Crucial role of neutrophils in the development of mechanical inflammatory hypernociception

Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan-induced mechanical hypernociception, which was determined using a modification of the Randall-Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE(2) levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan-induced hypernociception in a dose- and time-dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct-acting hypernociceptive mediators, PGE(2) and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan-induced TNF-alpha, IL-1 beta, and cytokine-induced neutrophil chemoattractant 1 (CINC-1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF-alpha, IL-1 beta, and CINC-1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct-acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL-1 beta produced PGE(2), and IL-1 beta-induced PGE(2) production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct-acting hypernociceptive mediators, such as PGE(2). Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.

IL-33 mediates antigen-induced cutaneous and articular hypernociception in mice

IL-33, a new member of the IL-1 family, signals through its receptor ST2 and induces T helper 2 (Th2) cytokine synthesis and mediates inflammatory response. We have investigated the role of IL-33 in antigen-induced hypernociception. Recombinant IL-33 induced cutaneous and articular mechanical hype rn ociception in a time- and dose-dependent manner. The hypernociception was inhibited by soluble (s) ST2 (a decoy receptor of IL-33), IL-1 receptor antagonist (IL-1ra), bosentan [a dual endothelin (ET)(A)/ETB receptor antagonist], clazosentan (an ETA receptor antagonist), or indomethacin (a cyclooxygenase inhibitor). IL-33 induced hypernociception in IL-18(-/-) mice but not in TNFR1(-/-) or IFN gamma(-/-) mice. The IL-33-induced hypernociception was not affected by blocking IL-15 or sympathetic amines (guanethidine). Furthermore, methylated BSA (mBSA)-induced cutaneous and articular mechanical hypernociception depended on TNFR1 and IFN gamma and was blocked by sST2, IL-1ra, bosentan, clazosentan, and indomethacin. mBSA also induced significant IL-33 and ST2 mRNA expression. Importantly, we showed that mBSA induced hypernociception via the IL-33 -> TNF alpha -> IL-1 beta -> IFN gamma -> ET-1 -> PGE(2) signaling cascade. These results therefore demonstrate that IL-33 is a key mediator of immune inflammatory hype rn ociception normally associated with a Th1 type of response, revealing a hitherto unrecognized function of IL-33 in a key immune pharmacological pathway that may be amenable to therapeutic intervention.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals. J. Leukoc. Biol. 87: 885-893; 2010.

Quercetin Reduces Inflammatory Pain: Inhibition of Oxidative Stress and Cytokine Production

Quercetin (1) is known to have both antioxidant and antinociceptive effects. However, the mechanism involved in its antinociceptive effect is not fully elucidated. Cytokines and reactive oxygen species have been implicated in the cascade of events resulting in inflammatory pain. Therefore, we evaluated the antinociceptive mechanism of 1 focusing on the role of cytokines and Oxidative stress. Intraperitoneal and oral treatments with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid and phenyl-p-benzoquinone and also the second phase of formalin- and carrageenin-induced mechanical hypernociception. Compound I also inhibited the hypernociception induced by cytokines (e.g., TNF alpha and CXCL1), but not by inflammatory mediators that directly sensitize the nociceptor such as PGE(2) and dopamine. On the other hand, 1 reduced carrageenin-induced IL-1 beta production as well as carrageenin-induced decrease of reduced glutathione (GSH) levels. These results suggest that I exerts its analgesic effect by inhibiting pro-nociceptive cytokine production and the oxidative imbalance mediation of inflammatory pain.

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans

Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA (+) background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA(+) background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA (-) background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA (+) background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

The role of hemocytes in the immunity of the spider Acanthoscurria gomesiana

Invertebrates protect themselves against microbial infection through cellular and humoral immune defenses. Since the available information on the immune system of spiders is scarce, the main goat of the present study was to investigate the role of hemocytes and antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria gomesiana. We previously described the purification and characterization of two AMPs from the hemocytes of naive spider A. gomesiana, gomesin and acanthoscurrin. Here we show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have reached the infection site, hemocytes may secrete coagulation cascade components and AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore, the main reactions involved in the spider immune defense might be coagulation and AMP secretion. (C) 2007 Elsevier Ltd. All rights reserved.

In vivo hydroquinone exposure alters circulating neutrophil activities and impairs LPS-induced lung inflammation in mice

Hydroquinone (HQ) is an environmental contaminant which causes immune toxicity. In this study, the effects of exposure to low doses of HQ on neutrophil mobilization into the LPS-inflamed lung were investigated. Male Swiss mice were exposed to aerosolized vehicle (control) or 12.5, 25 or 50 ppm HQ (1 h/day for 5 days). One hour later, oxidative burst, cell cycle. DNA fragmentation and adhesion molecules expressions in circulating neutrophils were determined by flow cytometry, and plasma malondialdehyde (MDA) levels were measured by HPLC. Also, 1 h later the last exposures, inflammation was induced by LPS inhalation (0.1 mg/ml/10 min) and 3 h later, the numbers of leukocytes in peripheral blood and in the bronchoalveolar lavage fluid (BALF) were determined using a Neubauer chamber and stained smears; adhesion molecules expressed on lung microvessel endothelial cells were quantified by immunohistochemistry; myeloperoxidase (MPO) activity was measured in the lung tissue by colorimetric assay; and cytokines in the BALF were determined by ELISA. In vivo HQ exposure augmented plasma MDA levels and oxidative activity of neutrophils, but did not cause alterations in cell cycle and DNA fragmentation. Under these conditions, the number of circulating leukocytes was not altered, but HQ exposure reduced LPS-induced neutrophil migration into the alveolar space, as these cells remained in the lung tissue. The impaired neutrophil migration into BALF may not be dependent on reduced cytokines secretions in the BALF and lung endothelial adhesion molecules expressions. However, HQ exposure increased the expression of beta(2) and beta(3) integrins and platelet-endothelial cell adhesion molecule-1 (PECAM-1) in neutrophils, which were not further enhanced by fMLP in vitro stimulation, indicating that HQ exposure activates circulating neutrophils, impairing further stimulatory responses. Therefore, it has been shown, for the first time, that neutrophils are target of lower levels of in vivo HQ exposure, which may be considered in host defense in infectious diseases. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Age-related changes in cerebellar phosphatase-1 reduce Na,K-ATPase activity

We evaluated whether changes in protein content and activity of PP-1 and PP-2A were the mechanism underneath the basal age-related reduction in alpha(2/3)-Na,K-ATPase activity in rats cerebella and whether this occurred through the cyclic GMP-PKG pathway. PP1 activity, but not its expression, increased with age, whereas PP-2 was not changed. The activity Of ot2/3-Na,K-ATPase varied with age. and there was a negative association between the PP-1 and alpha(2/3)-Na,K-ATPase activities. In young rats, the inhibition of PP-1 and PP-2A by okadaic acid (OA) increased in a dose-dependent manner alpha(1)- and alpha(2/3)-Na,K-ATPase, but had no effect on Mg-ATPase activity. A direct stimulation of PKG with 8-Br-cyclic GMP did not surmount the effect of OA. This analogue of cyclic GMP inhibited PP-1 activity only, indicating that at least part of the increase in alpha(1)- and alpha(2/3)-Na,K-ATPase activity induced by OA was mediated by the cyclic GMP-PKG-PP-1 cascade. Taking into account that PP1 inhibition increased alpha(2/3)-Na,K-ATPase activity, we propose that an age-related increase in PP-1 activity due to a decrease in cyclic GMP-PKG modulation plays a role for the age-related reduction of alpha(2/3)-Na,K-ATPase activity in rat cerebellum. (C) 2007 Elsevier Inc. All rights reserved.

Leukotrienes Target F-actin/Cofilin-1 to Enhance Alveolar Macrophage Anti-fungal Activity

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-delta (PKC delta) and PI3K but not PKC alpha and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.

Exogenous insulin stimulates glycogen accumulation in Rhipicephalus (Boophilus) microplus embryo cell line BME26 via PI3K/AKT pathway

Ticks are obligatory blood-feeding arthropods and important vectors of both human and animal disease agents. Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis, development and cell survival. In such cascade, Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). To study the insulin-triggered pathway and its possible roles during embryogenesis we used a culture of embryonic Rhipicephalus microplus cells (BME26). Exogenous insulin elevated cell glycogen content in the absence of fetal calf serum (FCS) when compared to cells without treatment. Moreover, in the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were blocked. We observed an increase in the relative expression level of PI3K`s regulatory subunit (p85), as determined by qRT-PCR. In the presence of PI3K inhibitors these effects on transcription were also reversed. Additionally, treatment with Wortmannin increased the expression level of the insulin-regulated downstream target glycogen synthase kinase 3 beta (GSK3 beta). The p85 subunit showed elevated transcription levels in ovaries from fully engorged females, but was differentially expressed during tick embryogenesis. These results strongly suggest the presence of an insulin responsive machinery in BME26 cells, and its correlation with carbohydrate/glycogen metabolism also during embryogenesis. (C) 2009 Published by Elsevier Inc.

Angiotensin II modulates CD40 expression in vascular smooth muscle cells

The signalling pathway CD40/CD40L (CD40 ligand) plays an important role in atherosclerotic plaque formation and rupture. AngII (angiotensin II), which induces oxidative stress and inflammation, is also implicated in the progression of atherosclerosis. In the present study, we tested the hypothesis that AngII increases CD40/CD40L activity in vascular cells and that ROS (reactive oxygen species) are part of the signalling cascade that controls CD40/CD40L expression. Human CASMCs (coronary artery smooth muscle cells) in culture exposed to IL (interleukin)-1 beta or TNF-alpha (tumour necrosis factor-a) had increased superoxide generation and enhanced CD40 expression, detected by EPR (electron paramagnetic resonance) and immunoblotting respectively. Both phenomena were abolished by previous incubation with membrane-permeant antioxidants or cell transfection with P22(phox) antisense. AngII (50-200 nmol/l) induced an early and sustained increase in CD40 mRNA and protein expression in CASMCs, which was blocked by treatment with antioxidants. Increased CD40 expression led to enhanced activity of the pathway, as AngII-treated cells stimulated with recombinant CD40L released higher amounts of IL-8 and had increased COX-2 (cyclo-oxygenase-2) expression. We conclude that AngII stimulation of vascular cells leads to a ROS-dependent increase in CD40/CD40L signalling pathway activity. This phenomenon may be an important mechanism modulating the arterial injury observed in atherosclerosis-related vasculopathy.

Fragment mass distributions in the fission of heavy nuclei by intermediate- and high-energy probes

Recent experiments have shown that the multimode approach for describing the fission process is compatible with the observed results. Asystematic analysis of the parameters obtained by fitting the fission-fragment mass distribution to the spontaneous and low-energy data has shown that the values for those parameters present a smooth dependence upon the nuclear mass number. In this work, a new methodology is introduced for studying fragment mass distributions through the multimode approach. It is shown that for fission induced by energetic probes (E > 30 MeV) the mass distribution of the fissioning nuclei produced during the intranuclear cascade and evaporation processes must be considered in order to have a realistic description of the fission process. The method is applied to study (208)Pb, (238)U, (239)Np and (241)Am fission induced by protons or photons.

Spallation product distributions and neutron multiplicities for accelerator-driven system using the CRISP code

Neutron multiplicities for several targets and spallation products of proton-induced reactions in thin targets of interest to an accelerator-driven system obtained with the CRISP code have been reported. This code is a Monte Carlo calculation that simulates the intranuclear cascade and evaporationl fission competition processes. Results are compared with experimental data, and agreement between each other can be considered quite satisfactory in a very broad energy range of incitant particles and different targets.

Cascades of Hopf bifurcations from boundary delay

We consider a 1-dimensional reaction-diffusion equation with nonlinear boundary conditions of logistic type with delay. We deal with non-negative solutions and analyze the stability behavior of its unique positive equilibrium solution, which is given by the constant function u equivalent to 1. We show that if the delay is small, this equilibrium solution is asymptotically stable, similar as in the case without delay. We also show that, as the delay goes to infinity, this equilibrium becomes unstable and undergoes a cascade of Hopf bifurcations. The structure of this cascade will depend on the parameters appearing in the equation. This equation shows some dynamical behavior that differs from the case where the nonlinearity with delay is in the interior of the domain. (C) 2009 Elsevier Inc. All rights reserved.