly higher in group I than in group H (p < 0.05) with no significant differences in other areas (p > 0.05, respectively). There were no significant correlations between i-NOS or c-NOS expression and exposure or asthina symptom duration (p > 0.05). No associations were found between i-NOS or c-NOS expression and the presence of specific IgE or IgG to TDI-human serum albumin conjugate (p > 0.05, respectively). In conclusion, i-NOS and c-NOS expressions were noted in EP, SM, VE, and MG in airway mucosa of TDI-induced asthma patients, which may contribute to airway, inflammation.
Nitric oxide (NO) functions as a messenger in transduction of the paracrine signal. It produces a rapid and relatively short-term response in target cells caused by a decrease in the Ca2+ level, as well as long-term effects as a result of induction of certain genes. Synthesis of NO occurs in cells of various types upon arginine oxidation by a specific monooxidase whose activity depends on calmodulin and external stimuli such as interferons, steroids, and tumor necrosis factors and other cytokines. In target cells, NO and its active derivatives, e.g., peroxynitrite, affect proteins containing heme, iron-sulfur centers, and active thiols. NO inhibits iron-sulfur enzymes and activates guanylate cyclase, transcription factors NF-κB and AP-1, and a system of MAP proteinases controlled by G protein H-Ras. Its action can initiate different, even opposite, effects in target cells, depending on additional factors of the redox and proliferative status, NO concentration, and other conditions. In addition to cytokines and other tissue hormones, NO influences the effector systems controlling proliferation, apoptosis, and differentiation of cells and their stress resistance.
1Basic Biology2Cell Trafficking3Cell Function4Immunomodulatory Capabilities5Basophilia and Eosinophilia as Disease Markers6Role in Infectious DiseasesKeywords:basophils and eosinophils;basophils and eosinophils-leukocytes with large cytoplasmic granules from CD34+ progenitor cells;May–Grünwald stain of human peripheral blood basophil;basophil adhesion;cell trafficking-basophils and eosinophil trafficking to inflammation sites through multistep process;basophil and eosinophil adhesion molecules and chemotaxis factors;IgE-mediated activation;immunomodulatory capabilities
The role of NO . catalase in the activation of partially purified soluble guanylate cyclase of rat liver by NaN3 and NH2OH was examined by electron spin resonance (ESR) spectroscopy. Equilibration of bovine liver catalase with NO resulted in formation of a paramagnetic species exhibiting a three-line ESR spectrum similar to that of NO . catalase. This paramagnetic complex produced concentration-dependent stimulation of preparations of partially purified guanylate cyclase that were devoid of detectable endogenous heme content. The stimulation of partially purified guanylate cyclase by NO . catalase was similar to that obtained with NO . hemoglobin and with NO . cytochrome P-420 prepared by reaction of hepatic microsomes of phenobarbital-treated rats with NO. By contrast, these same enzyme preparations did not respond to NO or catalase alone. Addition of hematin or hemoglobin plus a reducing agent to purified guanylate cyclase restored enzyme responsiveness to NO and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not to NaN3 or NH2OH. Responses to the latter agents were restored by catalase and potentiated by a H2O2-generating system. Formation of the NO . catalase complex was evident by ESR spectroscopy in test solutions containing NaN3 or nh2oh, catalase, and a glucose-glucose oxidase, H2O2-generating system. The presence of NO . catalase correlated well with the ability of test solutions to activate purified guanylate cyclase. These results provide evidence for catalase-dependent NO generation from NaN3 and NH2OH under conditions leading to guanylate cyclase activation. Preformed NO . hemoglobin or NO . cytochrome P-420 also activated heme-deficient partially purified guanylate cyclase. The ability of several preformed NO . heme protein complexes, but not NO, to stimulate heme-deficient guanylate cyclase supports the concept that formation of the paramagnetic nitrosyl . heme complex, mediated by either enzymatic or nonenzymatic reactions, is a common and essential step in the process by which NO or NO-forming compounds activate guanylate cyclase. In the absence of the NO ligand, both hemoglobin and catalase suppress the stimulatory effects of the corresponding NO . heme proteins on guanylate cyclase. Release of each heme protein from the NO . heme protein complex occurs more rapidly under aerobic compared to anaerobic conditions. However, hemoglobin is approximately 2000 times more effective as an inhibitor of NO . hemoglobin stimulation of guanylate cyclase than is catalase as an inhibitor of NO . catalase action. This finding may explain the more pronounced decline in the rate of cGMP generation in air in the presence of NO . hemoglobin compared to NO . catalase. The results imply that guanylate cyclase responses to activators that can form NO are determined by both the stimulatory activity of the endogenous heme acceptors of NO and the relative inhibitory effects of the unliganded heme proteins present.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) was established as the constitutive and elicited human umbilical vein endothelial cell-derived eosinophil viability-sustaining factor. Stimulation of endothelium cell monolayers with IL-1 alpha (5 U/ml) increased the 48-h elaboration of GM-CSF from a mean of 3.2 to a mean of 8.2 pM (P less than 0.05). Dexamethasone (100 nM) decreased the constitutive GM-CSF elaboration by 49% (P less than 0.001) but did not diminish production by IL-1 alpha-stimulated endothelium. However, eosinophil viability decreased by 21% in dexamethasone-pretreated IL-1 alpha-stimulated endothelial cell-conditioned medium (P less than 0.05), which suggested viability antagonism by glucocorticoids. After 24 h of culture, eosinophil viability for replicate cells in enriched medium alone or with 1 pM GM-CSF decreased from means of 43 and 75% to means of 21 and 54%, respectively, when dexamethasone was included (P less than 0.05). However, 10 pM GM-CSF, IL-3, or IL-5 protected the cells against dexamethasone and against endonuclease-specific DNA fragmentation. In this model system of eosinophil-tissue interactions, dexamethasone prevents the endothelial cells from inducing a pathobiologic phenotypic change in the eosinophil by suppression of GM-CSF elaboration to concentrations that are not cytoprotective. Cytokine priming by GM-CSF, IL-3, or IL-5 may account for the differential responsiveness of select eosinophilic disorders to glucocorticoids.
A metabolic pathway of activated macrophages (M phi) involving oxidation of the guanido nitrogens of L-arginine is required for inhibition of growth and respiration of some target cells. The goal of this study was to identify the M phi metabolite(s) that induce these injuries. The stable products of the L-arginine pathway, NO2- and NO3-, were incapable of causing cytostasis under coculture conditions. However, NO2- became cytostatic upon mild acidification, which favors its transformation into nitrogen oxides of greater reactivity. This suggested that NO. (and/or NO2), recently identified as an M phi metabolite of L-arginine, could be a mediator. Authentic NO. caused cytostasis and respiratory inhibition in L1210 cells in a dose-dependent manner. The mitochondrial lesions caused by NO. were confined to complex 1 and 2, a pattern of injury identical to that seen after coculture with activated M phi. Inclusion of NO. scavenger systems prevented cytostasis from developing in M phi-L1210 cocultures. Thus, M phi-generated NO. can account for L-arginine-dependent cytostasis and respiratory inhibition.
We have determined the influence of reference particle deformability and suspending buffer tonicity on the measurement of lymphocyte volume by an electronic particle volume analyzer. When the volume analyzer was standardized with latex spherules having a shape factor (fe) of 1.5, red cell volume was 96 cu micron and lymphocyte volume was 289 cu micron. The red cell volume corresponded closely to the true red cell volume; the true lymphocyte volume, however, was 218 cu micron when measured by the lymphocytocrit/lymphocyte count and 203 cu micron by wet lymphocyte weight and density (mean approximately 210 cu micron). The difference between the electronic volume (Ve) of 289 cu micron and true lymphocyte volume of 210 cu micron was due to the influence of lymphocyte deformability (shape factor) as it traverses the sizing aperture. Since the true volume equals the Ve/fe, the red cells with a shape factor near 1.0 were sized appropriately by this method. In contrast, the lymphocyte shape factor was 1.38; thus, the true lymphocyte volume was 289 cu micron/1.38 or 210 cu micron. The tonicity of the suspending solution also influenced the measurement of particle volume when osmotically inactive standard particles (e.g., latex spherules) were used as a reference. Whereas the true lymphocyte volume was 210 cu micron at 286 mosmole/liter, it was 194 cu micron at 330 and 229 cu micron at 250 mosmole/liter. The standard counting solution, Isoton, is hyperosmolar (330 mosmole/liter) and causes an 8% shrinkage of osmotically active cells.
CONGENERS of nitrogen monoxide (NO) are neuroprotective and neurodestructive1– 7. To address this apparent paradox, we considered the effects on neurons of compounds characterized by alternative redox states of NO: nitric oxide (NO.) and nitrosonium ion (NO+)8. Nitric oxide, generated from NO. donors or synthesized endogenously after NMDA (N-methyl-D-aspartate) receptor activation, can lead to neurotoxicity3,4. Here, we report that NO.-mediated neurotoxicity is engendered, at least in part, by reaction with superoxide anion (O.-
2), apparently leading to formation of peroxynitrite (ONOO−), and not by NO. alone. In contrast, the neuroprotective effects of NO result from downregulation of NMDA-receptor activity by reaction with thiol group(s) of the receptor's redox modulatory site1. This reaction is not mediated by NO. itself, but occurs under conditions supporting S-nitrosylation of NMDA receptor thiol (reaction or transfer of NO+). Moreover, the redox versatility of NO allows for its interconversion from neuroprotective to neurotoxic species by a change in the ambient redox milieu. The details of this complex redox chemistry of NO may provide a mechanism for harnessing neuroprotective effects and avoiding neurotoxicity in the central nervous system.
To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT-PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT-PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon-γ (IFN-γ) prior to mRNA extraction, RT-PCR yielded an iNOS-specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP-1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations.
The nitrovasodilator and nitric oxide donor molsidomine and its metabolite SIN-I dilate vascular smooth muscle and inhibit platelet activation by increasing intracellular concentrations of cyclic GMP We have therefore studied the effects of molsidomine and SIN-I on isolated human polymorphonuclear leucocytes (PMN)in vitro andex vivo.
In vitro molsidomine dose-dependently reduced-glucuronidase release and the generation of superoxide anions from non-activated and from FMLP- or PAF-stimulated human PMNs. SIN-1 was equally effective in reducing (-glucuronidase release and totally inhibited oxygen radical generation at a concentration of 580 mol l–1.In a double-blind, placebo-controlled, randomized trial we also studied-glucuronidase release and the generation of superoxide anions from isolated PMNs. Blood was drawn from 12 healthy volunteers before and 3 h after oral molsidomine (16 mg) or placebo. There was no statistically significant difference in-glucuronidase release and superoxide anion formation when the PMNs were isolated before or after molsidomine or placebo. This was the case for non-activated, as well as FMLP- or PAF-stimulated PMNs.Thus, the nitric oxide donors molsidomine and its metabolite SIN-I caused a dose-dependent inhibition of PMN functionsin vitro, but no significant inhibition when the PMNs were isolated after oral molsidomine.
Nitric oxide (NO) has been identified recently as a multifunctional mediator, produced by, and acting on, most cells of the body. Besides its function as endothelium-derived relaxing factor, as a neurotransmitter and as an immune defence molecule, evidence is accumulating that NO participates in inflammatory- and autoimmune-mediated tissue destruction. Modulation of NO synthesis and action represents a new approach to the treatment of inflammatory and autoimmune conditions.
Eosinophils are believed to injure tissues in a variety of allergic disease by virtue of their highly histotoxic contents and metabolites. They are readily observed in tissues during the allergic response yet the mechanisms governing the duration of tissue residence and route of removal remain obscure. We have previously reported in vitro and in vivo evidence that neutrophils undergo apoptosis (programmed cell death) and are recognized and ingested as intact cells by macrophages. We report that eosinophils, purified from the peripheral blood of asymptomatic healthy atopics, undergo apoptosis in vitro. After 72 to 96 h in culture, 57.0 +/- 6.2% (mean +/- SE) of the eosinophil population showed characteristic morphologic changes of apoptosis. Electrophoresis of the DNA from these cells demonstrated the typical "ladder" pattern of internucleosomal DNA cleavage, the hallmark of apoptosis-associated endonuclease activation. The rate of eosinophil apoptosis, slower than that reported for neutrophils, was delayed (by 80 +/- 6 h) in the presence of recombinant human IL-5, a cytokine previously reported to prolong eosinophil life in vitro but not known to modulate apoptosis. Aged, apoptotic eosinophils, but not fresh or aged preapoptotic eosinophils, were recognized and ingested as intact cells by macrophages. Apoptosis and ingestion by macrophages may represent a mechanism whereby the tissue longevity and removal of eosinophils is controlled.
We studied the effects and the mechanism of action of the cyclic GMP-lowering substance 6-anilino-5,8-quinolinedione (LY 83583) on cyclic GMP-mediated inhibition of platelet function. The activation of washed human platelets by thrombin was counteracted by 8-bromo-cyclic GMP and the direct activators of soluble guanylate cyclase, sodium nitroprusside and endothelium-derived relaxant factor (EDRF = nitric oxide). LY 83583 significantly antagonized the inhibitory effect of sodium nitroprusside and EDRF, but not that of 8-bromo-cyclic GMP, on thrombin-induced aggregation, ATP-release, adhesion to native endothelial cells and increase in concentration of free intracellular calcium ions. In accordance, increases in intracellular cyclic GMP by sodium nitroprusside and EDRF were attenuated by LY 83583. The inhibition of cyclic GMP-mediated effects on platelets by LY 83583 could be related to inhibition of platelet soluble guanylate cyclase, as the activation of the purified enzyme from platelets by sodium nitroprusside was directly inhibited by LY 83583. This effect of LY 83583 was attenuated in the presence of superoxide dismutase. Our findings support the hypothesis that sodium nitroprusside and EDRF inhibit platelet activation by stimulation of soluble guanylate cyclase via nitric oxide. Consequently, inhibition of nitric oxide-induced cyclic GMP formation by LY 83583, which may act by intracellular generation of superoxide anions, facilitates platelet activation.