Inhibition of Macrophage Functions by the C-Terminus of Murine S100A9 Is Dependent on B-1 Cells

The protein S100A9 plays a key role in the control of inflammatory response. the C-terminus of the murine S100A9 protein (mS100A9p) downregulates the spreading and phagocytic activity of adherent peritoneal cells. Murine peritoneal cells are constituted bymacrophages and B-1 cells, and the latter exert an inhibitory effect on macrophage functions by secreting interleukin-(IL-) 10. Here, we investigated the influence of B-1 cells on the inhibitory effect evoked by mS100A9p on macrophages. mS100A9p did not alter spreading and phagocytosis either by peritoneal macrophages obtained from mice deprived of B-1 cells or by bone marrow-derived macrophages (BMDM phi). Nevertheless, when BMDM phi were cocultivated by direct or indirect contact with B-1 cells treated with mS100A9p, the phagocytosis by BMDM phi was decreased, showing that the effect of mS100A9p on macrophages was modulated by B-1 cells and/or their secretory compounds. Furthermore, the inhibitory action of mS100A9p on phagocytosis by adherent peritoneal cells was abolished in cells obtained from IL-10 knockout mice. Taken together, the results show that mS100A9p has no direct inhibitory effect on macrophages; however, mS100A9p modulates B-1 cells, which in turn downregulates macrophages, at least in part, via IL-10. These data contribute to the characterization of S100A9 functions involving B-1 cells in the regulation of the inflammatory process.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao ButantanButantan Inst, Lab Pathophysiol, BR-05503000 São Paulo, BrazilHosp Sirio Libanes, Lab Neuromodulat & Expt Pain, BR-01308060 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Discipline Immunol, BR-04023900 São Paulo, BrazilCtr Univ Sao Camilo, Discipline Immunol, BR-04263200 São Paulo, BrazilUniv Paulista, Discipline Immunol, BR-04026002 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Discipline Immunol, BR-04023900 São Paulo, BrazilFAPESP: FAPESP-2002/08277-7Web of Scienc

Walker 256 tumor growth suppression by crotoxin involves formyl peptide receptors and lipoxin a(4)

We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A(4). Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A 4 and its natural analogue 15-epi-LXA(4) 4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A 4 and 15-epi-LXA 4, which might inhibit both tumor growth and formation of new vessels via FPRs.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (fellowship-CAPES)PAP (fellowship-Secretaria da Saude do Estado de Sao Paulo)FAPESP [07/52447-8]Guggenheim FoundationSpecial Laboratory of Pain and Signaling, Butantan Institute, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, BrazilCEIS/Department of Biology, Institute of Biosciences of Rio Claro, São Paulo State University (UNESP), Rio Claro, SP, BrazilLaboratory of Pathophysiology, Butantan Institute, Avenida Vital Brazil 1500, 05503-900 São Paulo, SP, BrazilDepartment of Natural Sciences, Mathematics and Education, Agricultural Sciences Center, Federal University of São Carlos, Rodovia Anhanguera Km 174, 13600-970 Araras, SP, BrazilLaboratory of Inflammation and Vascular Pharmacology, Federal University of São Paulo, Rua São Nicolau 210, 09913-030 Diadema, SP, BrazilDepartment of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes 1524, 05508-900 São Paulo, SP, BrazilDepartment of Pharmacology, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes 1524, 05508-900 São Paulo, SP, BrazilLaboratory of Inflammation and Vascular Pharmacology, Federal University of São Paulo, Rua São Nicolau 210, 09913-030 Diadema, SP, BrazilFAPESP: 07/52447-8Web of Scienc

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

Arthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation. The articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors. Plasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat). These results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluationCentro para el Estudio de Venenos y Animales Venenosos/[063-2010]/CEVAP/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Crotoxin Modulates Events Involved in Epithelial–Mesenchymal Transition in 3D Spheroid Model

Epithelial–mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer

Crotoxin Modulates Events Involved in Epithelial–Mesenchymal Transition in 3D Spheroid Model

Epithelial–mesenchymal transition (EMT) occurs in the early stages of embryonic development and plays a significant role in the migration and the differentiation of cells into various types of tissues of an organism. However, tumor cells, with altered form and function, use the EMT process to migrate and invade other tissues in the body. Several experimental (in vivo and in vitro) and clinical trial studies have shown the antitumor activity of crotoxin (CTX), a heterodimeric phospholipase A2 present in the Crotalus durissus terrificus venom. In this study, we show that CTX modulates the microenvironment of tumor cells. We have also evaluated the effect of CTX on the EMT process in the spheroid model. The invasion of type I collagen gels by heterospheroids (mix of MRC-5 and A549 cells constitutively prepared with 12.5 nM CTX), expression of EMT markers, and secretion of MMPs were analyzed. Western blotting analysis shows that CTX inhibits the expression of the mesenchymal markers, N-cadherin, α-SMA, and αv. This study provides evidence of CTX as a key modulator of the EMT process, and its antitumor action can be explored further for novel drug designing against metastatic cancer

Modulatory effect of fatty acids on fungicidal activity, respiratory burst and TNF-alpha and IL-6 production in J774 murine macrophages

The reported effects of different families of fatty acids (FA; SFA, MUFA, n-3 and n-6 PUFA) on human health and the importance of macrophage respiratory burst and cytokine release to immune defence led us to examine the influence of palmitic acid (PA), oleic acid (OA), linoleic acid, arachidonic acid, EPA and DHA on macrophage function. We determined fungicidal activity, reactive oxygen species (ROS) and cytokine production after the treatment of J774 cells with non-toxic concentrations of the FA. PA had a late and discrete stimulating effect on ROS production, which may be associated with the reduced fungicidal activity of the cells after treatment with this FA. OA presented a sustained stimulatory effect on ROS production and increased fungicidal activity of the cells, suggesting that enrichment of diets with OA may be beneficial for pathogen elimination. The effects of PUFA on ROS production were time-and dose-dependently regulated, with no evident differences between n-3 and n-6 PUFA. It was worth noting that most changes induced after stimulation of the cells with lipopolysaccharide were suppressed by the FA. The present results suggest that supplementation of the diet with specific FA, not classes of FA, might enable an improvement in host defence mechanisms or a reduction in adverse immunological reactions.FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPqCAPESCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Role of Translocator 18 KDa Ligands in the Activation of Leukotriene B4 Activated G-Protein Coupled Receptor and Toll Like Receptor-4 Pathways in Neutrophils

TSPO (Translocator 18 KDa; tryptophan-rich sensory protein oxygen sensor) is a constitutive outer mitochondrial membrane protein overexpressed in inflammatory cells during local or systemic processes. Despite its expression is characterized, role of TSPO in inflammation remains elusive. For this study, we investigated the role of TSPO ligands on neutrophil functions elicited by two different inflammatory pathways. Peritoneal neutrophils were isolated from male Balb-C mice, treated with TSPO ligand diazepam, Ro5-4864 or PK11195 (1,100 or 1000 nM; 2 h) and further stimulated with lipopolysaccharide from Escherichia coli (LPS), a binding for Toll-Like Receptor-4 (TLR4), or leukotriene B4 (LTB4), a G-protein coupled receptor (GPCR) ligand. LPS treatment did not lead to overexpression of TSPO on neutrophils, and pre-treatment with any TSPO ligand did not alter cytokine expression, adhesion molecule expression, or the production of reactive oxygen and nitrogen species caused by LPS stimulation. Conversely, all TSPO ligands impaired LTB4’s actions, as visualized by reductions in L-selectin shedding, β2 integrin overexpression, neutrophil chemotaxis, and actin filament assembly. TSPO ligands showed distinct intracellular effects on LTB4-induced neutrophil locomotion, with diazepam enhancing cofilin but not modifying Arp2/3 expression, and Ro5-4864 and PK11195 reducing both cofilin and Arp2/3 expression. Taken together, our data exclude a direct role of TSPO ligands in TLR4-elicited pathways, and indicate that TSPO activation inhibits GPCR inflammatory pathways in neutrophils, with a relevant role in neutrophil influx into inflammatory sites