Kefiran suppresses antigen-induced mast cell activation

Kefir is a traditional fermented milk beverage produced by kefir grains in the Caucasian countries. Kefiran produced by Lactobacillus kefiranofaciens in kefir grains is an exopolysaccharide having a repeating structure with glucose and galactose residues in the chain sequence and has been suggested to exert many health-promoting effects such as immunomodulatory, hypotensive, hypocholesterolemic activities. Here we investigated the effects of kefiran on mast cell activation induced by antigen. Pretreatment with kefiran significantly inhibited antigen-induced Ca(2+) mobilization, degranulation, and tumor necrosis factor-α production in bone marrow-derived mast cells (BMMCs) in a dose-dependent manner. The phosphorylation of Akt, glycogen synthase kinase 3β, and extracellular signal-regulated kinases (ERKs) after antigen stimulation was also suppressed by pretreatment of BMMCs with kefiran. These findings indicate that kefiran suppresses mast cell degranulation and cytokine production by inhibiting the Akt and ERKs pathways, suggesting an anti-inflammatory effect for kefiran.     

Role of serine esterases in mast cell activation

1. A variety of chymotryptic substrates and inhibitors prevented the release of histamine and prostaglandin D₂ from rat peritoneal mast cells stimulated with anti-IgE but not the calcium ionophore A23187 or a variety of polyamines.
2. The activity of the compounds was strikingly increased in cells reversibly permeabilized with ATP, indicating the importance of their effective incorporation into the cytosol.
3. The compounds produced a comparable inhibition of immunological, but not pharmacological, histamine release from human mast cells and basophils.
4. Treatment of rat mast cells with anti-IgE led to a marked increase in the total chymotryptic activity expressed by the cells.
5. Immunological, but not pharmacological, stimulation of permeabilized rat mast cells loaded with a fluorescent chymotryptic substrate led to a pronounced and rapid increase in fluorescence, indicating activation of the enzyme and hydrolysis of the substrate. These changes were attenuated by chymotryptic inhibitors.
6. In total, these data provide compelling evidence for the direct involvement of a serine protease in IgE-mediated histamine release from mast cells.

     

Marine brevetoxin induces IgE-independent mast cell activation

Brevetoxins (PbTx) are sodium channel neurotoxins produced by the marine dinoflagellate Karenia brevis during red tide blooms. Inhalation of PbTx in normal individuals and individuals with pre-existing airways disease results in adverse airway symptoms including bronchoconstriction. In animal models of allergic inflammation, inhalation of PbTx results in a histamine H₁-mediated bronchoconstriction suggestive of mast cell activation. How mast cells would respond directly to PbTx is unknown. We thus explored the activation of mouse bone marrow–derived mast cells (BMMCs) following exposure to purified PbTx-2. Following in vitro exposure to PbTx-2, we examined cellular viability, mast cell degranulation (β-hexosaminidase release), intracellular Ca²⁺ and Na⁺ flux, and the production of inflammatory mediators (IL-6). PbTx-2 induced significant cellular toxicity within 24 h as measured by LDH release and Annexin-V staining. However, within 1 h of exposure, PbTx-2 induced BMMC degranulation and an increase in IL-6 mRNA expression independent of the high-affinity IgE receptor (FcεRI) stimulation. Activation of BMMCs by PbTx-2 was associated with altered intracellular Ca²⁺ and Na⁺ levels. Brevenal, a naturally produced compound that antagonizes the activity of PbTx, prevented changes in intracellular Na⁺ levels but did not alter activation of BMMCs by PbTx-2. These findings demonstrate that PbTx-2 activates mast cells independent of FcεRI providing insight into critical events in the pathogenesis and a potential therapeutic target in brevetoxin-induced airway symptoms.     

Effects of tandospirone on second messenger systems and neurotransmitter release in the rat brain

• 1.1. We studied the effects of tandospirone, a novel serotonin (5-HT)_1A receptor-related anxiolytic, on the intracellular second messenger systems and neurotransmitter release.
• 2.2. Tandospirone inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes by activation of 5-HT_1A receptors and had high efficacy comparable to 5-HT_1A receptor agonists such as 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT).
• 3.3. Tandospirone suppressed carbachol-stimulated phosphatidyl-inositol metabolism (PI response), which was shown to be a 5-HT_1A receptor-mediated event.
• 4.4. Tandospirone did not affect the release of 5-HT, norepinephrine (NE), dopamine (DA) and acetylcholine (ACh) from rat brain slice preparations.
• 5.5. These findings suggested that tandospirone shows high agonistic efficacy on the postsynaptic 5-HT_1A receptors but does not affect the presynaptic autoreceptors located on nerve endings. The modulation of the second messenger system via postsynaptic 5-HT_1A receptors might be involved in the anxiolytic efficacy of tandospirone.

     

Studies of phosphorylation in rat mast cells (second report)--phosphatidylinositol kinase in rat mast cell granule membranes

Granules were isolated from sonicated purified rat mast cells on a Percoll gradient. The granules were shown to contain a highly active phosphatidylinositol kinase which catalyzes the formation of diphosphoinositide (DPI) from endogenous phosphatidylinositol in the granule membrane. The enzyme requires ATP and Mg2+ or Mn2+ for activity. DPI formation is almost completely dependent on MgCl2 or MnCl2, and maximal response was observed at 20 mM or 2 mM, respectively. The effects of the divalent cations are competitive. Ca2+, fluoride and cyclic AMP are inhibitory. The Km for ATP is 25 microM. The initial reaction is rapid, but the response ceases within a few min. The maximal response is seen at 28 degrees C.     

Bradykinin analogs antagonize bradykinin-induced second messenger production in a sensory neuron cell line

Bradykinin is the prime initiator of pain and the key initial activator of the inflammatory response at the site of tissue injury. The subsequent transfer of nociceptive information (pain sensation) into the central nervous system is then mediated via afferent type C dorsal root ganglion neurons. A recently developed hybrid cell line, F-11, shows many qualities characteristic of these pain-sensitive cells. In these neuronal hybrids, we have found that bradykinin induces sequential elevation in the concentrations of several second messengers involved in neuronal activation, including inositol trisphosphate (6.5-fold), intracellular calcium (2.7-fold), and cyclic GMP (20.5-fold). Importantly, the production of these second messengers is potently inhibited by several novel bradykinin antagonists that possess no intrinsic agonist activity. The same relative rank order of potency of inhibition of bradykinin-induced second messenger production was achieved in the inositol trisphosphate, calcium, and cyclic GMP assay systems, suggesting strongly that all three messenger systems are being activated by the same bradykinin receptor. The most potent antagonist was D-Arg0-Hyp3-Thi5,8-D-Phe7-bradykinin, which inhibited in a competitive manner, with pA2 values, upon Schild plot analysis, in the nanomolar range. These potent bradykinin antagonists may be useful in the characterization of bradykinin receptors and in the clinical management of pain and inflammation.     

Peptidergic pathway in human skin and rat peritoneal mast cell activation

The common pathway of heterogenous mast cell activation as mediated by antigens is through the cross-linking of IgE bound to FcϵRI receptors. The peptidergic pathway of mast cell activation, achieved by cationic secretagogues, is restricted to “serosal” mast cells, the experimental models being rat peritoneal and human skin mast cells. Cationic secretagogues include positively charged peptides but also various amines such as compound 48/80 and natural polyamines. An early intracellular event of this pathway is the activation of pertussis toxin-sensitive G proteins. The correlation observed between the ability of basic compounds to trigger mast cell exocytosis and their potency to activate purified G proteins strongly suggests that cationic compounds activate mast cell G proteins via a receptor-independent but membrane-assisted process. In this paper, alternative mechanisms are discussed. The consequence of G protein stimulation is the activation of phospholipase C with an increase in inositol triphosphates. Natural polyamines are relatively poor triggers of mast cells (10⁻⁴ to 10⁻² M). Neuropeptides such as substance P, neuropeptide Y or vasoactive intestinal peptide, peptidic hormones such as kinins, and venoms such as mastoparan and mast cell degranulating peptide, are all active in a concentration range from 10⁻⁷ to 10⁻⁴ M. The cationic anaphylatoxin C3a also stimulates mast cells at concentrations below precursor complement C3 blood levels. The component C3 of the complement system is one of only a few plasma proteins having activation fragments (i.e. C3a) that can be generated at micromolar levels. The effects of basic secretagogues defines a peptidergic pathway of mast cell activation, which represents a potentially toxic process considering the tissue effects caused by exogenous basic compounds such as venom peptides and certain amine containing drugs. Peptidergic activation of mast cells may also be a pathophysiological process having an important role in neurogenic inflammation and in diseases involving extensive activation of the blood complement cascade.     

Inhibition of anaphylaxis like reaction and mast cell activation by Sitagliptin

Mast cells stimulation activates degranulation process resulting in releasing of mediators, such as histamine. In this study, the effect of aqueous extract of sitagliptin, a selective dipeptidylpeptidase-4 inhibitor, on the mast cell-mediated allergic response was studied with the possible mechanisms of action, focusing on the histamine release and pro-inflammatory cytokine secretion in mast cells. Sitagliptin produced dose dependent inhibition in compound 48/80-induced systemic reactions. In addition, sitagliptin attenuated IgE-mediated skin allergic reaction. Sitagliptin dose-dependently reduced compound 48/80- and IgE-induced histamine release from mast cells. Sitagliptin decreased the secretion of pro-inflammatory cytokines, tumor necrosis factor-α, in mast cells. So, the finding of this study provides evidence that sitagliptin inhibits mast cell derived allergic reactions, and involvement of pro-inflammatory cytokine secretion in such effects.     

An antiallergic activity of disodium cromoglycate unrelated to mast cell activation

The pharmacological effects of disodium cromoglycate (DSCG) were studied in rats during the development of reactions to various allergens or carrageenin. DSCG (10 mg/kg and 100 mg/kg, i.v.) showed pronounced inhibitory effects on type I and type III (passive Arthus) allergic reactions. An immunological degranulation of mast cells and a significant decrease in tissue histamine content were observed in type I allergic reactions but not in type III allergic reactions characterized by an apparent infiltration of neutrophils. An antihistaminic agent, promethazine (1 mg/kg, i.v.) was effective only against type I allergic reactions and totally ineffective against type III allergic reactions. Thus, the results obtained above strongly suggest that DSCG exhibits at least two mechanisms of antiallergic action; one is related to mediator release from mast cells and the other is unrelated to mast cell activation.     

Alterations in receptor-coupled second messenger systems at up-regulated muscarinic receptors: analysis using primary cultured neurons

The effect of a long-term exposure (5 days) to atropine on muscarinic acetylcholine receptors and receptor-coupled second messenger systems was investigated using mouse cerebral cortical neurons in primary culture. The long-term exposure of neurons to atropine (10 nM) induced increases in both the Bmax and Kd values of [3H]quinuclidinyl benzilate (QNB) binding to muscarinic acetylcholine receptors. Alterations in muscarinic receptor-coupled second messenger systems, such as phosphoinositide (PI) hydrolysis and cyclic GMP (cGMP) formation following a long-term exposure to atropine, were also examined. Carbachol-stimulated PI hydrolysis was found to be decreased by the exposure to atropine in spite of the increase of muscarinic receptors. In addition, a long-term exposure to atropine had no effect on carbachol-stimulated cGMP formation as well as on the rightward shift of the carbachol competition curve of [3H]QNB binding in the presence of GTP. These results suggest that the up-regulation in muscarinic cholinergic receptors induced by long-term exposure to atropine may involve not only the increase in number of muscarinic receptors but also the decreased responsiveness in muscarinic receptor-coupled second messenger systems.     

Luteolin inhibits myelin basic protein-induced human mast cell activation and mast cell-dependent stimulation of Jurkat T cells

Background and purpose:

Allergic inflammation and autoimmune diseases, such as atopic dermatitis, psoriasis and multiple sclerosis (MS), involve both mast cell and T-cell activation. However, possible interactions between the two and the mechanism of such activations are largely unknown.

Experimental approach:

Human umbilical cord blood-derived cultured mast cells (hCBMCs) and Jurkat T cells were incubated separately or together, following activation with myelin basic protein (MBP), as well as with or without pretreatment with the flavonoid luteolin for 15 min. The supernatant fluid was assayed for inflammatory mediators released from mast cells and interleukin (IL)-2 release from Jurkat cells.

Key results:

MBP (10 μM) stimulates hCBMCs to release IL-6, IL-8, transforming growth factor (TGF)-β1, tumour necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), histamine and tryptase (n=6, P<0.05). Addition of mast cells to Jurkat cells activated by anti-CD3/anti-CD28 increases IL-2 release by 30-fold (n=3, P<0.05). MBP-stimulated mast cells and their supernatant fluid further increase Jurkat cell IL-2 release (n=3, P<0.05). Separation of mast cells and activated Jurkat cells by a Transwell permeable membrane inhibits Jurkat cell stimulation by 60%. Pretreatment of Jurkat cells with a TNF-neutralizing antibody reduces IL-2 release by another 40%. Luteolin pretreatment inhibits mast cell activation (n=3–6, P<0.05), Jurkat cell activation and mast cell-dependent Jurkat cell stimulation (n=3, P<0.05).

Conclusions and implications:

Mast cells can stimulate activated Jurkat cells. This interaction is inhibited by luteolin, suggesting that this flavonoid may be useful in the treatment of autoimmune diseases.     

Alterations of second messenger systems in the rat brain after 6-hydroxydopamine lesions of the medial forebrain bundle

We studied the sequential changes in second messenger systems in the striatum and substantia nigra (SN) after 6-hydroxydopamine lesions of the medial forebrain bundle in rats. The animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4 and 8 weeks postlesion. [³H]Phorbol-12,13-dibutyrate (PDBu), [³H]forskolin and [³H]rolipram were used to label protein kinase C (PKC), adenylyl cyclase and calcium/calmodulin-independent cyclic-AMP phosphodiesterase, respectively. The degeneration of nigrostriatal pathway produced a significant increase in [³H]PDBu binding in the ventromedial part of the ipsilateral striatum from 2 to 8 weeks postlesion. In the contralateral side, [³H]PDBu binding showed a transient increase in the SN only 4 weeks after lesioning. [³H]Forskolin binding showed a significant increase in the ipsilateral and contralateral striatum from 2 to 4 weeks postlesion. In the ipsilateral SN, a significant increase in [³H]forskolin binding was observed at 4 weeks after lesioning. However, no significant change in [³H]forskolin binding was observed in the contralateral SN during postlesion. On the other hand, [³H]rolipram binding showed no conspicuous alteration in the brain during postlesion. These results demonstrate that rats made hemiparkinsonism by unilateral 6-hydroxydopamine injection have a significant increase in [³H]PDBu and [³H]forskolin binding in the striatum and/or SN, whereas no significant change in [³H]rolipram binding is observed in these areas during postlesion. Our findings also suggest that the increase in [³H]forskolin binding is more pronounced than that in [³H]PDBu binding in the brain after unilateral 6-hydroxydopamine injection. Thus, our studies may provide valuable information concerning degeneration of the nigrostriatal pathway such as Parkinson’s disease.     

Confocal laser scanning microscopy to study molecular mechanism of mast cell activation

In the immune system, mast cells are a key cell type in the pathogenesis of immunoglobulin E (IgE)-dependent hypersensitivity reactions. Engagement of the high-affinity IgE receptors by multivalent antigens initiates the downstream activation of signal-transducing enzymes and evokes degranulation and cytokine production via an increase in the intracellular Ca2+ concentration. In addition, mast cells also play a prominent role in non-IgE-mediated hypersensitivity reactions. Mast cells are closely apposed to nerves in vivo and are likely to be regulated functionally by nerves. However, the molecular mechanisms for mast cell activation in an IgE-dependent and -independent manner have not been fully clarified. Confocal laser scanning microscopy has played an essential role in cell biology by allowing visualization of specific intracellular signaling molecules with high spatiotemporal resolution in living cells. We have studied intracellular movements of Ca2+ using a specific fluorescent probe and several types of signaling molecules using derivatives of green fluorescent protein in a living single mast cell using a microscopic strategy. We here describe our imaging analysis of the calcium signals to the nucleus, the movement of secretory granules in the degranulation process, and the nucleocytoplasmic shuttling of mitogen-activated protein kinase in mast cells. Further, we demonstrate that direct communication between mast cells and nerves occurs. These findings provide useful information from a new perspective to understand the molecular mechanisms of allergic reaction and inflammation.     

Zaltoprofen inhibits bradykinin-induced responses by blocking the activation of second messenger signaling cascades in rat dorsal root ganglion cells

Bradykinin interacts with the bradykinin B2 receptor on dorsal root ganglion (DRG) neurons, setting off a series of reactions inside the cells that ultimately make the vanilloid receptor 1 more sensitive to a normal stimulus by activating various enzymes coupled with second messenger signaling cascades. Zaltoprofen, a propionic acid derivative non-steroidal anti-inflammatory drug (NSAID), was proved to inhibit bradykinin-induced pain responses in vivo experimental systems more potently than indomethacin or other NSAIDs, but the molecular mechanisms underlying its action are not yet fully understood. Currently it appears unlikely that zaltoprofen binds to specific sites on the protein of the bradykinin B2 receptor, hence we have examined the effect of zaltoprofen on bradykinin-induced responses of adult DRG neurons to investigate possible interaction sites. Compared with several other NSAIDs, such as indomethacin, loxoprofen and diclofenac, zaltoprofen most potently inhibits bradykinin-enhancement of capsaicin-induced 45Ca2+ uptake into DRG neurons. Zaltoprofen also significantly inhibits bradykinin-induced 12-lipoxygenase (12-LOX) activity and the slow bradykinin-induced onset of substance P release from DRG neurons. These data indicate zaltoprofen may produce its analgesic effects through the inhibition of bradykinin B2 receptor-mediated bradykinin responses of not only cyclooxygenases (COXs) but also bradykinin induced 12-LOX inhibitors.