Hannu Kankaanranta

Inhibition by nitric oxide-donors of human polymorphonuclear leucocyte functions.

1 The study was designed to test the hypothesis that nitric oxide (NO)‐releasing compounds increase guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) production in human polymorphonuclear leucocytes (PMNs) and concomitantly inhibit PMN functions, i.e. leukotriene B4 (LTB4) synthesis, degranulation, chemotaxis and superoxide anion (O2−) release. The effects of two new NO‐releasing compounds, GEA 3162 and GEA 5024 were compared to 3‐morpholino‐sydnonimine (SIN‐1) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP). 2 GEA 3162 and GEA 5024 (1–100 μm) inhibited Ca ionophore A23187‐induced LTB4 and β‐glucuronidase release, chemotactic peptide FMLP‐induced chemotaxis and opsonized zymosan‐triggered chemiluminescence dose‐dependently in human PMNs. SIN‐1 and SNAP were weaker inhibitors. 3 Cellular cyclic GMP production was increased after exposure to NO‐donors concomitantly with the inhibition of PMN functions. No alterations in the levels of adenosine 3′:5′‐cylic monophosphate (cyclic AMP) were detected. 4 The results suggest that NO, possibly through increased cyclic GMP, inhibits the activation of human PMNs and may thus act as a local modulator in inflammatory processes.

Increased alveolar nitric oxide concentration in asthmatic patients with nocturnal symptoms.

Nocturnal asthma symptoms and impaired lung function at night are related to inflammatory activity in the peripheral lung compartment. Exhaled nitric oxide (NO) measurement at multiple exhalation flow rates can be used to separately assess alveolar and bronchial NO production and inflammation. The authors hypothesised that asthmatic patients with nocturnal symptoms have a higher alveolar NO concentration than those with only daytime symptoms. The authors asked 40 patients with newly-diagnosed steroid-naïve asthma about their nocturnal asthma symptoms through the use of a written questionnaire. Alveolar NO concentration and bronchial NO flux were assessed in the 40 asthmatics and 40 healthy controls. Nineteen of the 40 patients reported nocturnal symptoms. Patients with nocturnal symptoms had a higher alveolar NO concentration (1.7±0.3 (mean±sem) parts per billion (ppb)) than patients without nocturnal symptoms (0.8±0.3 ppb, p=0.012) or healthy controls (1.0±0.1 ppb, p=0.032). Bronchial NO flux was higher both in patients with (2.4±0.4 nL·s−1, p<0.001) and without (2.6±0.4 nL·s−1, p<0.001) nocturnal symptoms, compared to controls (0.7±0.1 nL·s−1). Nocturnal symptoms in asthmatic patients are related to a higher alveolar nitric oxide concentration. The results suggest that assessment of alveolar nitric oxide concentration can be used to detect the parenchymal inflammation in asthmatic patients with nocturnal symptoms.

Inhaled fluticasone decreases bronchial but not alveolar nitric oxide output in asthma

Exhaled nitric oxide (NO) concentration is a noninvasive measure of airway inflammation and is increased in asthma. Inhaled glucocorticoids decrease exhaled NO concentration, but the relative contributions of alveolar and bronchial levels to the decrease in exhaled NO concentration are unknown. Alveolar NO concentration and bronchial NO flux can be separately approximated by measuring exhaled NO at several exhalation flow rates. The effect of steroid treatment on alveolar and bronchial NO output in asthma was studied. Alveolar NO concentration and bronchial NO flux were assessed in 16 patients with asthma before and during treatment with inhaled fluticasone for 8 weeks and in 16 healthy controls. Before the treatment, asthmatics had increased bronchial NO flux (mean+/-SEM: 3.6+/-0.4 versus 0.7+/-0.1 nL x s(-1), p<0.001) but normal alveolar NO concentration (1.2+/-0.5 versus 1.0+/-0.2 parts per billion (ppb), p>0.05) compared with controls. Inhaled fluticasone decreased bronchial NO flux from 3.6+/-0.4 to 0.7+/-0.1 nL x s(-1) (p<0.01) but had no effect on alveolar NO concentration (before: 1.2+/-0.5; after: 1.2+/-0.1 ppb, p>0.05). The forced expiratory volume in one second improved, whereas asthma symptom score and serum levels of eosinophil cationic protein and eosinophil protein X decreased during the treatment. In conclusion, inhaled fluticasone decreases bronchial but not alveolar nitric oxide output simultaneously with clinical improvement in patients with asthma.

ANTI-INFLAMMATORY ACTIVITIES OF EMBLICA OFFICINALIS GAERTN LEAF EXTRACTS

Abstract— Emblica officinalis Gaertn, a tree growing in subtropical and tropical parts of China, India, Indonesia and the Malay Peninsula, has been used for anti‐inflammatory and antipyretic treatments of rural populations in these areas. In the present study, we examined the effects of Emblica officinalis extracts on carrageenan‐ and dextran‐induced rat hind paw oedema. Anti‐inflammatory activity was found in the water fraction of methanol extract of the plant leaves. The effects of the same fraction were tested on the synthesis of mediators of inflammation such as leukotriene B4 (LTB4), platelet‐activating factor (PAF) and thromboxane B2 (TXB2), and on LTB4‐ and N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine (FMLP)‐induced migration of human polymorphonuclear leucocytes (PMNs) in‐vitro. The water fraction of the methanol extract inhibited migration of human PMNs in relatively low concentrations. It did not inhibit LTB4 or PAF synthesis in human PMNs or TXB2 synthesis in human platelets during clotting, suggesting that the mechanism of the anti‐inflammatory action found in the rat paw model does not involve inhibition of the synthesis of the measured lipid mediators.

Induction of Nitric Oxide Synthesis by Probiotic Lactobacillus rhamnosus GG in J774 Macrophages and Human T84 Intestinal Epithelial Cells

Backgrounds and Aims: Probiotic Lactobacillus rhamnosus GG (LGG) has proved to be beneficial in the treatment of viral- and antibiotic-associated diarrhea but the mechanisms of action remain unknown. Nitric oxide (NO) is involved in the protective mechanisms in the gastrointestinal tract and may contribute to some of the beneficial effects of probiotics. The aim of the present study was to investigate if induction of NO synthesis is involved in the cellular actions of LGG.Methods: NO synthesis and its regulation were measured in cultures of J774 murine macrophages and human T84 colon epithelial cells. NO production was measured as its metabolite nitrite accumulated into the culture medium. Inducible nitric oxide synthase (iNOS) protein and iNOS mRNA were detected by Western blot and RT-PCR, respectively.Results: In J774 macrophages, LGG induced a low level production of NO in the presence of gamma interferon (IFNγ) and it was inhibited by NOS inhibitors, cycloheximide and by a NF-kappa B inhibitor pyrrolidinedithiocarbamate. Accordingly, LGG and IFNγ-stimulation increased iNOS mRNA and protein levels. T84 cells produced NO in response to LGG when first primed with a combination of IL-1β, TNFα and IFNγ. Lipoteichoic acid (LA), an antigenic structure in gram-positive bacteria, also induced NO formation in J774 cells in the presence of IFNγ suggesting that LA may be the active component in LGG.Conclusions: LGG induces NO production in J774 macrophages and in human T84 colon epithelial cells through induction of iNOS by a mechanism involving activation of transcription factor NF-κB. Induction of iNOS and low-level synthesis of NO may be involved in the protective actions of LGG in the gastrointestinal tract.

Radical releasing properties of nitric oxide donors GEA 3162, SIN-1 and S-nitroso-N-acetylpenicillamine

The nitric oxide (NO)-, superoxide anion (O2.-)- and peroxynitrite (ONOO-)-releasing properties of 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162) were characterized and compared with the known NO-donors 3-morpholino-sydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine. All the three compounds released NO in aqueous solutions in a dose-dependent manner as measured by ozone-chemiluminescence. GEA 3162 produced more NO than SIN-1, but less than S-nitroso-N-acetylpenicillamine during a 45 min incubation time. SIN-1 reduced nitro blue tetrazolium and the effect was inhibitable by superoxide dismutase. Reduction of nitro blue tetrazolium was not detected in the solutions of GEA 3162 and S-nitroso-N-acetylpenicillamine suggesting that SIN-1 but not GEA 3162 and S-nitroso-N-acetylpenicillamine release O2.- in their decomposition process. Formation of ONOO- in solutions of GEA 3162, SIN-1 and S-nitroso-N-acetylpenicillamine was estimated indirectly by measuring the formation of nitrotyrosine. The data indicate that ONOO- was produced in the presence of SIN-1 but not in solutions of GEA 3162 and S-nitroso-N-acetylpenicillamine. The results suggest that GEA 3162 produces negligible amounts of O2.- and ONOO- as compared to SIN-1. This adds the value of GEA 3162 as an useful tool in NO research and could well explain the earlier findings on the superior NO-like biological activity of oxatriazole derivatives as compared to SIN-1.

Nitric oxide-releasing compounds inhibit neutrophil adhesion to endothelial cells

In the present work, we demonstrated that chemically different nitric oxide (NO)-releasing compounds inhibit tumor necrosis factor alpha (TNF-alpha)-induced polymorphonuclear leukocyte adhesion to endothelial cells in vitro. Two mesoionic oxatriazole derivatives GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3(3, 4-dichlorophenyl)-chloride) and GEA 3175 (1,2,3,4-oxatriazolium, -3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt) were compared to the earlier-known NO donor SIN-1 (3-morpholino-sydnonimine). GEA 3162 (3-10 microM) and GEA 3175 (10-30 microM) inhibited human polymorphonuclear leukocyte adhesion to B(4) endothelial cells in a dose-dependent manner being more potent than SIN-1. In the present model, leukocytes rather than endothelial cells seemed to be the target of the effect of NO. Flow cytometric analysis showed that NO-releasing compounds did not alter TNF-alpha induced CD11/CD18 surface expression in polymorphonuclear leukocytes. The inhibitory action of NO-releasing compounds on adhesion paralleled with the increased synthesis of cGMP in polymorphonuclear leukocytes. Analogues of cGMP inhibited polymorphonuclear leukocyte adhesion indicating a role for cGMP in the action of NO donors. The results suggest that exogenous NO in the form of NO-releasing compounds inhibits polymorphonuclear leukocyte adhesion to endothelial cells, which may be implicated in the regulation of leukocyte migration and leukocyte-mediated tissue injury.

Increased bronchial nitric oxide production in patients with asthma measured with a novel method of different exhalation flow rates

The concentration of nitric oxide (NO) in exhaled air is increased in patients with asthma, suggesting that measuring fractional exhaled NO concentration (FENO) may be used to monitor asthmatic airway inflammation. However, increased FENO is not specific for asthma, as other inflammatory lung diseases may also increase FENO. To augment the specificity of FENO measurement, we tested a novel theoretical modelling of pulmonary NO dynamics that allows the approximation of alveolar NO concentration and bronchial NO flux separately by measuring FENO at several exhalation flow rates. We measured FENO at four exhalation flow rates in 10 steroid-naive asthmatics, 5 patients with extrinsic allergic alveolitis, and in 10 healthy controls. Both the asthmatics and the patients with alveolitis had significantly higher FENO than the healthy controls. The increased NO concentration originated from the bronchial level in the asthmatics and from the alveolar level in the patients with alveolitis. In the second part of the study we assessed the repeatability of FENO test, within-day and day-to-day (during two weeks) variation in FENO, and the effects of mouth pressure and cigarette smoking on FENO in healthy volunteers. Repeatability of 10 subsequent measurements was high (coefficient of variation (CV) 4.6% ± 0.4%), and no diurnal variation was found. The day-to-day variation during a 2-week period gave a CV of 10.6% ± 1.0%. The magnitude of mouth pressure (5-20 cmH2O in adults, 5-40 cmH2O in children) during measurement had no effect on FENO. Smoking a cigarette caused a small and transient but statistically significant increase in FENO at 1 and 5 min after smoking. In conclusion, FENO measurement is highly repeatable with low day-to-day variation among healthy subjects. Our results also suggest that the present novel method of measuring FENO at several exhalation flow rates can be used to approximate alveolar and bronchial contributions to FENO separately and thus increase the clinical value of this test.

Beclomethasone, budesonide and fluticasone propionate inhibit human neutrophil apoptosis

Inhaled glucocorticoids are widely used to treat chronic obstructive pulmonary disease without much evidence of efficiency in this disease where neutrophils may contribute to the pathophysiology. This prompted us to test the effects of several currently used inhaled and systemic glucocorticoids on human neutrophil apoptosis. Beclomethasone, budesonide, dexamethasone, fluticasone propionate, hydrocortisone and prednisolone inhibited apoptosis in a concentration-dependent manner as assessed by flow cytometric analysis, annexin-V binding and morphological analysis. The maximal inhibition of apoptosis was 50-60%. The order of potency was fluticasone propionate (EC(50) 0.6+/-0.2 nM) approximately equal to budesonide (EC(50) 0.8+/-0.2 nM)> dexamethasone approximately equal to prednisolone approximately equal to beclomethasone approximately equal to hydrocortisone. The inhibitory effects of glucocorticoids were reversed by mifepristone. Moreover, glucocorticoids slightly enhanced the inhibitory effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil apoptosis. The present data suggests that budesonide and fluticasone propionate prolong human neutrophil survival by inhibiting apoptosis at clinically relevant drug concentrations via an effect on glucocorticoid receptor.

Inhibition of classical PKC isoenzymes downregulates STAT1 activation and iNOS expression in LPS-treated murine J774 macrophages

Proinflammatory cytokines and bacterial products trigger inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in inflammatory and tissue cells. In inflammation, NO acts as an important mediator having both proinflammatory and destructive effects. Protein kinase C (PKC) is a family of serine–threonine protein kinase isoenzymes involved in signal transduction pathways related to inflammatory responses. The aim of the present study was to investigate the role of classical PKC (cPKC) isoenzymes in the regulation of iNOS expression and NO production in murine J774 macrophages and the mechanisms involved. RO318220 (inhibits PKCβ, PKCγ and PKCɛ), GÖ6976 (inhibits cPKC isoenzymes PKCα and PKCβ) and LY333531 (inhibits PKCβ) reduced lipopolysaccharide (LPS)‐induced NO production and iNOS expression in a dose‐dependent manner as did 6 h pretreatment with 1 μM phorbol 12‐myristate 13‐acetate (PMA) (which was shown to downregulate PKC expression). PKC inhibitors also reduced LPS‐induced iNOS mRNA levels, but they did not affect the half‐life of iNOS mRNA. PKC inhibitors did not alter LPS‐induced activation of NF‐κB as measured by electrophoretic mobility shift assay. All PKC inhibitors used and pretreatment with 1 μM PMA inhibited signal transducer and activator of transcription 1 (STAT1) activation as measured by the translocation of STAT1α from the cytosol to the nucleus by Western blot. In addition, inhibition of STAT1 activation by AG‐490, an inhibitor of JAK‐2, also reduced NO production. These results suggest that cPKC isoenzymes, especially PKCβ, mediate the upregulation of iNOS expression and NO production in activated macrophages in an NF‐κB‐independent manner, possibly through the activation of transcription factor STAT1.