Heloisa H.A. Ferreira

Nitric oxide modulates eosinophil infiltration in antigen-induced airway inflammation in rats.

The influence of nitric oxide (NO) on eosinophil infiltration into the airways was investigated in rats actively sensitized with ovalbumin. The animals were treated chronically with the NO synthase inhibitor, N omega-Nitro-L-arginine methyl ester (L-NAME; 75 mumol rat-1 day-1), for 4 weeks. Bronchoalveolar lavage was performed at 6, 24, 48 and 72 h after intratracheal injection of ovalbumin. Intratracheal challenge of the sensitized rats with ovalbumin caused a significant increase in total leucocyte infiltration in bronchoalveolar lavage fluid both 24 and 48 h post-ovalbumin injection. Neutrophils and eosinophils peaked, respectively, at 24 h (29%) and 48 h (30%) in bronchoalveolar lavage fluid whereas the mononuclear cell did not differ significantly from the counts in non-sensitized rats at any time. At both 6 and 24 h post-ovalbumin injection, the chronic treatment of the animals with L-NAME affected neither the total nor the differential leucocyte content. However, at 48 h post-ovalbumin challenge, the total cell count was reduced by approximately 48% in the L-NAME-treated animals and this was associated with a marked inhibition (81%) of the eosinophil influx. Histological examination of the lungs from these animals (48 h post-ovalbumin challenge) also showed a prominent reduction (69.5%; P < 0.05) of the eosinophil infiltration in the respiratory segments. Our results demonstrate that NO plays a pivotal role in the eosinophil infiltration in airways of actively sensitized rats.

Inhibition of eosinophil chemotaxis by chronic blockade of nitric oxide biosynthesis

The effect of chronic N omega-nitro-L-arginine methyl ester (L-NAME) treatment on the in vivo eosinophil migration induced by bradykinin, platelet-activating factor (PAF), lipopolysaccharide and carrageenin has been investigated in the rat using the pleurisy model. The in vitro (microchemotaxis chamber) eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), PAF and zymosan-activated serum was also evaluated in the rat. The eosinophils were obtained from the peritoneal cavity of male Wistar rats and isolated on a discontinuous metrizamide gradient. Chronic inhibition of nitric oxide biosynthesis was achieved by adding L-NAME to the drinking water to give an intake of approximately 75 mumol/rat/day for 4 weeks. Rats treated chronically with L-NAME developed a significant level of hypertension (163 +/- 4.8 mmHg; P < 0.01) compared with animals which received either the same dose of the inactive enantiomer D-NAME (124 +/- 3.2 mmHg) or tap water alone (119 +/- 1.6 mmHg). The intrapleural injection of bradykinin (50 micrograms), PAF (1 microgram), lipopolysaccharide (0.25 microgram) and carrageenin (125 micrograms) into untreated rats in vivo induced a significant level of eosinophil migration by 24 h post-injection. This migration was markedly reduced in L-NAME-treated rats. Eosinophils obtained from untreated rats showed a significant level of migration in vitro in response to fMLP (5 X 10(-8) M), PAF (10(-8) M) and zymosan-activated serum (27 microliters). In contrast, the migration induced by these chemotactic agents was markedly reduced in cells isolated from animals treated chronically with L-NAME. L-Arginine (5.5 mM), but not D-arginine (5.5 mM), restored the ability of eosinophils from L-NAME-treated animals to migrate in response to fMLP. Our results indicate that nitric oxide plays a major role in the in vivo and ex vivo migration of eosinophils.

Hydrogen sulfide inhibits oxidative stress in lungs from allergic mice in vivo.

Recent studies show that endogenous hydrogen sulfide (H(2)S) plays an anti-inflammatory role in the pathogenesis of airway inflammation. This study investigated whether exogenous H(2)S may counteract oxidative stress-mediated lung damage in allergic mice. Female BALB/c mice previously sensitized with ovalbumin (OVA) were treated with sodium hydrosulfide (NaHS) 30 min before OVA challenge. Forty eight hours after antigen-challenge, the mice were killed and leukocyte counting as well as nitrite plus nitrate concentrations were determined in the bronchoalveolar lavage fluid, and lung tissue was analysed for nitric oxide synthase (NOS) activity, iNOS expression, superoxide dismutase (SOD), catalase, glutathione reductase (GR) and glutathione peroxidase (GPx) activities, thiobarbituric acid reactive species and 3-nitrotyrosine containing proteins (3-NT). Pre-treatment of OVA-sensitized mice with NaHS resulted in significant reduction of both eosinophil and neutrophil migration to the lungs, and prevented the elevation of iNOS expression and activity observed in the lungs from the untreated allergic mice, although it did not affect 3-NT. NaHS treatment also abolished the increased lipid peroxidation present in the allergic mouse lungs and increased SOD, GPx and GR enzyme activities. These results show, for the first time, that the beneficial in vivo effects of the H(2)S-donor NaHS on allergic airway inflammation involve its inhibitory action on leukocyte recruitment and the prevention of lung damage by increasing endogenous antioxidant defenses. Thus, exogenous administration of H(2)S donors may be beneficial in reducing the deleterius impact of allergic pulmonary disease, and might represent an additional class of pharmacological agents for treatment of chronic pulmonary diseases.

Nitric oxide has a role in regulating VLA-4-integrin expression on the human neutrophil cell surface.

Recent research demonstrates that the beta1 integrins may be involved in neutrophil migration. Here, we investigate the role of nitric oxide in the expression and function of the very late antigen-4 (VLA-4) and Mac-1 integrins on human neutrophils. Human blood neutrophils were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME) and their adhesion to fibronectin (FN) and serum observed. Adhesion of neutrophils to FN and serum increased significantly following incubation with 0.1mM L-NAME by 65.5 and 44.6%, respectively. Increased adhesions to FN and serum were abolished by a VLA-4-specific monoclonal antibody, HP2/1, and a Mac-1-specific monoclonal antibody, ICRF 44, respectively. The microfilament- and microtubule-depolymerizing agents, dihydrochalasin B and nocodazole, were also able to reverse L-NAME-induced adhesion to both FN and serum. L-NAME induced a discrete increase in the expression of CD49d (VLA-4, 25.3+/-4.8%), but not CD11b, on the neutrophil cell surface, as detected by flow cytometry. Results indicate that NO has a role in regulating VLA-4 and Mac-1 function on the human neutrophil cell surface and that this modulation in integrin function is accompanied by cytoskeletal rearrangements and changes in the ability of the neutrophil to adhere to the extracellular matrix.

Chronic Mild Prenatal Stress Exacerbates the Allergen-Induced Airway Inflammation in Rats

The effects of chronic mild prenatal stress on leukocyte infiltration into the airways was investigated in rat offspring. The chronic prenatal stress consisted of transitory and variable changes in the rats living conditions. Offspring at adult age were actively sensitized (day 0) and intratracheally challenged (day 14) with ovalbumin. Bronchoalveolar lavage was performed in the offspring at 48 h after intratracheal challenge with ovalbumin. A significant increase in total leukocyte infiltration was observed in the non-stressed offspring group and this was associated with a marked recruitment of eosinophils without a significant effect on the influx of neutrophils and mononuclear cells. In the prenatal stressed offspring, the counts of both total leukocyte and eosinophils, as well as mononuclear cells, was increased by 50% compared to the non-stressed offspring. We provide here the first experimental evidence that chronic mild unpredictable prenatal stress produces a marked increase in the allergen-induced airway inflammation in the rat offspring.

Role of nitric oxide on in vitro human eosinophil migration.

Eosinophils purified from the rat peritoneal cavity have been found to contain nitric oxide synthase (NOS) functionally coupled to a cyclic GMP transduction pathway that is involved in in vitro eosinophil migration, but no studies on cell locomotion have been done with purified human eosinophils. Therefore, this study was carried out to investigate the effects of N(omega) -nitro-L-arginine methyl ester (L-NAME; a non-selective NOS inhibitor), 1-(2-trifluoromethylphenyl) imidazole (TRIM; a type I/type II NOS inhibitor), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective type II NOS inhibitor), and 1H-[1,2,4]-oxidiazolo[4,3-a] quinoxalin-1-one (ODQ; a soluble guanylate cyclase inhibitor) on human eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Human eosinophils were purified from peripheral blood of healthy volunteers using a Percoll gradient followed by an immunomagnetic cell separator. Chemotaxis was evaluated using a 48-well microchemotaxis chamber. The fMLP (1.0 x 10(-7) M)-induced eosinophil migration was reduced significantly by l-NAME (0.1 and 1.0 mM), whereas the inactive enantiomer N(omega)-nitro-D-arginine methyl ester (D-NAME) had no effect. The inhibition by l-NAME was restored by sodium nitroprusside (0.25 mM). The NOS inhibitors AMT and TRIM (0.05 to 0.25 mM each) also markedly attenuated fMLP-induced chemotaxis. Additionally, ODQ (0.01 to 0.5 mM) concentration-dependently inhibited fMLP-induced migration, and the inhibition was restored by 2.0 mM dibutyryl cyclic GMP. In conclusion, this study demonstrates that human eosinophils present a nitric oxide-cyclic GMP pathway that is involved in the in vitro locomotion of this cell type.

Expression of nitric oxide synthases and in vitro migration of eosinophils from allergic rhinitis subjects.

The expression of nitric oxide (NO) synthases and the role of the NO cyclic GMP pathway on the migration of eosinophils from untreated patients with allergic rhinitis were investigated. Inducible NO synthase was strongly expressed in eosinophils from healthy individuals, but not in eosinophils from allergic rhinitis patients. The neuronal isoform was observed in eosinophils from each group studied, whereas no staining for the endothelial isoform was detected in either group. The chemotaxis to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 x 10(-7) M) and eotaxin (100 ng/ml) was significantly potentiated in allergic rhinitis eosinophils. In both groups, N(omega)-nitro-L-arginine methyl ester (L-NAME, 1.0 mM) or 1H(1,2,4)-oxadiazolo(4,3,-a)quinoxalin-1-one (ODQ, 0.2 mM) markedly reduced the chemotaxis. The selective iNOS inhibitor N-(3-(aminomethyl)benzyl)acetamidine (1400 W, 0.1-1.0 mM) significantly reduced the chemotaxis of eosinophils from healthy but not from allergic rhinitis subjects. The inhibition by L-NAME was restored by 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetyl-penicillamine, whereas the inhibition by ODQ was restored by dibutyryl cyclic GMP. In conclusion, both endothelial and inducible NO synthase isoforms are absent in allergic rhinitis eosinophils, suggesting that the NO cyclic GMP pathway in this cell type is maintained through the activity of a neuronal isoform.

Increased glutathione levels contribute to the beneficial effects of hydrogen sulfide and inducible nitric oxide inhibition in allergic lung inflammation

OBJECTIVE The interaction between nitric oxide (NO) and hydrogen sulfide (H2S) in the airways could have significant implications for the pathogenesis and therapeutic effects of both on lung diseases. In this study we investigated whether the beneficial effects of H2S on asthma could be comparable to that inhibition of inducible NO synthase (iNOS). METHODS Female BALB/C mice sensitized with ovalbumin (OVA) received either the H2S donor sodium hydrosulfide (NaHS, 14μmol/kg) or the iNOS inhibitor 1400W (1mg/kg), 30min before each OVA challenge during six days. On the first, second and sixth days, the leucocyte infiltration in lung parenchyma and bronchoalveolar lavage was evaluated. The aconitase activity (a sensor of O2 formation) and lipid peroxidation, as well as levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined in the lung tissues. RESULTS OVA-challenge caused a significant and time-dependent increase in the eosinophil number in the airways, which was accompanied by a significant decrease of aconitase activity and GSH/GSSG ratio along with enhanced lipid peroxidation in the lungs. Treatment with NaHS or 1400W significantly attenuated the airways eosinophilia that was paralleled by an increase in aconitase activity and decrease of lipid peroxidation. NaHS or 1400W treatments also reversed the decreased GSH/GSSG ratio seen after OVA-challenge. CONCLUSIONS The present study shows for the first time that the increased GSH/GSSG ratio caused by either H2S supplementation or iNOS-inhibition is a potential mechanism protecting airways against oxidative stress and inflammatory lung diseases.

Modulation of Eosinophil Functions by Nitric Oxide: Cyclic GMPdependent and -independent Mechanisms

Recruitment of eosinophils into tissues is a feature of a variety of allergic diseases, including asthma and nasal allergy. Eosinophils secrete several preformed granule proteins (eosinophil peroxidase, major basic protein, eosinophil cationic protein and eosinophil-derived neurotoxin) and newly-generated substances (oxygen-derived toxic metabolites, lipid mediators, cytokines and chemokines), which may contribute to the exacerbation of the allergic diseases. In the past decade, NO has been recognized as a major immunomodulatory mediator of inflammatory responses, particularly in the lung, where it is believed to play a pivotal role in modulating pulmonary eosinophilia and airways hyperresponsiveness in both allergic animals and humans, as evidenced by functional, biochemical and immunohistochemical studies. The NOcGMP signaling cascade was initially implicated in the modulation of eosinophil functions; however, additional studies have demonstrated that direct cGMP-independent mechanisms may also play important roles in eosinophil functions. Much progress in understanding the influence of NO on eosinophil functions has been achieved with the use of selective and non-selective NOS inhibitors, as well as NO-donor compounds, along with NOS isoform gene knock-out mice. However, these studies have resulted in numerous controversies and conflicting findings, possibly as a consequence of the diversity of experimental models used, animal species employed, methods of immunization and challenge with allergens, amongst others. The present review summarizes the role of NO in modulating, in vivo and in vitro, eosinophil adhesion, chemotaxis, airways hyperresponsiveness and apoptosis, outlining the conflicting findings in the literature, with emphasis on the allergic inflammatory responses.

Fibromyalgia and vibratory platform exercise: Cortisol improvement and women´s quality of life

OBJECTIVES: The main objective of this pilot study was to evaluate the effects of an exercise protocol, using vibratory platform, in quality of life and the cortisol rhythmicity in fibromyalgic women. METHODS: Twenty women, which 10 were healthy and 10 were diagnosed with fibromyalgia. All of them performed an exercise program on a vibratory platform and were evaluated for salivary cortisol production throughout the day before and after the treatment. The volunteers with fibromyalgia were also evaluated with a specific questionnaire for quality of life before and after the treatment. Salivary samples were collected at 6 a.m., noon, 6 p.m. and before bedtime, 10 p.m. Cortisol was quantified using immunoenzymatic assay. RESULTS: Women with fibromyalgia improved their quality of life and started to have an adjustment in the cortisol rhythmicity after exercise. Even considering the limitations of the volunteers’ number who participated in this pilot, a relationship between the improvement of the quality of life and the adjustment in the cortisol rhythmicity produced by the exercise program practiced could be assumed.