Ana L.A. Pires

Soluble Factors Released by Toxoplasma gondii-Infected Astrocytes Down-Modulate Nitric Oxide Production by Gamma Interferon-Activated Microglia and Prevent Neuronal Degeneration

ABSTRACT The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-γ) in the central nervous system (CNS). However, IFN-γ-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-γ-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-γ-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-γ-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E2 (PGE2) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE2 and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE2 secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.

Alloxan Diabetes Reduces Pleural Mast Cell Numbers and the Subsequent Eosinophil Influx Induced by Allergen in Sensitized Rats

Alloxan damages insulin-producing cells and has been used as an inducer of experimental diabetes in several animal species. In this study, administration of alloxan (40 mg/kg, i.v.) to rats was followed by a selective and time-dependent reduction in the number of pleural mast cells (50 +/- 2.2%, p < 0.01; mean +/- SEM), while mononuclear cell and eosinophil counts were not altered. As compared to naive rats, the reduction in mast cell numbers was first noted 48 h following alloxan administration and remained unaltered for at least 60 days. It is noteworthy, that the depletion in the mast cell population was not accompanied by alterations in the total amount of histamine stored per cell. Sensitized rats turned diabetic by alloxan treatment performed 72 h before challenge showed a less pronounced antigen-induced mast cell degranulation compared to nondiabetic rats. Moreover, rats injected with alloxan 72 and 48 but not 24 h before challenge, reacted to allergenic challenge with 50% reduction in the number of eosinophils recruited to the pleural cavity within 24 h. We found that the less pronounced eosinophil accumulation did not relate to an intrinsic cell locomotor abnormality since eosinophils from diabetic rats presented similar chemotactic responses to LTB4 and PAF in vitro as compared to matching controls. Insulin (3 IU/rat) restored basal levels of mast cells and reversed the subsequent inhibition of allergen-induced pleural eosinophilia, suggesting a causative relationship between these phenomena. Treatment with insulin also significantly increased the number of mast cells in the pleural cavity of naive rats (from 637 +/- 57 to 978 +/- 79 x 10(3) cells/cavity, p < 0.001). Consistently, previous depletion of mast cells by means of local treatment with compound 48/80 significantly reduced the antigen-induced eosinophil recruitment in sensitized animals. We conclude that the reduction in the pleural mast cell population noted in alloxan-treated rats could be directly implicated in the diminished pleural eosinophil influx following allergen challenge. This hyporesponsiveness is independent of an intrinsic abnormality of cell chemotaxis, but can be imitated by local mast cell depletion.

Nebulized lidocaine prevents airway inflammation, peribronchial fibrosis, and mucus production in a murine model of asthma.

Background: Evidence suggests that nebulized lidocaine is beneficial in asthma therapy, but to what extent and the mechanisms underlying this effect remain poorly understood. The aim of this study was to assess the impact of lidocaine treatment using a murine model of allergic asthma characterized by expression of pivotal features of the disease: inflammation, mucus production, and lung remodeling. Methods: A/J mice sensitized with ovalbumin were treated with inhaled lidocaine or vehicle immediately after ovalbumin intranasal challenges. Lung function, total and differential leukocytes in bronchoalveolar lavage fluid, peribronchial eosinophil density, interleukin (IL)-4, IL-5 and eotaxin-1 levels, epithelial mucus, collagen, extracellular-matrix deposition, matrix metalloproteinase-9 activity, and GATA-3 expression were evaluated. Between five and eight animals per group were used. Results: Inhaled lidocaine inhibited ovalbumin-induced airway hyperreactivity to methacholine, and accumulation of lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid 24 h after the last allergen provocation. Lidocaine administration also prevented other pathophysiological changes triggered by ovalbumin in lung tissue, including peribronchial eosinophil and neutrophil infiltration, subepithelial fibrosis, increased content of collagen and mucus, matrix metalloproteinase-9 activity, and increased levels of IL-4, IL-5, IL-13, and eotaxin-1. Furthermore, inhaled lidocaine inhibited lung tissue GATA-3 expression in ovalbumin-challenged mice. We also demonstrated that lidocaine inhibited the expression of GATA-3 in ovalbumin-stimulated T cells in vitro. Conclusions: Inhaled lidocaine prevents eosinophilic inflammation, overproduction of mucus, and peribronchial fibrosis in a murine model of asthma, and impaired airway hyperreactivity, possibly by inhibiting allergen-evoked GATA-3 expression and the subsequent up-regulation of proinflammatory cytokines and chemokines.

Macrophage migration inhibitory factor is essential for allergic asthma but not for Th2 differentiation.

Macrophage migration inhibitory factor (MIF) is increased in asthmatic patients and plays a critical role in the pathogenesis of asthma. We show here that mice lacking MIF failed to develop airway hyper‐responsiveness (AHR), tissue eosinophilia, and mucus metaplasia. Analysis of the bronchoalveolar fluids revealed a substantial reduction of IL‐13, eotaxin and cysteinyl‐leukotrienes. The lack of these cardinal features of asthma in MIF–/– mice occurs regardless of high concentrations of IL‐4 in the lung and OVA‐specific IgE in the serum. Antigen‐specific lymphocyte proliferation and IL‐13 production were similarly increased in the draining lymph nodes of OVA‐immunized and challenged MIF–/– mice compared to WT, but were reduced in the spleen of MIF–/–, thus indicating differential roles of MIF in these compartments. Stimulation of naive CD4+ cells with anti‐CD3 antibody demonstrated that MIF–/– cells produced increased amounts of IFN‐γ and IL‐4 compared to WT CD4+ cells. Finally, treatment of sensitized BALB/c mice with neutralizing anti‐MIF antibody abrogated the development of ARH and airway inflammation without affecting the production of Th2 cytokines or IgE. The present study demonstrates that MIF is required for allergic inflammation, adding important elements to our knowledge of asthma pathogenesis and suggesting that neutralization of MIF might be of therapeutic value in asthma.

Modulation of eotaxin formation and eosinophil migration by selective inhibitors of phosphodiesterase type 4 isoenzyme.

This study was undertaken to investigate the possible contribution of the blockade of eotaxin generation to the anti‐eosinophilotactic effect of phosphodiesterase (PDE) type 4 inhibitors. In some experiments, the putative synergistic interaction between PDE type 4 inhibitors and the β2‐agonist salbutamol was also assessed. Sensitized guinea‐pigs aerosolized with antigen (5% ovalbumin, OVA) responded with a significant increase in eotaxin and eosinophil levels in the bronchoalveolar lavage fluid (BALF) at 6 h. Eosinophil recruitment was inhibited by both PDE type 4 inhibitors rolipram (5 mg kg−1, i.p.) and RP 73401 (5 mg kg−1, i.p.) treatments. In contrast, only rolipram inhibited eotaxin production. Sensitized rats intrapleurally challenged (i.pl.) with antigen (OVA, 12 μg cavity−1) showed a marked eosinophil infiltration at 24 h, preceded by eotaxin generation at 6 h. Intravenous administration of a rabbit anti‐mouse eotaxin antibody (0.5 mg kg−1) significantly reduced allergen‐evoked eosinophilia in this model. Local pretreatment with rolipram (40 μg cavity−1) or RP 73401 (40 μg cavity−1) 1 h before challenge reduced eosinophil accumulation evaluated in the rat pleural effluent, but only the former was active against eotaxin generation. The inhibitors of PDE type 3 (SK&F 94836) and type 5 (zaprinast) failed to alter allergen‐evoked eosinophil recruitment in rats. Local injection of β2‐agonist salbutamol (20 μg cavity−1) inhibited both eosinophil accumulation and eotaxin production following pleurisy. The former was better inhibited when salbutamol and rolipram were administered in combination. Treatment with rolipram and RP 73401 dose‐dependently inhibited eosinophil adhesion and migration in vitro. These effects were clearly potentiated by salbutamol at concentrations that had no effect alone. Our findings indicate that although rolipram and RP 73401 are equally effective in inhibiting allergen‐induced eosinophil infiltration only the former prevents eotaxin formation, indicating that PDE 4 inhibitors impair eosinophil accumulation by mechanisms independent of eotaxin production blockade.

Effectiveness of Cissampelos sympodialis and its isolated alkaloid warifteine in airway hyperreactivity and lung remodeling in a mouse model of asthma

BACKGROUND Cissampelos sympodialis Eichl. (Menispermaceae) is a plant found in Northeastern and Southeast of Brazil and hot water infusion of C. sympodialis root bark is largely used in the indigenous and folk medicine to treat several inflammatory disorders, including asthma. Asthma is a chronic inflammatory allergic disease characterized by airway hyperreactivity (AHR), eosinophil tissue infiltration and lung remodeling. The aim of this study was to evaluate the therapeutic effect of C. sympodialis and its isolated alkaloid warifteine on allergen triggered airway hyperreactivity (AHR) and lung remodeling in murine model of asthma. METHODOLOGY/PRINCIPAL FINDINGS The oral pre-treatment with C. sympodialis or warifteine inhibited allergen-induced AHR to inhaled methacholine and IL-13 levels in the bronchoalveolar lavage (BAL). In order to investigate the therapeutic potential of C. sympodialis and warifteine, animals were treated 1h after the last ovalbumin (OVA) challenge in sensitized animals. Similarly to the pre-treatment, post-treatment with warifteine was effective to inhibit significantly AHR to inhaled methacholine and to reduce IL-13 levels in the BAL. In addition, oral pre- or post-treatments with C. sympodialis or warifteine reduced OVA-induced eosinophil tissue infiltration, mucus production and subepithelial fibrosis to values similar to nonallergic controls. CONCLUSIONS Our data show the anti-allergic and immunoregulatory properties of C. sympodialis, acting mostly through the active compound warifteine, to inhibit the airway hyperreactivity and lung remodeling through a mechanism at least partially dependent of IL-13 and eosinophil inhibition. Therefore placing warifteine as an interesting therapeutic candidate in allergic inflammation and corroborating the folk medicine use of C. sympodialis as anti-allergic plant.

Bradykinin Induces Eosinophil Accumulation in the Rat Pleural Cavity

Intrathoracic injections of bradykinin (1-100 micrograms/cavity) induced a dose-dependent increase in the number of eosinophils recovered from the rat pleural cavity 24 h later. Eosinophilia by bradykinin was preceded by a marked pleural neutrophil influx within 6 h and was absent only 72 h following stimulation. Bradykinin (10(-9)-10(-5) M) failed to induce in vitro eosinophil chemotaxis, indicating that its in vivo effect must be mediated by an intermediate messenger. BW 755C (25 mg/kg) and the more selective lipoxygenase inhibitor BW A4C (20 micrograms/cavity) suppressed the pleural eosinophilia induced by bradykinin (50 micrograms/cavity), whereas the platelet-activating factor (PAF)-acether antagonist BN 52021 was inactive. We conclude that bradykinin is able to attract eosinophil in vivo by a mechanism independent of PAF-acether and sensitive to the blockage of the lipoxygenase pathway.

Interference of cetirizine with the late eosinophil accumulation induced by either PAF or compound 48/80

1 The effect of topical or systemic treatment with the histamine H1‐receptor antagonist, cetirizine, on the rat pleural eosinophil accumulation induced by PAF or compound 48/80 was investigated. The number of pleural resident eosinophils increased 6 h after the intrathoracic (i.t.) injection of PAF (1 μg/cavity), peaked within 24 h and persisted significantly augmented for at least 96 h. Compound 48/80 (25 μg/cavity) also produced a long lasting pleural eosinophilia but this was first noted only 24 h after stimulation. 2 Intraperitoneal pretreatment with cetirizine inhibited eosinophilia induced by either PAF (ED50 = 19 mg kg−1) or compound 48/80 (ED50 = 14 mg kg−1) whereas meclizine, another histamine H1‐receptor antagonist, was inactive. 3 Administered locally, cetirizine (5 and 15 μg/cavity) also dose‐dependently inhibited both PAF‐ and compound 48/80‐induced eosinophilia, providing evidence that its inhibitory effect is not due to any action upon circulating eosinophils. Consistent with this result, incubation of isolated peritoneal eosinophils with cetirizine failed to modify in vitro eosinophil migration caused by PAF. 4 Since the late eosinophilia induced by PAF may depend on the synthesis of a transferable protein mediator, cetirizine was administered to donor or recipient rats in order to determine whether it interferes with the generation or with the expression of this protein. We showed that only the treatment of recipient rats abolished the transfer of the eosinophilotactic activity, indicating that cetirizine does not modify the generation but inhibits the expression of this activity. 5 Our findings indicate that cetirizine is able to inhibit eosinophil accumulation by acting locally. The mechanism is neither related to its recognized ability to antagonize histamine H1‐receptors nor to a direct action upon circulating eosinophils.

Suppressive effects of nitric oxide‐releasing prednisolone NCX‐1015 on the allergic pleural eosinophil recruitment in rats

Background The addition of a nitric oxide (NO)‐releasing moiety to prednisolone was shown to enhance the anti‐inflammatory activity of this glucocorticoid in some experimental conditions, but its effectiveness in the context of eosinophilic inflammation remains to be elucidated.

Long-lasting inhibitory activity of the hetrazepinic BN 50730 on exudation and cellular alterations evoked by PAF and LPS.

1 Inhibitory effects of the hetrazepinic derivative BN 50730 on the rat pleural inflammatory response, triggered by PAF or lipopolysaccharides (LPS), were examined. The type of pharmacological blockade exerted by this antagonist in in vitro assays of eosinophil chemotaxis and platelet aggregation were also investigated. 2 Intrathoracic injection of PAF (1 μg per cavity) caused a 4 fold increase in the extravasated protein within 15min and led to a marked eosinophil accumulation 24 h post‐challenge. BN 50730 (0.5–10 μg per cavity) inhibited exudation by PAF dose‐dependently without modifying the response induced by histamine, bradykinin or 5‐hydroxytryptamine (5‐HT). 3 The kinetics of the inhibitory effect on exudation revealed that the actions of WEB 2086 and BN 52021 (10 μg per cavity) were over within 2 and 4h respectively, whereas BN 50730 (10 μg per cavity) retained 80% of its inhibitory activity for 4 days. 4 Oral treatment with BN 50730 (10–20 mg kg−1, 1 h beforehand) suppressed the leucocyte accumulation and late eosinophilia observed 6 and 24 h after PAF respectively, but did not modify the eosinophilia induced by leukotriene B4 (LTB4) or bradykinin. BN 50730 also failed to reduce the eosinophil accumulation induced by LPS but drastically inhibited the neutrophil influx. 5 The pre‐incubation of rat peritoneal eosinophils for 10 min with BN 50730 (30nM‐1 μm) dose‐dependently inhibited the chemotaxis induced by PAF (0.1 μm) in vitro. The IC50 values for BN 52021, WEB 2086 and BN 50730 in this system were 5, 5 and 0.05 μm respectively. 6 In separate assays, rat peritoneal eosinophils and rabbit washed platelets were preincubated with BN 50730 or WEB 2086 (1 μm) then subjected to a series of at least two consecutive washings in order to remove the antagonist from the receptor environment. Under such conditions, only the cells pretreated with WEB 2086 recovered the sensitivity to the lipid. 7 We conclude that BN 50730 is a potent, specific and long‐acting PAF antagonist and its effect seems to result from a high affinity and non‐competitive interaction of the drug with the PAF receptor.