IL-6 and TNF-alpha production during active canine visceral leishmaniasis

We investigated the production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) during canine visceral leishmaniasis (VL) to gain a better understanding of the role of such multi-functional cytokines in parasite resistance. IL-6 and TNF-alpha levels were measured by capture ELISA in sera from 8 healthy dogs from a non-endemic area (control group) and in sera from 16 dogs from Aracatuba, SP, Brazil, an area endemic for leishmaniosis. The dogs from the endemic area were selected by positive ELISA serology against total Leishmania chagasi antigen, positive spleen imprints for Leishmania, and the presence of at least three clinical signs associated with active visceral leishmaniasis (fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotory difficulty, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy).Enhanced systemic IL-6 production was found in sera from dogs with the active disease compared to healthy dogs (t-test, P < 0.05). In contrast, TNF-alpha did not differ between the two groups studied. There was no correlation between IL-6 production and anti-leishmanial antibody titers in the sera. Our findings suggest that IL-6 is a good marker of active disease during leishmaniasis, and that other cytokines may be involved in the hypergammaglobulinemia characteristic of canine visceral leishmaniasis. (c) 2006 Published by Elsevier B.V.

MKK3/6-p38 MAPK signaling is required for IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells

Coupled bone turnover is directed by the expression of receptor-activated NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1 beta and TNF-alpha-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1 beta and TNF-alpha-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1 beta treatment and subsequently reduced similar to 70%-90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1 beta-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4-5-fold by IL-1 beta or TNF-alpha treatment. IL-1 beta-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1 beta and TNF-alpha-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1 beta and TNF-alpha-induced RANKL expression in bone marrow stromal cells.

TNF-alpha activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism

Human monocytes activated by recombinant tumor necrosis factor alpha (TNF-alpha) exhibited significant fungicidal activity on the yeast cells of a highly virulent strain of Paracoccidioides brasiliensis. This process was significantly inhibited in the presence of catalase (CAT - a scavenger of H2O2), but not in the presence of superoxide-dismutase (SOD - a scavenger of superoxide anion) or N-G-monomethyl-L- arginine (N-G-MMLA - a nitric oxide inhibitor). Furthermore, there was a direct association between the intracellular killing of the fungus and the production of H2O2 by activated cells. These results strongly suggest a role for H2O2 in the killing of highly virulent strains of P. brasiliensis by TNF-alpha-activated human monocytes.

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

TNF-alpha acts in the hypothalamus inhibiting food intake and increasing the respiratory quotient - Effects on leptin and insulin signaling pathways

Acting in the hypothalamus, tumor necrosis factor-alpha (TNF-alpha) produces a potent anorexigenic effect. However, the molecular mechanisms involved in this phenomenon are poorly characterized. In this study, we investigate the capacity of TNF-alpha to activate signal transduction in the hypothalamus through elements of the pathways employed by the anorexigenic hormones insulin and leptin. High dose TNF-a promotes a reduction of 25% in 12 h food intake, which is an inhibitory effect that is marginally inferior to that produced by insulin and leptin. In addition, high dose TNF-a increases body temperature and respiratory quotient, effects not reproduced by insulin or leptin. TNF-alpha, predominantly at the high dose, is also capable of activating canonical pro-inflammatory signal transduction in the hypothalamus, inducing JNK, p38, and NF kappa B, which results in the transcription of early responsive genes and expression of proteins of the SOCS family. Also, TNF-a activates signal transduction through JAK-2 and STAT-3, but does not activate signal transduction. through early and intermediary elements of the insulin/leptin signaling pathways such as IRS-2, Akt, ERK and FOXO1. When co-injected with insulin or leptin, TNF-a, at both high and low doses, partially impairs signal transduction through IRS-2, Akt, ERK and FOXO1 but not through JAK-2 and STAT-3. This effect is accompanied by the partial inhibition of the anorexigenic effects of insulin and leptin, when the low, but not the high dose of TNF-alpha is employed. In conclusion, TNF-alpha, on a dose-dependent way, modulates insulin and leptin signaling and action in the hypothalamus. (c) Published by Elsevier B.V.

Anti-inflammatory activity of Alchornea triplinervia ethyl acetate fraction: Inhibition of H2O2, NO and TNF-alpha

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of hydrogen peroxide, nitric oxide and TNF-alpha production in peritoneal macrophages by ethyl acetate fraction from Alchornea glandulosa

The effects of Alchornea glandulosa ethyl acetate fraction (AGF) on hydrogen peroxide (H2O2), nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages activated with lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) were investigated. Analysis by thin layer chromatography (TLC) of AGF showed several constituents, including flavonoids, which may have anti-inflammatory activity. Inhibitory effects of the fraction in H2O2 and NO production ranged from 8.59 +/- 7.84% to 70.56 +/- 4.16% and from 16.06 +/- 3.65% to 38.73 +/- 3.90%, respectively. The TNF-alpha production was only partially inhibited in the tested concentrations (12.21 +/- 6.23%-15.16 +/- 0.96%). According to these results, it is suggested that AGF has anti-inflammatory activity. This medicinal plant may have therapeutic potential in the control of inflammatory disorders.

EPIDERMAL GROWTH-FACTOR AND TRANSFORMING GROWTH-FACTOR-ALPHA CHARACTERISTICS OF HUMAN ORAL-CARCINOMA CELL-LINES

This study examined the expression of epidermal growth factor (EGF) cell-surface receptors, the response to exogenous ligand and the autocrine production of transforming growth factor a (TGF-a) in normal and carcinoma-derived human oral keratinocytes. One of eight malignant cell lines overexpressed EGF receptors, while the remainder expressed receptor numbers similar to normal cells. Exogenous EGF stimulated incorporation of tritiated thymidine in a dose-dependent manner. In keratinocytes expressing normal numbers of EGF receptors, the cellular response to exogenous EGF correlated positively with total EGF receptor number. SCC-derived keratinocytes produced more TGF-a than normal cells. There was no statistical correlation between the autocrine production of TGF-a, EGF cell-surface receptor expression and cellular response to exogenous EGF. While the growth-stimulatory effects of exogenous TGF-cl were inhibited by the addition of a neutralising antibody, the presence of this antibody in conditioned medium failed to produce a similar decrease in growth. The results indicate that overexpression of EGF receptors is not an invariable characteristic of human oral squamous carcinoma-derived cell lines. Further, the contribution of TGF-a to the growth of normal and carcinoma-derived human oral keratinocytes in vitro may be less significant than previously documented.

Downregulation of nuclear factor-kappa B (NF-kappa B) pathway by silibinin in human monocytes challenged with Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular basis for defining the pineal gland and pinealocytes as targets for tumor necrosis factor

The pineal gland, the gland that translates darkness into an endocrine signal by releasing melatonin at night, is now considered a key player in the mounting of an innate immune response. Tumor necrosis factor (TNF), the first pro-inflammatory cytokine to be released by an inflammatory response, suppresses the translation of the key enzyme of melatonin synthesis (arylalkylamine-N-acetyltransferase, Aanat). Here, we show that TNF receptors of the subtype 1 (TNF-R1) are expressed by astrocytes, microglia, and pinealocytes. We also show that the TNF signaling reduces the level of inhibitory nuclear factor kappa B protein subtype A (NFKBIA), leading to the nuclear translocation of two NFKB dimers, p50/p50, and p50/RelA. The lack of a transactivating domain in the p50/p50 dimer suggests that this dimer is responsible for the repression of Aanat transcription. Meanwhile, p50/RelA promotes the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide, which inhibits adrenergically induced melatonin production. Together, these data provide a mechanistic basis for considering pinealocytes a target ofTNF and reinforce the idea that the suppression of pineal melatonin is one of the mechanisms involved in mounting an innate immune response. © 2011 Carvalho-Sousa, da Silveira Cruz-Machado, Tamura, Fernandes, Pinato, Muxel, Cecon and Markus.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Associação entre expressão de receptores TLR2 e TLR4 e produção de TNF-alpha e IL-10 por monócitos de gestantes portadoras de pré-eclâmpsia

Preeclampsia (PE) is a pregnancy specific syndrome characterized by a systemic inflammatory response, with higher intensity than that observed in normal pregnancy. Cells of the immune system, such as monocytes and granulocytes are endogenously activated and secrete high levels of free radicals and inflammatory cytokines. The objective of this study was to assess the activation state of monocytes from pregnant women with preeclampsia by endogenous expression of TLR2 e TLR4 receptors and to correlate the expression of TLR2 and TLR4 on monocytes surface of pregnant women with PE with the production of tumor necrosis factor-alpha (TNF- and interleukin-10 (IL-10) by these cells stimulated or not with peptidoglycan (PG) and lipopolysaccharide (LPS), as agonists agents of TLR2 and TLR4, respectively. We evaluated 15 pregnant women with PE, 15 normotensive pregnant women (NT) and 15 non-pregnant (NP). Peripheral blood monocytes were incubates in the presence or absence of LPS or PG. The supernatant obtained after 18h of culture was aspirated and used for TNF- and IL-10 determination by enzyme immunoassay (ELISA). The endogenous expression of TLR2 and TLR4 receptors was evaluated by flow cytometry. Our results showed significant highly concentrations of TNF- and TLR4 expression in monocytes of preeclamptic women when compared with NT and NP. Normal pregnant women presented higher levels of IL-10 in comparison with PE and NP groups. TLR2 expression was similar in the three groups studied. Therefore, our study highlights the important role of TLR4 in PE and the consequent high production of TNF- by monocytes of these patients, as well as the potential mechanism involving low levels of IL-10 in the pathophysiology of the disease. These observations demonstrate the strong link between the pathology of PE and the immune system of these patients

TNF-alpha and IL-6 immunohistochemistry in rat renal tissue experimentaly infected with Leptospira interrogans serovar Canicola

Leptospirosis is a public health problem worldwide and its etiology remains unclear. Its pathogenesis involves a complex interaction between host and infecting microorganism. The inflammatory reaction that controls the infection process also underscores many pathophysiological events occurring in leptospirosis. We investigated the presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in renal tissues by immunohistochemical and histopathological examination in animals experimentally inoculated with Leptospira serovar Canicola. All the tests were carried out 2, 7, 14, 21 or 28 days after inoculation. Although TNF-alpha and IL-6 had been detected in tissues throughout the observation period, these cytokines appeared more intensely during the initial phase of infection. Therefore, both TNF-alpha and IL-6 were associated with the immunopathogenesis of leptospirosis. This profile suggests a high immunocellular response throughout the early infection stages followed by subsequent humoral response.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-kappa B in the rat hippocampus

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-kappa B (NF-kappa B) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-kappa B binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-kappa B activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-kappa B, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-alpha (Tnf-alpha), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF alpha B activation and increased NOS and alpha 2/3-Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-kappa B activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-kappa B activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain. (c) 2011 Wiley Periodicals, Inc.