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.

Prostaglandin E(2)-loaded microspheres as strategy to inhibit phagocytosis and modulate inflammatory mediators release

PGE(2), an arachidonic acid metabolite produced by various type of cells regulates a broad range of physiological activities in the endocrine, cardiovascular, gastrointestinal, and immune systems, and is involved in maintaining the local homeostasis. In the immune system, PGE(2) is mainly produced by APCs and it can suppress the Th1-mediated immune responses. The aim of this study was to develop PGE(2)-loaded biodegradable MS that prolong and sustain the in vivo release of this mediator. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the MS. We determined their diameters, evaluated the in vitro release of PGE(2), using enzyme immunoassay and MS uptake by peritoneal macrophages. To assess the preservation of biological activities of this mediator, we determined the effect of PGE(2) released from MS on LPS-induced TNF-alpha release by murine peritoneal macrophages. We also analyzed the effect of encapsulated PGE(2) on inflammatory mediators release from HUVECs. Finally, we studied the effect of PGE(2) released from biodegradable MS in sepsis animal model. The use of this formulation can provide an alternative strategy for treating infections, by modulating or inhibiting inflammatory responses, especially when they constitute an exacerbated profile. (C) 2008 Elsevier B.V. All rights reserved.

Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc gamma RIIB expression on B cells

Background: Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results: Maternal immunization with OVA showed increased levels of Fc gamma RIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-gamma-secreting T cells and IL-4 and IL-12-secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced Fc gamma RIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion: Maternal immunization upregulates the inhibitory Fc gamma RIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

KeaA, a Dictyostelium kelch-domain protein that regulates the response to stress and development

Background: The protein kinase YakA is responsible for the growth arrest and induction of developmental processes that occur upon starvation of Dictyostelium cells. yakA-cells are aggregation deficient, have a faster cell cycle and are hypersensitive to oxidative and nitrosoative stress. With the aim of isolating members of the YakA pathway, suppressors of the death induced by nitrosoative stress in the yakA-cells were identified. One of the suppressor mutations occurred in keaA, a gene identical to DG1106 and similar to Keap1 from mice and the Kelch protein from Drosophila, among others that contain Kelch domains. Results: A mutation in keaA suppresses the hypersensitivity to oxidative and nitrosoative stresses but not the faster growth phenotype of yakA-cells. The growth profile of keaA deficient cells indicates that this gene is necessary for growth. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. A morphological analysis of keaA deficient cells during multicellular development indicated that, although the mutant is not absolutely deficient in aggregation, cells do not efficiently participate in the process. Gene expression analysis using cDNA microarrays of wild-type and keaA deficient cells indicated a role for KeaA in the regulation of the cell cycle and pre-starvation responses. Conclusions: KeaA is required for cAMP signaling following stress. Our studies indicate a role for kelch proteins in the signaling that regulates the cell cycle and development in response to changes in the environmental conditions.

Glypican-3 reexpression regulates apoptosis in murine adenocarcinoma mammary cells modulating PI3K/Akt and p38MAPK signaling pathways

Glypican-3 (GPC3) is a proteoglycan involved in proliferation and cell survival. Several reports demonstrated that GPC3 is downregulated in some tumors, such as breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their invasive and metastatic capacities, associated with a decrease of their motility and an increase of their cell death. We demonstrated that GPC3 inhibits canonical Wnt signaling, as well as it activates non canonical pathway. Now, we identified signaling pathways responsible for the pro-apoptotic role of GPC3 in LM3 cells. We found for the first time that GPC3 inhibits the PI3K/Akt anti-apoptotic pathway while it stimulates the p38MAPK stress-activated one. We report a concomitant modulation of CDK inhibitors as well as of pro- and anti-apoptotic molecules. Our results provide new clues regarding the mechanism involved in the modulation induced by GPC3 of mammary tumor cell growth and survival.

Phagocytosis and Nitric Oxide Levels in Rheumatic Inflammatory States in Elderly Women

Background Very few studies have investigated, in the elderly, the effect of rheumatic inflammatory states on phagocyte function and free radical production. The objective of this article is to evaluate phagocytosis by neutrophils and the production of nitric oxide (.NO) by monocytes in elderly women recruited among patients of the Brazilian Public Health System. Methods: Forty patients aged more than 60 years with rheumatic inflammatory diseases were studied. Phagocytosis was measured by flow cytometry. .NO production was measured by the total nitrite assay and conventional inflammation markers were determined. Data were analyzed with the Mann Whitney nonparametric test and P<0.05 was considered significant. Results. C-reactive protein levels and white blood cell counts were significantly higher in inflammation than in the control group (P<0.05). The phagocytosis fluorescence intensity per neutrophil and the percentual of neutrophils expressing phagocytosis were significantly higher (P<0.05) in the test than in the control group. Furthermore, there was significant .NO overproduction by monocytes, (P<0.05). Conclusion: Phagocytosis and .NO production are affected by rheumatic states. This suggests that the increased .NO levels may play a part in the increased oxidative stress in rheumatic diseases in elderly women. J. Clin. Lab. Anal. 25:47-51, 2011. (C) 2011 Wiley-Liss, Inc.

Phagocytosis, Oxidative Burst, and Produced Reactive Species are Affected by Iron Deficiency Anemia and Anemia of Chronic Diseases in Elderly

Iron and oxidative stress have a regulatory interplay. During the oxidative burst, phagocytic cells produce free radicals such as hypochlorous acid (HOCl). Nevertheless, scarce studies evaluated the effect of either iron deficiency anemia (IDA) or anemia of chronic disease (ACD) on phagocyte function in the elderly. The aim of the present study was to determine the oxidative burst, phagocytosis, and nitric oxide ((aEuro cent)NO) and HOCl, reactive species produced by monocytes and neutrophils in elderly with ACD or IDA. Soluble transferrin receptor, serum ferritin, and soluble transferrin receptor/log ferritin (TfR-F) index determined the iron status. The study was constituted of 39 patients aged over 60 (28 women and 11 men) recruited from the Brazilian Public Health System. Oxidative burst fluorescence intensity per neutrophil in IDA group and HOCl generation in both ACD and IDA groups were found to be lower (p < 0.05). The percentages of neutrophils and monocytes expressing phagocytosis in ACD group were found to be higher (p < 0.05). There was an overproduction of (aEuro cent)NO from monocytes, whereas the fundamental generation of HOCl appeared to be lower. Phagocytosis, oxidative burst, and (aEuro cent)NO and HOCl production are involved in iron metabolism regulation in elderly patients with ACD and IDA.

Thalidomide treatment down-regulates SDF-1 alpha and CXCR4 expression in multiple myeloma patients

The chemokine stromal-derived factor-1 alpha (SDF-1 alpha) and its receptor CXCR4 are critically involved in directional migration and homing of plasma cells in multiple myeloma. Here, we show that the expression of SDF-1 alpha and CXCR4 was significantly down-regulated in patients treated with thalidomide (n = 10) as compared to newly diagnosed MM patients (n = 31) and MM patients treated with other drugs (n = 38). SDF-1 alpha and CXCR4 expression was also significantly decreased in a RPMI 8226 cell line treated with 10 and 20 mu mol/L of thalidomide. Our findings indicate that thalidomide therapy induces down-regulation of CXCR4 and its ligand SDF-1 alpha in multiple myeloma. (c) 2008 Elsevier Ltd. All rights reserved.

Effect of fish oil containing parenteral lipid emulsions on neutrophil chemotaxis and resident-macrophages` phagocytosis in rats

Background & aims: There is scarce information about immune function and parenteral. fish oil (FO). The influence of a new parenteral. lipid emulsion (LE) containing fish oil (SMOF) was experimentally evaluated on neutrophils` chemotaxis and macrophages` phagocytosis. Methods: Adult mate Lewis rats (n = 40) were randomized into five groups; one non-surgical. control and four to receive parenteral LE or saline infusion through jugular vein catheterization: SMOF (mixture of 30% medium-chain triglycerides, 30% soybean, 25% olive and 15% fish oils); MCT/LCT (physical mixture of 50% medium-chain triglycerides and 50% soybean oil); MCT/LCT/FO (80% MCT/LCT supplemented with 20% FO) and SS (saline). In the 5th experimental day and after intravenous colloidal carbon injection, blood and tissue (liver, lung and spleen) samples were collected and immunological analyses were performed. Results: LE didn`t influence neutrophil chemotaxis. SMOF didn`t influence phagocytosis (p > 0.05) while MCT/LCT and MCT/LCT/FO LE increased the number of liver and lung resident macrophages that had engaged in phagocytosis compared with CO-NS and SS (p < 0.05). Only MCT/LCT/FO increased the number of spleen resident macrophages that had engaged in phagocytosis (p < 0.05). Conclusions: LE, independently of composition, had no influence on neutrophils` chemotaxis, but showed different effect on phagocytosis by macrophages. SMOF LE had neutral effect while fish oil LE enriched with MCT/LCT LE increased resident-macrophages` phagocytosis. (c) 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss-or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi. J. Leukoc. Biol. 86: 989-998; 2009.

Local renin-angiotensin system regulates left ventricular hypertrophy induced by swimming training independent of circulating renin: a pharmacological study

Introduction. This study addressed the role of the local renin-angiotensin system (RAS) in the left ventriular hypertropy (LVH) induced by swimming training using pharmacological blockade. Materials and methods. Female Wistar rats treated with enalapril maleate (60 mg.kg(-1).d(-1), n = 38), losartan (20 mg.kg(-1).d(-1), n = 36) or high salt diet (1% NaCl, n = 38) were trained by two protocols (T1: 60-min swimming session, 5 days per week for 10 weeks and T2: the same T1 protocol until the 8(th) week, then 9(th) week they trained twice a day and 10(th) week they trained three times a day). Salt loading prevented activation of the systemic RAS. Haemodynamic parameters, soleus citrate synthase (SCS) activity and LVH (left ventricular/body weight ratio, mg/g) were evaluated. Results. Resting heart rate decreased in all trained groups. SCS activity increased 41% and 106% in T1 and T2 groups, respectively. LVH was 20% and 30% in T1 and T2 groups, respectively. Enalapril prevented 39% of the LVH in T2 group (p < 0.05). Losartan prevented 41% in T1 and 50% in T2 (P < 0.05) of the LVH in trained groups. Plasma renin activity (PRA) was inhibited in all salt groups and it was increased in T2 group. Conclusions. These data provide evidence that the physiological LVH induced by swimming training is regulated by local RAS independent from the systemic, because the hypertrophic response was maintained even when PRA was inhibited by chronic salt loading. However, other systems can contribute to this process.

Effects of Cold Stress, Corticosterone and Catecholamines on Phagocytosis in Mice: Differences between Resting and Activated Macrophages

Objective: We subjected mice to acute cold stress and studied the effect on phagocytosis by peritoneal macrophages mediated by 3 types of phagocytic receptors: Fc gamma, complement receptors 3 (CR3) and mannose and beta-glucan receptors. Methods: Mice were subjected to a cold stress condition (4 C for 4 h), and then peritoneal macrophages were harvested and phagocytosis assays performed in vitro. Results: We found a striking difference between resting and lipopolysaccharide (LPS)-activated macrophages (by intraperitoneal injection of LPS 4 days before the stress experiment): for resting macrophages cold stress caused a decrease in phagocytosis mediated by Fc gamma or mannose receptors, while for activated macrophages we observed an increase in phagocytosis by the 3 types of receptors. These effects were associated with an increase in plasma concentrations of corticosterone and catecholamines following the cold stress. In order to verify whether these hormone changes could account for the observed effects on phagocytosis, we performed in vitro assays by incubating macrophages harvested from nonstressed animals with these hormones for 4 h at 37 degrees C and measuring their phagocytic capacity. The following experiments were done: (a) with resting (nonactivated) macrophages; (b) with macrophages previously activated in vitro by incubation with LPS; (c) with macrophages previously activated in vivo by intraperitoneal injection of mice with LPS, 4 days before harvesting the cells. We found that for resting macrophages, corticosterone decreased phagocytosis mediated by Fc gamma and mannose and beta-glucan receptors, but catecholamines had no effect. For macrophages activated either in vivo or in vitro, catecholamines caused an increase in phagocytosis (excluding mannose receptors) while corticosterone had no effect. Conclusion: The above findings suggest that stress can regulate phagocytosis in different ways, depending on the kind of phagocytic receptor involved, the level of stress hormones and the physiological state of the macrophages. Copyright (C) 2010 S. Karger AG, Basel

Effects of Acute Cold Stress on Phagocytosis of Apoptotic Cells: The Role of Corticosterone

Background and Aims: Stress can alter many aspects of the immune response, and many studies have been conducted on the effects of stress on inflammatory processes, but little is known about its influence on the resolution of inflammation in tissue homeostasis, which includes the clearance of apoptotic cells by macrophages in a non-phlogistic way. In the present study, we investigated the effect of acute cold stress on the phagocytosis of apoptotic cells by macrophages. Methods: Mice were submitted to acute cold stress (4 degrees C for 4 h) and the capacity of peritoneal macrophages to phagocyte apoptotic thymocytes and to secrete anti-inflammatory cytokines was evaluated. Plasma corticosterone and catecholamine levels were investigated to assess their effect on the phagocytic capacity of macrophages in vitro. Results: We showed that acute cold stress decreases phagocytosis of apoptotic cells at the inflammatory site by lipopolysaccharide-activated macrophages but did not affect resting macrophages. The inhibitory effect on phagocytosis is accompanied by a reduced level of TGF-beta and higher IL-10 secretion. After stress, plasma concentrations of corticosterone increased 6-fold, epinephrine 2-fold and norepinephrine 1.7-fold compared to control mice. In vitro experiments showed that the decrease in phagocytosis after stress could be attributed, at least in part, to the effects of corticosterone; epinephrine and norepinephrine had no effect. Conclusions: The current study shows that acute cold stress decreases phagocytosis of apoptotic cells from an inflammatory environment by macrophages, and this inhibition is mediated by the intracellular glucocorticoid receptor. Copyright (C) 2009 S. Karger AG, Basel

IL-4 regulates susceptibility to intestinal inflammation in murine food allergy

Cardoso CR, Provinciatto PR, Godoi DF, Ferreira BR, Teixeira G, Rossi MA, Cunha FQ, Silva JS. IL-4 regulates susceptibility to intestinal inflammation in murine food allergy. Am J Physiol Gastrointest Liver Physiol 296: G593-G600, 2009. First published January 8, 2009; doi:10.1152/ajpgi.90431.2008.-Allergies involve a state of immediate hypersensitivity to antigens, including food proteins. The mechanism underlying the initiation and development of allergic responses involves IL-4 that directly induces the differentiation of committed effector Th2 lymphocytes. Although it is clear that Th2 responses play a pivotal role in the development of allergic responses, it remains unclear which mechanisms are involved in the development of the intestinal damages observed in food allergy. Accordingly, this work aimed to study the role of Th2/IL-4-dependent responses in the development of food allergy and intestinal pathology. C57BL/6 wild-type (WT) and IL-4(-/-) mice were sensitized with peanut proteins, challenged with peanut seeds, and followed for the development of food allergy and intestinal inflammation. Results demonstrated that exposure to peanut seeds led to weight loss in WT but not in IL-4(-/-) mice that preserved gut integrity with no signs of mucosal inflammation. These animals presented increased levels of IgG2a in sera, suggesting a role for allergic antibodies in the pathogenesis of WT animals. Most importantly, results also showed that lack of IL-4 modulated gut mucosal response in food allergy through diminished expression of TNF-alpha mRNA, increased Th1 IFN-gamma, IL-12p40, regulatory cytokines, and Foxp3, demonstrating their relevance in the control of allergic inflammatory processes, especially in the intestine. Finally, this study highlighted some of the complex mechanisms involved in the pathogenesis of allergic responses to food antigens in the gut, thereby providing valuable tools for directing novel therapeutic or preventive strategies to the control of allergic enteropathy.

Bradykinin regulates calpain and proinflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells

Yogi A, Callera GE, Tostes R, Touyz RM. Bradykinin regulates calpain and proinflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells. Am J Physiol Regul Integr Comp Physiol 296: R201-R207, 2009. First published September 17, 2008; doi: 10.1152/ajpregu.90602.2008.-Transient receptor potential melastatin-7 (TRPM7) channels have recently been identified to be regulated by vasoactive agents acting through G protein-coupled receptors in vascular smooth muscle cells (VSMC). However, downstream targets and functional responses remain unclear. We investigated the subcellular localization of TRPM7 in VSMCs and questioned the role of TRPM7 in proinflammatory signaling by bradykinin. VSMCs from Wistar-Kyoto rats were studied. Cell fractionation by sucrose gradient and differential centrifugation demonstrated that in bradykinin-stimulated cells, TRPM7 localized in fractions corresponding to caveolae. Immunofluorescence confocal microscopy revealed that TRPM7 distributes along the cell membrane, that it has a reticular-type intracellular distribution, and that it colocalizes with flotillin-2, a marker of lipid rafts. Bradykinin increased expression of calpain, a TRPM7 target, and stimulated its cytosol/membrane translocation, an effect blocked by 2-APB (TRPM7 inhibitor) and U-73122 (phospholipase C inhibitor), but not by chelerythrine (PKC inhibitor). Expression of proinflammatory mediators VCAM-1 and cyclooxygenase-2 (COX-2) was time-dependently increased by bradykinin. This effect was blocked by Hoe-140 (B(2) receptor blocker) and 2-APB. Our data demonstrate that in bradykinin-stimulated VSMCs: 1) TRPM7 is upregulated, 2) TRPM7 associates with cholesterol-rich microdomains, and 3) calpain and proinflammatory mediators VCAM-1 and COX2 are regulated, in part, via TRPM7- and phospholipase C-dependent pathways through B2 receptors. These findings identify a novel signaling pathway for bradykinin, which involves TRPM7. Such phenomena may play a role in bradykinin/B(2) receptor-mediated inflammatory responses in vascular cells.