Lysophosphatidylinositol Promotes Chemotaxis and Cytokine Synthesis in Mast Cells with Differential Participation of GPR55 and CB2 Receptors.

Mast cells (MCs) are the main participants in the control of immune reactions associated with inflammation, allergies, defense against pathogens, and tumor growth. Bioactive lipids are lipophilic compounds able to modulate MC activation. Here, we explored some of the effects of the bioactive lipid lysophosphatidylinositol (LPI) on MCs. Utilizing murine bone marrow-derived mast cells (BMMCs), we found that LPI did not cause degranulation, but slightly increased FcεRI-dependent ß-hexosaminidase release. However, LPI induced strong chemotaxis together with changes in LIM kinase (LIMK) and cofilin phosphorylation. LPI also promoted modifications to actin cytoskeleton dynamics that were detected by an increase in cell size and interruptions in the continuity of the cortical actin ring. The chemotaxis and cortical actin ring changes were dependent on GPR55 receptor activation, since the specific agonist O1602 mimicked the effects of LPI and the selective antagonist ML193 prevented them. The LPI and O1602-dependent stimulation of BMMC also led to VEGF, TNF, IL-1α, and IL-1ß mRNA accumulation, but, in contrast with chemotaxis-related processes, the effects on cytokine transcription were dependent on GPR55 and cannabinoid (CB) 2 receptors, since they were sensitive to ML193 and to the specific CB2 receptor antagonist AM630. Remarkably, GPR55-dependent BMMC chemotaxis was observed towards conditioned media from distinct mouse and human cancer cells. Our data suggest that LPI induces the chemotaxis of MCs and leads to cytokine production in MC in vitro with the differential participation of GPR55 and CB2 receptors. These effects could play a significant role in the recruitment of MCs to tumors and the production of MC-derived pro-angiogenic factors in the tumor microenvironment.

Valproic acid restricts mast cell activation by Listeria monocytogenes.

Mast cells (MC) play a central role in the early containment of bacterial infections, such as that caused by Listeria monocytogenes (L.m). The mechanisms of MC activation induced by L.m infection are well known, so it is possible to evaluate whether they are susceptible to targeting and modulation by different drugs. Recent evidence indicates that valproic acid (VPA) inhibits the immune response which favors L.m pathogenesis in vivo. Herein, we examined the immunomodulatory effect of VPA on L.m-mediated MC activation. To this end, bone marrow-derived mast cells (BMMC) were pre-incubated with VPA and then stimulated with L.m. We found that VPA reduced MC degranulation and cytokine release induced by L.m. MC activation during L.m infection relies on Toll-Like Receptor 2 (TLR2) engagement, however VPA treatment did not affect MC TLR2 cell surface expression. Moreover, VPA was able to decrease MC activation by the classic TLR2 ligands, peptidoglycan and lipopeptide Pam3CSK4. VPA also reduced cytokine production in response to Listeriolysin O (LLO), which activates MC by a TLR2-independent mechanism. In addition, VPA decreased the activation of critical events on MC signaling cascades, such as the increase on intracellular Ca2+ and phosphorylation of p38, ERK1/2 and -p65 subunit of NF-κB. Altogether, our data demonstrate that VPA affects key cell signaling events that regulate MC activation following L.m infection. These results indicate that VPA can modulate the functional activity of different immune cells that participate in the control of L.m infection.

Mast Cell-Tumor Interactions: Molecular Mechanisms of Recruitment, Intratumoral Communication and Potential Therapeutic Targets for Tumor Growth.

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.

Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells.

BACKGROUND: Huntington's disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases. METHODS: Bone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Toll-like receptor (TLR) 4. Degranulation was assessed by measuring the secretion of ß-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-KitWsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed. RESULTS: R6/1 BMMCs showed normal ß-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4-induced anti-inflammatory cytokines (like IL-10 and TGF-ß). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-KitWsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice. CONCLUSIONS: Our data show that mHtt expression causes an impaired production of pro- and anti-inflammatory mediators triggered by TLR-4 receptor in MCs in vitro and in vivo, which could contribute to the aberrant immunophenotype observed in HD.

Mast Cells Localize in Hypoxic Zones of Tumors and Secrete CCL-2 under Hypoxia through Activation of L-Type Calcium Channels.

Hypoxia is a condition that together with low pH, high amounts of reactive oxygen species (ROS), and increased adenosine levels characterize tumor microenvironment. Mast cells (MCs) are part of tumor microenvironment, but the effect of hypoxia on the production of MC-derived cytokines has not been fully described. Using the hypoxia marker pimonidazole in vivo, we found that MCs were largely located in the low-oxygen areas within B16-F1 mice melanoma tumors. In vitro, hypoxia promoted ROS production, a ROS-dependent increase of intracellular calcium, and the production of MCP 1 (CCL-2) in murine bone marrow-derived MCs. Hypoxia-induced CCL-2 production was sensitive to the antioxidant trolox and to nifedipine, a blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). Simultaneously with CCL-2 production, hypoxia caused the ROS-dependent glutathionylation and membrane translocation of the α1c subunit of Cav1.2 LVDCCs. Relationship between ROS production, calcium rise, and CCL-2 synthesis was also observed when cells were treated with H2O2 In vivo, high CCL-2 production was detected on hypoxic zones of melanoma tumors (where tryptase-positive MCs were also found). Pimonidazole and CCL-2 positive staining diminished when B16-F1 cell-inoculated animals were treated with trolox, nifedipine, or the adenosine receptor 2A antagonist KW6002. Our results show that MCs are located preferentially in hypoxic zones of melanoma tumors, hypoxia-induced CCL-2 production in MCs requires calcium rise mediated by glutathionylation and membrane translocation of LVDCCs, and this mechanism of CCL-2 synthesis seems to operate in other cells inside melanoma tumors, with the participation of the adenosine receptor 2A.

TLR4 Receptor Induces 2-AG-Dependent Tolerance to Lipopolysaccharide and Trafficking of CB2 Receptor in Mast Cells.

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.

Immunoglobulin E induces VEGF production in mast cells and potentiates their pro-tumorigenic actions through a Fyn kinase-dependent mechanism.

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.

Lyn kinase controls TLR4-dependent IKK and MAPK activation modulating the activity of TRAF-6/TAK-1 protein complex in mast cells.

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-ß-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.

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells.

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.

Fyn kinase controls FcepsilonRI receptor-operated calcium entry necessary for full degranulation in mast cells.

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.