Influence of Selenium on Mast Cell Mediator Release

Selenium supplementation still enhanced the immune response even in individuals who, according to current standards, would be considered as not being overtly selenium deficient. Mast cells are granulated cells that play a pivotal role in allergic reactions. In this study, we investigated the modulatory effect of sodium selenite on mediator release and degranulation of murine mast cell line (MC/9). Cells were pre-treated with selenium selenite (1, 2, 3 μg/ml) for 24 h and controls left untreated. Then, cells were sensitized overnight with anti-dinitrophenyl (DNP) IgE and challenged with DNP/HSA for degranulation induction. The histamine and prostaglandin D2 (PGD2) were measured by ELISA, and β-hexosaminidase was measured by spectrophotometery method. Selenium-treated cells revealed significant decrease in concentration of PGD2 (P?=?0.019) and β-hexosaminidase (P?=?0.009). In addition, a slight reduction of histamine release by the selenium-treated cells was observed, based on our intracellular and extracellular assessments. The most inhibitory effect of selenium supplementation on mediator release of MC/9 cells was obtained in the presence of 3 μg/ml of sodium selenite. The results of the present study demonstrate beneficial effects of supplemental selenium in attenuating clinical manifestations of allergy and asthma.     

Mast Cell Survival and Mediator Secretion in Response to Hypoxia

Tissue hypoxia is a consequence of decreased oxygen levels in different inflammatory conditions, many associated with mast cell activation. However, the effect of hypoxia on mast cell functions is not well established. Here, we have investigated the effect of hypoxia per se on human mast cell survival, mediator secretion, and reactivity. Human cord blood derived mast cells were subjected to three different culturing conditions: culture and stimulation in normoxia (21% O₂); culture and stimulation in hypoxia (1% O₂); or 24 hour culture in hypoxia followed by stimulation in normoxia. Hypoxia, per se, did not induce mast cell degranulation, but we observed an increased secretion of IL-6, where autocrine produced IL-6 promoted mast cell survival. Hypoxia did not have any effect on A23187 induced degranulation or secretion of cytokines. In contrast, cytokine secretion after LPS or CD30 treatment was attenuated, but not inhibited, in hypoxia compared to normoxia. Our data suggests that mast cell survival, degranulation and cytokine release are sustained under hypoxia. This may be of importance for host defence where mast cells in a hypoxic tissue can react to intruders, but also in chronic inflammations where mast cell reactivity is not inhibited by the inflammatory associated hypoxia.     

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

Objectives

We analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.

Methods

Electrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O₂ ^.-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.

Results

EFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O₂ ^- release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.

Conclusion

While both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.     

Growth Response to Hormones by a New Rat Ovary Cell Line

SEVERAL endocrine cell lines established in recent years show a functional response to hormones in vitro¹ but, except for one mammary cell line², none of them exhibits the normal hormone requirement for growth in vivo. We have now isolated a rat ovarian cell line whose growth in vitro is markedly stimulated by bovine luteinizing hormone (LH-NIH-B7), a pituitary gonadotrophin and by dexamethasone, a synthetic glucocorticoid. This cell line provides the first permanent in vitro system for studying the growth stimulation of gonadal cells by hormones.     

Inhibitory Effects of Apple Polyphenol on Induced Histamine Release from RBL-2H3 Cells and Rat Mast Cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC₅₀ values for histamine release were 30 μg/ml and 25 μg/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

In Vivo Release of Dopamine and Its Metabolites from Rat Striatum in Response to Domoic Acid

The microdialysis technique was used to examine the effect of the neurotoxin domoate, an analog of glutamic acid, on striatal dopamine activity. Our results show that the intracerebral administration of different concentrations of domoate (100 and 500 M) produced increases in the extracellular levels of dopamine associated to decreases in the extracellular levels of its metabolites dihydroxyphenylacetate and homovanillate from rat striatum. These changes seem to be related according to a time sequence, indicating a possible effect on the metabolism of dopamine. Changes were also observed in locomotor activity (cycling behavior, sniffing around and chewing) in rats during the domoate infusion. The physiological mechanism by which domoate increased dopamine release remains to be worked out.     

Utilization of High-Density Lipoprotein Sphingomyelin by the Developing and Mature Brain in the Rat

Utilization of very long chain saturated fatty acids by brain was studied by injecting 20-day-old and adult rats with high-density lipoprotein containing [stearic or lignoceric acid-14C, (methyl-3H)choline]sphingomyelin. Labeling was followed for 24 h. Very small amounts of 14C were recovered in the brain of all rats, and there was no preferential uptake of lignoceric acid. Approximately 20% of the entrapped 14C was located in the form of unchanged sphingomyelin 24 h after injection. This result shows that the rat brain utilizes very little very long chain fatty acids (greater than or equal to 20 C atoms) from high-density lipoprotein sphingomyelin, even during the myelinating period. The [3H]choline moiety from sphingomyelin was recovered in brain phosphatidylcholine in a higher proportion in comparison with the 14C uptake. The brain 3H increased throughout the studied period in all experiments, but was much higher in the myelinating brain than in the mature brain. From the radioactivity distribution in liver and plasma lipids, it is clear that the choline 3H in the brain originates from either double-labeled phosphatidylcholine of lipoproteins or tritiated lysophosphatidylcholine bound to albumin, both synthesized by the liver.     

Human Mas-Related G Protein-Coupled Receptors-X1 Induce Chemokine Receptor 2 Expression in Rat Dorsal Root Ganglia Neurons and Release of Chemokine Ligand 2 from the Human LAD-2 Mast Cell Line

Primate-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are highly enriched in dorsal root ganglia (DRG) neurons and induce acute pain. Herein, we analyzed effects of MRGPR-X1 on serum response factors (SRF) or nuclear factors of activated T cells (NFAT), which control expression of various markers of chronic pain. Using HEK293, DRG neuron-derived F11 cells and cultured rat DRG neurons recombinantly expressing human MRGPR-X1, we found activation of a SRF reporter gene construct and induction of the early growth response protein-1 via extracellular signal-regulated kinases-1/2 known to play a significant role in the development of inflammatory pain. Furthermore, we observed MRGPR-X1-induced up-regulation of the chemokine receptor 2 (CCR2) via NFAT, which is considered as a key event in the onset of neuropathic pain and, so far, has not yet been described for any endogenous neuropeptide. Up-regulation of CCR2 is often associated with increased release of its endogenous agonist chemokine ligand 2 (CCL2). We also found MRGPR-X1-promoted release of CCL2 in a human connective tissue mast cell line endogenously expressing MRGPR-X1. Thus, we provide first evidence to suggest that MRGPR-X1 induce expression of chronic pain markers in DRG neurons and propose a so far unidentified signaling circuit that enhances chemokine signaling by acting on two distinct yet functionally co-operating cell types. Given the important role of chemokine signaling in pain chronification, we propose that interruption of this signaling circuit might be a promising new strategy to alleviate chemokine-promoted pain.     

Exposure to Ebselen Changes Glutamate Uptake and Release by Rat Brain Synaptosomes

We investigated effects of Ebselen, diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ on [³H]glutamate uptake and release by brain synaptosomes. Ebselen after acute exposure inhibited K⁺-stimulated [³H]glutamate release by brain synaptosomes. (PhSe)₂ and (PhTe)₂ did not change [³H]glutamate release by brain synaptosomes. Ebselen, (PhSe)₂ and (PhTe)₂ had no significantly effects on [³H]glutamate uptake after acute exposure. In vitro, Ebselen (100 M) inhibited [³H]glutamate release and uptake. (PhSe)₂ had no significant effect, while (PhTe)₂ (100 M) inhibited [³H]glutamate uptake by brain synaptosomes. In vitro, (PhSe)₂, (PhTe)₂ and Ebselen caused a significant inhibition of [³H]glutamate uptake by brain synaptic vesicles in vitro. The results demonstrated that organochalcogenides have a rather complex effect on glutamate homeostasis depending on the compound and the schedule of exposition. We propose that the neuroprotective action of Ebselen can be related, in addition to its glutathione peroxidase-like and antilipoperoxidative activity, to a direct interaction with the glutamatergic system by reducing K^ï-evoked glutamate release.     

Mast cell survival and activation by IgE in the absence of antigen: a consideration of the biologic mechanisms and relevance

Mast cells are not only major effector cells in allergy and host defense against parasites and bacteria but also important cellular components in other immune responses. Recent studies on the effects of monomeric IgE on mast cell survival and activation have made an impact on our view of the IgE binding to its high-affinity receptors, Fc epsilonRI. Traditionally, IgE binding to Fc epsilonRI has been considered as a passive action of "sensitization" before receptor aggregation by Ag. However, recent studies indicate that at high concentrations some monoclonal IgEs have effects on mast cells similar to or identical to those induced by IgE+Ag stimulation. These effects may be due to induction of Fc epsilonRI aggregation by these IgEs in the absence of Ag. This review will synthesize recent findings of the heterogeneity of IgEs in their ability to induce survival and activation events, their mechanisms, the potential in vivo significance of IgE-Fc epsilonRI interactions, and the implications of the mouse studies to human diseases.     

Uptake and Calcium-Dependent Release of Ethylenediamine (1,2-Diaminoethane) by Rat Brain Slices

Abstract: The uptake of [¹⁴C]ethylenediamine into slices of rat brain and its subsequent evoked release have been studied. An active uptake process was demonstrated by comparing uptake at 37 and 4°C. This uptake showed a K_m of 1.36 m M , was partly sodium-dependent and was reduced by nipecotic acid. Release could be readily evoked by 30 m M potassium, and by electrical stimulation, the release in both cases being calcium-dependent. In view of these findings and the reported interactions of ethylenediamine with γ-aminobutyric acid-related mechanisms, it might be of interest to determine whether this simple diamine occurs endogenously in the mammalian brain.     

rMCP-2, the Major Rat Mucosal Mast Cell Protease,an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins

Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2, -3 and 4. rMCP-2, the most abundant of these enzymes, has been shown to increase the permeability of the intestinal epithelium, most likely by cleavage of cell adhesion and junction proteins and thereby play a role in intestinal parasite clearance. However, no target for this effect has yet been identified. To address this question we here present its extended cleavage specificity. Phage display analysis showed that it is a chymase with a specificity similar to the corresponding enzyme in mice, mMCP-1, with a preference for Phe or Tyr in the P1 position, and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junction molecules. To see if these proteins were also susceptible to cleavage in their native conformation we cleaved 5 different recombinant cell adhesion and cell junction proteins. Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.     

Adenosine Formation and Release by Embryonic Chick Neurons and Glia in Cell Culture

Adenosine formation and release were studied in 48-h-old cultured ciliary ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxyglucose and 2 micrograms/ml oligomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15-40% of the fall in ATP. Dilazep (3 X 10(-6) M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80-90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of alpha, beta-methylene ADP that inhibited ecto-5'-nucleotidase by 80-90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto-5'-nucleotidase was excluded.     

J-Shaped Dose-Response Relationship for Tumor Induction by Caffeic Acid in the Rat Forestomach,Modeled by Non-Monotonic Dose Response for DNA Damage and Cell Proliferation

Dose-response relationships for chemical tumor induction can be described with stochastic carcinogenesis models by incorporation of dose-dependent parameters. We used a two-stage mutational model with clonal expansion of intermediate cells to mimic the observed U-shaped (more precisely: J-shaped) dose response for cell division in the forestomach of rats treated with caffeic acid. The question was whether biologically acceptable model parameters for the rates of mutation and cell turnover could be found to fit the experimental data, including tumor incidence in controls and at high dose. Using rates of cell turnover in proportion to the experimental labeling indices for DNA replication, model parameters could fit the tumor incidences within the 95% confidence limits of the experimental data, and the J-shape was retained quantitatively. However, when the smaller confidence limits derived from historical data on the spontaneous forestomach tumor incidence were taken into account, the original model could no longer accommodate all data and had to be expanded. For that, mutation rates were considered to be proportional to both the level of DNA damage and the rate of cell division, and both factors were allowed to be dose dependent. For the DNA damage, an increment over background was used at the higher, cytotoxic dose levels. This model improvement resulted in biologically acceptable parameters to fit all available data, demonstrating its usefulness for further analyses of hormetic dose response.     

ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes

Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.     

Intranasal Administration of Lentiviral miR-135a Regulates Mast Cell and Allergen-Induced Inflammation by Targeting GATA-3

Mast cell (MC) degranulation is the foundation of the acute phase of allergic rhinitis (AR). Previously, downregulation of GATA binding protein 3 (GATA-3) was shown to suppress MC activation in an AR mouse model. Binding of microRNA-135a (miR-135a) to GATA-3 was also observed, and overexpression of this miRNA decreased GATA-3 mRNA and protein expression. However, the effects of miR-135a on MCs during AR are currently unknown. In the present study, we utilized a lentiviral (LV) vector to intranasally administer miR-135a to ovalbumin (OVA)-sensitized AR mice. Following miR-135a treatment, the total serum IgE concentration observed during AR was significantly reduced. In the nasal mucosa, the expression of T-box expressed in T cells (T-bet) was higher, whereas that of GATA-3 was lower in the AR mice following miRNA treatment. Notably, during AR, the ratio of type 1 T-helper cells (Th1) to type 2 (Th2) cells in the spleen is unbalanced, favoring Th2. However, administering miR-135a to the AR mice appeared to balance this ratio by increasing and decreasing the percentage of Th1 and Th2 cells, respectively. MiR-135a also appeared to strongly suppress the infiltration of eosinophils and MCs into the nasal mucosa, and it was specifically localized in the MCs, suggesting that its influence is modulated through regulation of GATA-3 in these cells. Additional work identifying the full therapeutic potential of miR-135a in the treatment of AR and diseases involving allergen-induced inflammation is warranted.