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Obesity is one of the main health problems worldwide. It is a disease that is associated with excessive food consumption, although it is also associated with a process of chronic inflammation and with a group of disorders known as metabolic syndrome. Some of the most distinctive characteristics of obesity are that the individual's adipocytes are hypertrophied, have an irregular adipokine secretion profile, have increased recruitment of inflammatory cells, and altered metabolic homeostasis; which eventually results in the development of various important pathologies or conditions, such as insulin resistance and mitochondrial dysfunction. This review aims to present the current knowledge on inflammation of adipose tissue associated with obesity.

Mast cells (MCs) are tissue-resident immune cells that are important players in diseases associated with chronic inflammation such as cancer. Since MCs can infiltrate solid tumors and promote or limit tumor growth, a possible polarization of MCs to pro-tumoral or anti-tumoral phenotypes has been proposed and remains as a challenging research field. Here, we review the recent evidence regarding the complex relationship between MCs and tumor cells. In particular, we consider: (1) the multifaceted role of MCs on tumor growth suggested by histological analysis of tumor biopsies and studies performed in MC-deficient animal models; (2) the signaling pathways triggered by tumor-derived chemotactic mediators and bioactive lipids that promote MC migration and modulate their function inside tumors; (3) the possible phenotypic changes on MCs triggered by prevalent conditions in the tumor microenvironment (TME) such as hypoxia; (4) the signaling pathways that specifically lead to the production of angiogenic factors, mainly VEGF; and (5) the possible role of MCs on tumor fibrosis and metastasis. Finally, we discuss the novel literature on the molecular mechanisms potentially related to phenotypic changes that MCs undergo into the TME and some therapeutic strategies targeting MC activation to limit tumor growth.

La industria alimentaria ha incrementado su tasa de producción por alimentos siempre disponibles, listos para su consumo o que requieren de una mínima preparación, pero sin que aumente su calidad y sólo para satisfacer la demanda de una sociedad moderna. La característica que tienen en común estos alimentos, es el uso de un producto derivado del almidón del maíz denominado Jarabe de Maíz con un alto contenido de Fructosa (JMAF), el almidón es ampliamente usado en la fabricación de este tipo de alimentos procesados, y cuyo consumo desmedido está asociado con el incremento en la tasa de obesidad y de trastornos metabólicos. El metabolismo de la fructosa, conlleva a la disminución de la cantidad de energía disponible en el organismo. Simultáneamente, la producción de metabolitos ocasiona cambios en las vías de señalización celular promoviendo un incremento en la cantidad de lípidos como los triglicéridos y el colesterol de muy baja densidad. Los altos niveles de estas moléculas favorecen condiciones que encaminan a la obesidad y a la resistencia a la insulina, propiciando el desarrollo de la diabetes mellitus tipo 2, hiperuricemia e inflamación que por lo general son la base para el origen del cáncer y el inicio de las enfermedades neurológicas. Esta revisión se centra en la relación entre la fructosa y estas enfermedades y su posible mecanismo de acción.

Today, international attention is focused on sustainability and respect for the environment; there is an irretrievable wear and tear on nature, reflecting the excessive and inappropriate use of natural resources. Mexico committed itself in 2015 to the UN to take action to meet the 2030 Agenda for Sustainable Development goals. It seeks to determine the importance of environmental accounting in Mexico for the fulfillment of some of these objectives and what its current situation is. A bibliographic search was done on information from recognized data sources; rigorously, selecting the most relevant research in the accounting field, as well as a revision of the Financial Reporting Standards and ISOS that regulate the ecological issue. Concluding that environmental accounting plays an important role in two of the objectives of the 2030 agenda; ecological accounts and sustainable work is a little-seen issue in Mexico that is evolving, so it is important to encourage a citizen's awareness of the measures necessary to prevent and compensate for the damage, it is not an obligation that only falls on the government is a matter for all Mexicans.

Activation of high affinity receptor for IgE (FceRI) by IgE/antigen complexes in mast cells (MCs) leads to the release of preformed pro-inflammatory mediators stored in granules by a Ca2+-dependent process known as anaphylactic degranulation. Degranulation inhibition has been proposed as a strategy to control allergies and chronic inflammation conditions. Cannabinoids are important inhibitors of inflammatory reactions but their effects on IgE/Ag-mediated MCs responses are not well described. In this study, we analyzed the effect of the endocannabinoid anandamide (AEA), the selective CB2 receptor agonist HU308, and the GPR55 receptor agonist lysophosphatidylinositol (LPI) on FceRI-induced activation in murine bone marrow-derived mast cells (BMMCs). Our results show that AEA, HU380 and LPI inhibited FceRI-induced degranulation in a concentration-dependent manner. This effect was mediated by CB2 and GPR55 receptor activation through a mechanism insensitive to pertussis toxin. Degranulation inhibition was prevented by CB2 and GPR55 antagonism, but not by CB1 receptor blockage. AEA also inhibited calcium-dependent cytokine mRNA synthesis induced by FceRI crosslinking, without affecting early phosphorylation events. In addition, AEA, HU308 and LPI inhibited intracellular Ca2+ rise in response to IgE/Ag. CB2 and GPR55 receptor antagonism could not prevent the inhibition produced by AEA and HU308, but partially blocked the one caused by LPI. These results indicate that AEA inhibits IgE/Ag-induced degranulation through a mechanism that includes the participation of CB2 and GPR55 receptors acting in close crosstalk, and show that CB2-GPR55 heteromers are important negative regulators of FceRI-induced responses in MCs.

El Factor de crecimiento transformante β (TGF-β) es una citocina pleiotrópica implicada en distintas condiciones patológicas, como desórdenes autoinmunes, alergias y en los últimos años, en el cáncer. Esta citocina ejerce efectos supresores de tumores que las células cancerosas deben evadir para lograr la progresión del tumor. Sin embargo, paradójicamente, el TGF-β también modula procesos inflamatorios que favorecen la progresión del tumor, como el reclutamiento de células del sistema inmune al sitio del mismo; entre estas células se encuentran las células cebadas (CCs), las cuales, a su vez también participan en la regulación del tumor, a través de la secreción de distintos mediadores proinflamatorios, proangiogénicos y factores de crecimiento. En esta revisión se describen algunos avances en la comprensión del papel del TGF-β en la regulación de las CCs y la contribución de éstas en el desarrollo y la metástasis de tumores sólidos. El entendimiento de la función del TGF-β y de las células cebadas durante el desarrollo del cáncer es fundamental para el diseño de nuevas terapias que inhiban la progresión del tumor.

La sobreeducacion, definida como el rubro de profesionistas en puestos de trabajo que requieren de menor educacion a la adquirida, ha sido un fenomeno que ha impactado en los ultimos anos en el mercado laboral mexicano. Por ello, esta investigacion tiene como objetivo responder las siguientes preguntas: ?cual es el efecto de la sobreeducacion e informalidad en el ingreso de los profesionistas?, ?cual es la probabilidad de estar sobreeducado, por tipo de carreras, en el mercado labo ral de los profesionistas?, ?cual es la probabilidad de estar en el mercado informal, a pesar de tener una carrera profesional?, ?cual es la probabilidad de no estar sobreeducado? La base de datos se tomo de la Encuesta Nacional de Ocupacion y Empleo (enoe) 2016-iv trimestre. La metodologia propuesta es generar una ecuacion tipo minceriana y un modelo tipo probit multinomial. Los principales resultados revelan que estar en un mercado sobreeducado, reduce el ingreso en 24.6 %, mientras que estar en el mercado informal lo reduce en 44.5%. La probabilidad de estar sobreeducado en forma global, es de 33 %, mientras que estar en el mercado informal es de 14 %.

Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow–derived mast cells from wild-type and Fyn-deficient mice. Fyn−/− cells showed higher LPS-induced secretion of preformed and de novo–synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn−/− cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn−/− cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/β, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation–associated protein (AHNAK, desmoyokin). LPS-induced PKCα/β activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn−/− MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/β necessary for the secretion of TNF by VAMP3+ carriers.

Mast cells (MCs) participate in allergic diseases by producing pro-inflammatory mediators and regulatory cytokines. After high affinity IgE (FceRI) receptor activation, they release tryptase, b-hexosaminidase and other preformed pro-inflammatory substances by anaphylactic degranulation. Also, upon stimulation through Toll-like receptor 4 (TLR4), they release pro-inflammatory mediators (such as Tumor Necrosis Factor, TNF), by piecemeal degranulation, orchestrating protective reactions against pathogens. Reactions involved in anaphylactic degranulation require specific vesicular-associated membrane proteins (VAMPs) involved in the latest step of exocytosis. However, the particular roles of VAMP proteins on piecemeal secretion observed after TLR4 receptor activation are not known. Utilizing RBL-2H3 cells differentiated to a pro-inflammatory phenotype, we investigated the role of VAMP8 and VAMP3 on lipopolysaccharide (LPS)-induced mediator secretion in MCs. Confocal microscopy images showed that, in non-stimulated cells, TNF localizes in VAMP3 and VAMP8-positive vesicles. After TLR4 stimulation, we observed an increase in TNF and VAMP8 co-localization, without any change on TNF and VAMP3 co-localization. Remarkably, VAMP8 blockade with specific siRNA impaired TNF secretion after LPS-challenge, while VAMP3 siRNA-mediated down regulation did not. Our data indicate that, although TNF is maintained in VAMP3-containing vesicles, TLR4 triggering leads to the secretion of TNF in VAMP8-containing carriers. Our results contribute to the description of the molecular events involved in late steps of TNF secretion induced by innate immune stimuli in that important immune cell type.

Mast Cells (MCs) initiate allergic responses but also participate in innate inflammatory reactions triggered by infection, tissue damage and cancer. MCs are recruited to affected sites where they release preformed and neo-formed mediators of inflammation. Bioactive lipids (such as sphingosine-1-phosphate, S1P) have demonstrated to induce MC chemotaxis. Recently, α-lysophosphatidilinositol (LPI) has awakened scientific interest due to the discovery of its endogenous receptor, the orphan receptor GPCR55. This receptor has been shown to couple to Gα12/13 pathway, controlling actin polimeryzation and cell migration. In this study, we aimed to characterize the role of LPI in the activation and migration of MCs. Using murine bone marrow-derived MCs, we observed that low concentrations of this lipid (10 nM and 100 nM) induces maximal ERK 1/2 phosphorylation at 15 and 10 minutes, respectively. Also, we found that LIMK (an important G12/13 and Rho-dependent kinase) is phosphorylated after 5 and 10 min after 10 nM or 100 nM LPI, respectively. Both ERK 1/2 and LIMK phosphorylation patterns were reproduced with the selective agonist of GPCR55, O-1602, at equi-potent concentrations to those of the LPI. Using Boyden chamber assays, we observed that LPI provocked higher chemotaxis of MCs when compared with S1P. Finally, using confocal microscopy, we observed that LPI triggered depolymerization of cortical actin, as well as the formation of filopodia between 15 and 30 minutes post stimulation. We concluded that GPR55 expressed in MCs induces important changes on MAPK and LIMK activation and its ligand, LPI, is a potent chemotactic factor of MCs.

Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow–derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow–derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.

Activation of high affinity receptor for IgE (FcεRI) by IgE/antigen complexes in mast cells (MCs) leads to the release of preformed pro-inflammatory mediators stored in granules by a Ca

Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl2) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl2-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl2-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

Effects of immunoglobulin E (IgE)-dependent sensitization on the response to bacterial lipopolysaccharide (LPS) were analyzed in mast cells. Murine bone marrow-derived mast cells (BMMCs) were sensitized or not with IgE before stimulation with LPS. TLR4 and co-receptors expression was analyzed by flow cytometry and RT-PCR, TNF-α production by ELISA, IKK and IκB activation by western blot or immunoprecipitation. NFκB nuclear translocation was determined by EMSA. IgE-sensitized BMMCs secreted larger amounts of TNF-α than non-sensitized cells shortly after LPS challenge. No change in TLR4, CD14 or MD-2 expression was detected after the IgE-dependent sensitization process, whereas TLR4-dependent phosphorylation of IKK and IκB was augmented. IgE-dependent sensitization increased basal NFκB activity. Endotoxin tolerance was not affected by the IgE-dependent sensitization process. IgE-induced sensitization primes mast cells for higher response to LPS through pre-activation of NFκB transcription factor. IgE-dependent sensitization does not modify events leading to endotoxin tolerance.

Although presence of mast cells (MCs) to sites with low oxygen concentration (such as inflamed tissues with compromised blood flux and solid tumors) has been reported, the effects of hypoxia on the cytokine profile produced by MCs under those conditions has not been fully explored. We used the murine B16 melanoma model to quantify the presence of MCs in normoxic or hypoxic zones of the tumor and, bone marrow-derived mast cells (BMMCs) subjected to a 1% O2 atmosphere to characterize the production of specific cytokines and chemokines by this cell type in hypoxic conditions. Hypoxic areas of melanoma tumors were identified with hypoxyprobe and the number of MCs was determined by confocal analysis of tryptase positive cells. Using this methodology, important MCs presence in hypoxic zones of the tumor was detected. In vitro, BMMCs subjected to hypoxia were able to produce VEGF and the potent chemokine CCL2. Chemokine production in hypoxia was sensitive to the antioxidant Trolox and the IKK inhibitor BAY117085. CCL2 production was also induce by monomeric IgE by a mechanism also sensitive to Trolox and BAY117085. CCL2 production triggered by hypoxia or monomeric IgE was sensitive to calcium chelator BAPTA and the L-type calcium channel blocker Nifedipine. Our results show that MCs are importantly recruited to hypoxic sites of melanoma tumors and that hypoxia induces the production of the chemokine CCL2 by a mechanism that require the production of reactive oxygen species (ROS) and intracellular calcium rise through the activation of L-type calcium channels. Also, data indicate that monomeric IgE triggers a very similar response than hypoxia does to lead to the production of CCL2 in mast cells.

Mast cells are important effectors in allergic reactions since they produce a number of pre-formed and de novo synthesized inflammatory compounds in response to high affinity IgE receptor (FcɛRI) crosslinking. IgE/Antigen-dependent degranulation and cytokine synthesis of this cell type have been recognized as relevant pharmacological targets for the control of deleterious inflammatory reactions. Despite the relevance of allergic diseases worldwide, pharmacological control of mast cell degranulation has not been achieved. In this work we analyzed the effect of the natural xanthone jacareubin on the main activation parameters of this cell type utilizing bone marrow-derived mast cells (BMMCs). Jacareubin, the xanthone obtained from the heartwood of the tropical tree Callophyllum brasilense, showed no toxicity when tested on bone marrow-derived mast cells (BMMCs) and did not activate degranulation by itself. However, jacareubin inhibited IgE/Ag-induced degranulation (determined as the secretion of β-hexosaminidase) in a dose-response manner (IC50 = 46 nM). Jacareubin blocked FcɛRI-dependent reactive oxygen species (ROS) formation, intracellular calcium mobilization and calcium-dependent protein kinase C (PKC) phosphorylation. Detailed analysis suggest that at least one of the molecular actions of jacareubin relies on the inhibition of plasma membrane calcium channels involved in degranulation of mast cells. It is proposed to jacareubin as a natural drug with antioxidant activity and a regulator of calcium channels, with prophylactic potential for the treatment of allergies produced by mast cells.

Binding of monomeric Immunoglobulin E (IgE) to the high affinity IgE receptor (FcεRI) on mast cells induces a sensitization process which increases cell survival, augments membrane receptor expression and diminishes activation threshold. Although IgE-dependent sensitization is fundamental for allergic reactions, little is known about the influence of locally produced mediators on the outcome of a posterior allergen challenge. Since Transforming Growth Factorβ (TGFβ) is an important immunomodulator present in most of the tissues where mast cells reside, we decided to analyze the consequences of TGFβ exposure during the sensitization step of mast cells on a posterior IgE-antigen stimulation. Bone Marrow-derived Mast Cells (BMMCs) were sensitized with IgE in the presence or absence of TGFβ. Then, antigen was added and the secretion of the angiogenic cytokine Vascular Endothelial Growth Factor (VEGF) was determined. BMMCs sensitized with IgE+TGFβ showed an increased antigen-induced VEGF secretion compared to those sensitized with IgE alone. Sensitization with IgE+TGFβ did not modify membrane FcεRI receptor expression neither altered antigen-induced degranulation of the cells. Although both IgE and IgE+TGFβ sensitized cells showed an increase in VEGF mRNA stabilization after antigen addition, VEGF mRNA half-life was longer in IgE+TGFβ sensitized cells. p38 MAPK inhibitor SB202196 blocked VEGF mRNA stabilization after antigen addition specially on IgE+TGFβ sensitized cells. These findings suggest that TGFβ presence during the sensitization phase of mast cells can induce modifications to the FcεRI signal transduction system, provoking increased VEGF mRNA stabilization and protein secretion after IgE-antigen stimulation through a p38 MAPK-dependent mechanism.

Some cancer cell lines release soluble factors that activate the endothelial cells in vitro; also endothelial activation in vivo includes an increased expression of adhesion molecules on the apical membrane, and an increased permeability, which may contribute to the extravasation process of circulating cells. We have adapted the Miles assay into a protocol that uses IgE/antigen complex and VEGF-1 as controls. The Miles assay comprises the intradermic injection of a pro-inflammatory agent into the skin and the intravenous introduction of a dye; the increase in vascular permeability will allow for the extravasation of the dye and thus the skin will be stained. The dye is then extracted from the dissected skin and quantified by spectrophotometry. The use of localized treatments will allow for testing a larger number of experimental samples in the same animal. With this model, the effects of tumoral soluble factors (TSFs) on endothelial permeability can be studied, as well as the signaling pathways involved. It can also serve to study the interactions between endothelial, immune, and cancer cells during the extravasation process.

Some cancer cell lines release soluble factors that activate the endothelial cells in vitro; also endothelial activation in vivo includes an increased expression of adhesion molecules on the apical membrane, and an increased permeability, which may contribute to the extravasation process of circulating cells. We have adapted the Miles assay into a protocol that uses IgE/antigen complex and VEGF-1 as controls. The Miles assay comprises the intradermic injection of a pro-inflammatory agent into the skin and the intravenous introduction of a dye; the increase in vascular permeability will allow for the extravasation of the dye and thus the skin will be stained. The dye is then extracted from the dissected skin and quantified by spectrophotometry. The use of localized treatments will allow for testing a larger number of experimental samples in the same animal. With this model, the effects of tumoral soluble factors (TSFs) on endothelial permeability can be studied, as well as the signaling pathways involved. It can also serve to study the interactions between endothelial, immune, and cancer cells during the extravasation process.

Background High concentrations of plasmatic IgE have been related to distinct systemic inflammatory conditions that frequently predispose individuals to hypersensitivity reactions. Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce. MC are an important component in tumor microenvironment where they seem to secrete a number of immunomodulatory and angiogenic mediators, such as the Vascular Endothelial Growth Factor (VEGF) by not well-described mechanisms. In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth. Methods For in vitro studies, murine bone marrow-derived mast cells (BMMCs) were used. Pharmacological inhibitors and phosphorylation of key elements controlling VEGF secretion and protein translation were used to characterize the mechanism of VEGF production triggered by IgE. In vivo, the effect of a single i.v. administration of monomeric IgE on B16 melanoma tumor weight, intratumoral blood vessel formation and tumor-associated MC was assessed in four groups of mice: MC-proficient (WT), MC-deficient (Wsh), Wsh reconstituted with MC derived from WT mice (Wsh Rec WT) and Wsh reconstituted with MC derived from Fyn −/− mice (Wsh Rec Fyn −/−). Results Monomeric IgE induced VEGF secretion through a Fyn kinase-dependent mechanism and modulated de novo protein synthesis modifying the activity of the translational regulator 4E-BP1 in BMMCs. In vivo, monomeric IgE increased melanoma tumor growth, peritumoral MC and blood vessel numbers in WT but not in Wsh mice. The positive effects of IgE on melanoma tumor growth were reproduced after reconstitution of Wsh mice with WT but not with Fyn −/− BMMCs. Conclusion Our data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.

Mast cells (MCs) control allergic reactions and contribute to protective innate immune responses through TLR4 activation. The tyrosine kinase Lyn is important to the high affinity IgE receptor (FcεRI) signal transduction system in MCs, but its role on the TLR4 signalling cascade is still elusive. Here, we characterized several TLR4-triggered responses in bone marrow-derived mast cells (BMMCs) from wild-type (WT) and Lyn−/− mice. We found that Lyn−/− MCs secreted lower amounts of TNF-α after LPS challenge when compared with WT cells. Lyn−/− BMMCs showed less MAPK, IκB phosphorylation and NF-κB nuclear translocation after TLR-4 triggering than WT cells. LPS-induced MAPK and inhibitor of IκB kinase (IKK) phosphorylation were importantly reduced in the absence of Lyn. A constitutive interaction between TNF receptor associated factor 6 (TRAF-6) and phosphorylated TGF-β-activated kinase (TAK-1) was observed in Lyn−/− BMMCs and this complex was insensitive to LPS addition. Lyn kinase was activated and associated to TRAF-6 shortly after LPS addition in WT MCs. Analyzing two local MC-dependent innate immune responses in vivo, we found that Lyn positively controls early TNF-α production and immune cell recruitment after an intraperitoneal injection of LPS. Our results indicate that Lyn plays a positive role in TLR4-induced production of TNF-α in MCs controlling the activity of the TRAF-6/TAK-1 protein complex.

Mast cells (MCs) express the high affinity IgE receptor (Fc RI) in their plasma membrane. This receptor binds monomeric IgE and, after antigen recognition, induces Ca2+-dependent degranulation of mast cells. Fc RI receptor signal transduction system is regulated by two Srcfamily kinases, Fyn and Lyn. Fyn kinase positively regulates degranulation but its role on the molecular mechanism that mobilizes intracellular Ca2+ has not been explored in detail. In this work, we analyzed by fluorometric techniques and pharmacological tools Ca2+ mobilization in bone marrow-derived mast cells(BMMCs)obtained from WT and Fyn–/– mice stimulated by IgEantigen. We observed that Fyn–/– BMMCs showed a reduction in extracellular Ca2+ influx when stimulated with IgE-antigen, compared to WT cells. The calcium channel blocker 2aminoethoxydiphenyl-borane (2-APB) was able inhibit maximal Ca2+ rise in WT but not in Fyn–/– cells. Ca2+ entry through 2-APB sensitive channels was important for full degranulation, NF B activation and mRNA accumulation of IL-2 and CCL-2 in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channel after IgE-antigen stimulation, but it is not related to protein tyrosine phosphorylation. Our results strongly suggest that Fyn kinase activate TRPC calcium channels that contribute to receptor operated calcium entry in mast

IgE-antigen-dependent crosslinking of the high affinity IgE receptor (FcepsilonRI) on mast cells leads to degranulation, leukotriene synthesis and cytokine production. Calcium (Ca(2+)) mobilization is a sine qua non requisite for degranulation, allowing the rapid secretion of stored pro-inflammatory mediators responsible for allergy symptoms. Fyn is a Src-family kinase that positively controls FcepsilonRI-induced mast cell degranulation. However, our understanding of the mechanism connecting Fyn activation to secretion of pre-synthesized mediators is very limited. We analyzed FcepsilonRI-dependent Ca(2+) mobilization in bone marrow-derived mast cells (BMMCs) differentiated from WT and Fyn -/- knock out mice. Fyn -/- BMMCs showed a marked defect in extracellular Ca(2+) influx after FcepsilonRI crosslinking but not after thapsigargin addition. High concentrations of Gadolinium (Gd(3+)) partially blocked FcepsilonRI-induced Ca(2+) influx in WT cells but, in contrast, completely inhibited Ca(2+) mobilization in Fyn -/- cells. Low concentrations of an inhibitor of the canonical transient receptor potential (TRPC) Ca(2+) channels (2-aminoethoxyphenyl-borane, 2-APB) blocked FcepsilonRI-induced maximal Ca(2+) rise in WT but not in Fyn -/- cells. Ca(2+) entry through Fyn-controlled, 2-APB sensitive channels was found to be important for full degranulation and IL-2 mRNA accumulation in WT cells. Immunoprecipitation assays showed that Fyn kinase interacts with TRPC 3/6/7 channels after IgE-antigen stimulation, but its association is not related to protein tyrosine phosphorylation. Results indicate Fyn kinase mediates the receptor-dependent activation of TRPC channels that contribute to degranulation in FcepsilonRI-stimulated mast cells.