Suppression of Brain Mast Cells Degranulation Inhibits Microglial Activation and Central Nervous System Inflammation

Brain inflammation has a critical role in the pathophysiology of brain diseases. Microglia, the resident immune cells in the brain, play an important role in brain inflammation, while brain mast cells are the “first responder” in the injury rather than microglia. Functional aspects of mast cell-microglia interactions remain poorly understood. Our results demonstrated that site-directed injection of the “mast cell degranulator” compound 48/80 (C48/80) in the hypothalamus induced mast cell degranulation, microglial activation, and inflammatory factor production, which initiated the acute brain inflammatory response. “Mast cell stabilizer” disodium cromoglycate (cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced microglial activation, inhibition of MAPK and AKT pathways, and repression of protein expression of histamine receptor 1 (H₁R), histamine receptor 4 (H₄R), protease-activated receptor 2 (PAR2), and toll-like receptor 4 (TLR4) in microglia. We also demonstrated that C48/80 had no effect on microglial activation in mast cell-deficient Kit^W-sh/W-sh mice. These results implicate that activated brain mast cells trigger microglial activation and stabilization of mast cell inhibits microglial activation-induced central nervous system (CNS) inflammation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.     

Application of laser interference microscopy (LIM) for investigation of the protective effect of prolyl-glycyl-proline (Pro-Gly-Pro) on mast cells

The effect of peptide prolyl-glycyl-proline (Pro-Gly-Pro) on morphometric parameters of mast cells upon their activation by compound 48/80 or synacten was investigated. Cell image, obtained by the method of laser interference microscopy (LIM), is a distribution of the optical path difference of light (OPD). It evaluates the changes of the individual components of cytoplasm (maxOPD) and the total distribution of OPD (“dry mass”). The changes of “dry mass” in cytoplasm correlate with the changes of the secreted histamine amount (?0.86). Preliminary incubation of mast cells with Pro-Gly-Pro (6 × 10^?5 M) did not change the area, the state of the individual components of the cytoplasm (nucleus) and “dry mass” (histamine vesicles) in cells. The “dry mass” (histamine vesicles) and maxOPD decreased while the release of histamine increased upon the activation of mast cells by compound 48/80 (0.02 mg/mL). Preincubation of cells with Pro-Gly-Pro had no effect. Activation of cells by synacten (2 and 20 μM) led to the increase of the cell area and the reduction of maxOPD and “dry mass” (histamine vesicles). Preincubation of the cells with Pro-Gly-Pro prevented these changes. So, the protective effect of Pro-Gly-Pro was observed in the case of the activation of mast cells by synacten but not by compound 48/80.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using H NMR

¹H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events folowing exocytosis, activate glycolysis.     

Activation and inhibition of mast cells degranulation affect their morphometric parameters

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.     

Simultaneous detection of histamine release and lactate production in rat mast cells induced by compound 48/80 using 1H NMR.

1H NMR spectroscopy was used to evaluate histamine release and lactate production in intact mast cells isolated from rats. The resonance lines of the aromatic histamine protons in mast cells, detected by the selective spin-excitation technique, were broader and located in a lower magnetic field than those in free histamine solution. When exocytosis of mast-cell granules was induced by compound 48/80, free histamine appeared, with a corresponding decrease in the amount of histamine in the mast cells; the lactate signal was also detected in the spectrum. On the addition of compound 48/80, there was a further release of histamine from mast cells, accompanied by further production of lactate. This result indicates that the mechanisms which induce the exocytosis of granules, and/or the events following exocytosis, activate glycolysis.     

Histamine release from mouse and rat mast cells cultured with supernatants from chronic murine graft-vs-host splenocytes.

There is growing interest in studying pathways of mast cell activation. In a mouse model of chronic graft-vs-host disease (cGVHD) extensive mast cell activation and degranulation occurs in vivo coincident with the development of dermal fibrosis. An interesting feature of this model is that the mast cell reaction is slow to develop, occurring over a period of weeks and waning by 300 days. The aim of our work was to investigate the effects of supernatants from splenocytes of such cGVHD mice (cGVHD sups) on mouse and rat peritoneal mast cells cocultured with 3T3 skin fibroblasts. We found that cGVHD sups are able to release histamine from both mouse and rat cultured mast cells in a slow fashion. Histamine release became evident only after 5-8 days of coculture of the mast cells with the cGVHD supernatants and thereafter decreased to basal levels. Mast cell activation due to cGVHD supernatants was a noncytotoxic event as demonstrated by mast cell counts in the cocultures and by the ability of mast cells to exclude trypan blue. Mast cells that had been activated by incubation with the cGVHD sups were as responsive to stimulation with either anti-IgE antibodies or compound 48/80 as were mast cells incubated with control sups. Supernatants from mice early in GVHD (Days 11-28) were most active in promoting histamine release. Supernatants from spleens of mice which had GVHD for 290 days and where the mast cells had returned to full granulation in vivo were inactive. This is the first in vitro study demonstrating slow mast cell histamine release instituted by other cells, namely the splenocytes of cGVHD mice.     

Histamine release by pharmacological agents in the absence of external free Ca2+

The involvement of extracellular free Ca2+ in histamine release was investigated in rat peritoneal mast cells. Incubation of non-antigenized cells in a media with high extracellular potassium did not increase histamine release. Secretion induced by A23187 and compound 48/80 in the presence of Ca2+ requires metabolic energy. In the absence of external free Ca2+ (2.5 microM) histamine release induced by A23187 is reduced but not abolished. Secretion induced by compound 48/80 is independent of extracellular Ca2+. These results lead us to suggest that mast cell plasma membranes probably lack voltage-gated Ca2+ channels and that external Ca2+ may not be an absolute requisite for histamine secretion.     

Pulsed Low-Frequency Magnetic Fields Induce Tumor Membrane Disruption and Altered Cell Viability

Tumor cells express a unique cell surface glycocalyx with upregulation of sulfated glycosaminoglycans and charged glycoproteins. Little is known about how electromagnetic fields interact with this layer, particularly with regard to harnessing unique properties for therapeutic benefit. We applied a pulsed 20-millitesla (mT) magnetic field with rate of rise (dB/dt) in the msec range to cultured tumor cells to assess whether this affects membrane integrity as measured using cytolytic assays. A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure. Heparinase treatment, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. We further examined a non-neoplastic human primary cell line (lung lymphatic endothelial cells) as a typical normal host cell from the lung cancer microenvironment and found no effect of field exposure on membrane integrity. The field exposure was also sufficient to alter proliferation of tumor cells in culture, but not that of normal lymphatic cells. Pulsed magnetic field exposure of human breast cancer cells that express a sialic-acid rich glycocalyx also induced protease release, and this was partially abrogated by sialidase pretreatment, which removes cell surface anionic sialic acid. Scanning electron microscopy showed that field exposure may induce unique membrane “rippling” along with nanoscale pores on A549 cells. These effects were caused by a short exposure to pulsed 20-mT magnetic fields, and future work may examine greater magnitude effects. The proof of concept herein points to a mechanistic basis for possible applications of pulsed magnetic fields in novel anticancer strategies.     

Influence of a macrolide antibiotic, roxithromycin, on mast cell growth and activation in vitro

BACKGROUND: Long-term administration of macrolide antibiotics is recognized to be able to favorably modify the clinical condition of inflammatory diseases, such as diffuse panbronchiolitis and cystic fibrosis. However, the precise mechanisms by which macrolide antibiotics could improve clinical conditions of the patients are not well understood. AIM: The present study was designed to examine the influence of macrolide antibiotics on effector cell functions responsible for inflammation through the choice of roxithromycin (RXM) and mast cell. METHODS: Mast cells were induced by long-term culture of splenocytes from BALB/c mice. RXM was added to the cultures at seeding and then every 4-5 days, when the culture medium was replaced with a fresh one. The influence of RXM on mast cell growth was evaluated by counting the number of cells grown on the 16th day. We also examined the influence of RXM on mast cell activation by examining histamine release and inflammatory cytokine secretion. RESULTS AND CONCLUSION: RXM could not inhibit mast cell growth, even when splenocytes were exposed to 100 microg/ml of RXM throughout the entire culture periods. RXM also could not suppress histamine release from cultured mast cells in response to non-immunological and immunological stimulations. However, RXM could suppress inflammatory cytokine, interleukin-1beta, interleukin-6, granulocyte macrophage-colony stimulating factor and tumor necrosis factor-alpha, secretions induced by concanavalin A stimulation at a concentration of as little as 0.5 microg/ml. These results may suggest that RXM modulated the ability of mast cells to secrete inflammatory cytokines and results in improvement of clinical condition of chronic inflammatory diseases.     

The effect of 2450 MHz microwave radiation on histamine secretion by rat peritoneal mast cells

Isolated rat peritoneal mast cells actively secrete histamine in response to reaginic or chemical stimulation. Mast cells were irradiated in a waveguide microwave exposure chamber at 2450 MHz with power absorptions of 8.2 and 41.0 mW/g for periods up to 3 h. These levels of microwave absorption caused no change in the morphological characteristics or viability of the cells. Irradiated mast cells were stimulated with compound 48/80, a potent, noncytotoxic histamine releasing agent. The dose response curves showed that neither prior nor simultaneous irradiation of mast cells at 37°C affected 48/80-induced secretion. However, microwave power absorptions of 41.0 mW/g inhibited secretion at 44.0°C. Precise measurements of the effect of heat on secretion indicated that this level of inhibition could have been produced by a radiation induced increase in cell temperature between 0.4 and 0.9°C above ambient levels. Alternatively, the heat stress produced at 44°C may have sensitized the cells to the electromagnetic effects of the microwave radiation. Rat peritoneal mast cells can therefore be useful as a model for the study of functioning secretory cells during microwave irradiation and can also be used to monitor the synergistic effects of cell heating during in vitro exposure.     

Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action.

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanistically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inflammatory responses.     

Participation of mast cell 5-hydroxytryptamine in the vasoconstrictor effect of neurotensin in the rat perfused hindquarter

Neurotensin (NT) (1 X 10(-8) - 1.5 X 10(-6) g ml-1) caused a transient, dose-dependent increase in perfusion pressure in the rat perfused hindquarter. The vasoconstrictor effect of NT was associated with a short-lived, dose-dependent release of histamine and 5-hydroxytryptamine (5-HT) in the hindquarter effluent. Compound 48/80, a classical mast cell secretagogue, also elicited a vasoconstrictor effect in, and release of histamine from, the rat hindquarter. The vasoconstrictor effect and the release of histamine and 5-HT evoked by NT were much smaller in hindquarters derived from rats pretreated with compound 48/80 for 4 days to cause mast cell depletion than in hindquarters derived from control rats. The mast cell inhibitor cromoglycate (4 mg ml-1) inhibited by about 50% the histamine releasing effect and vasoconstriction produced by the lowest concentrations of NT utilized. The histamine releasing effect of compound 48/80 was more sensitive to blockade by cromoglycate than that of NT. The steroidal antiinflammatory and antiallergic drug dexamethasone did not affect the histamine and 5-HT releasing effect of NT. The vasoconstrictor effects of NT, compound 48/80 and 5-HT were markedly reduced by the 5-HT receptor antagonist methysergide (1 X 10(-7) g ml-1). Histamine (1 X 10(-6) - 10(-4) g ml-1) evoked a decrease in perfusion pressure in hindquarters pre-exposed to noradrenaline. The results suggest the participation of mast cell 5-HT in the vasoconstrictor effect of NT in the rat perfused hindquarter.     

Morphological and functional characteristics of peritoneal mast cells from young rats

To study why neonatal and young rats are resistant to the effects of some secretagogues, such as compound 48/80 and 2.5-S nerve growth factor, we examined peritoneal mast cells from 14–15-day-old rats (young rats) and compared them to peritoneal mast cells from adults. Peritoneal mast cells from young rats contain approximately one-tenth of the amount of histamine observed in adult peritoneal mast cells. However, both cell populations contained similar low levels of the mucosal mast cell-associated protease rat mast cell protease II. Histochemical analysis of peritoneal mast cells from young rats using safranin O and berberine sulphate suggested that only a portion of the granules of these cells contained heparin. At an ultrastructural level the young rat peritoneal mast cell contains relatively few granules. The majority of mast cells from young rats have a bilobed or indented nucleus which is only rarely observed in adult cells. Functionally, the young rat peritoneal mast cell demonstrates a significantly reduced histamine release in response to the connective tissue mast cellspecific secretagogues compound 48/80 and 2.5-S nerve growth factor. In contrast, the percent histamine release in response to the neurotransmitter substance P, which degranulates both connective tissue mast cells and intestinal mucosal mast cells, was similar in the adult cells and the young rat cells. This study demonstrates substantial differences between the young rat and adult peritoneal mast cells which may explain the ability of very young animals to withstand large doses of certain secretagogues.     

Mast cell degranulating (MCD) peptide: a prototypic peptide in allergy and inflammation.

The solid phase synthesis of mast degranulating peptide (MCD peptide) raised the possibility of preparing analogs and examining the pharmacology and the proposed role of this peptide as a potential agent in allergy and inflammation. MCD peptide, a cationic 22-amino acid residue peptide with two disulfide bridges, causes mast cell degranulation and histamine release at low concentrations and has anti-inflammatory activity at higher concentrations. Because of these unique immunologic properties, MCD peptide may serve as a useful tool for studying secretory mechanisms of inflammatory cells such as mast cells, basophils, and leukocytes, leading to the design of compounds with therapeutic potential.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Observations of Forsythia koreana methanol extract on mast cell-mediated allergic reactions in experimental models

To explore effects of Forsythia koreana methanol extract (FKME) on mast cell-mediated allergic and inflammatory properties, the effect of FKME was evaluated on compound 48/80-induced systemic anaphylaxis, ear swelling, and anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-induced passive cutaneous anaphylaxis (PCA). In addition, the effect of FKME was investigated on the histamine release from rat peritoneal mast cells (RPMCs) stimulated by compound 48/80, which promotes histamine release. The human mast cell line HMC-1 was stimulated by phorbol 12-myristate 13-acetate plus calcium ionophore A23187. Activated HMC-1 can produce several proinflammatory and chemotactic cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8. Cytokine levels in the culture supernatant were measured by an enzyme-linked immunosorbent assay. Cytotoxicity by FKME was determined by a 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. FKME inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling in mice. When 1 g/kg FKME was pretreated or posttreated with mice, compound 48/80-induced mice morality was 50 and 66.7%, respectively. One gram per kilogram of FKME pretreatment inhibited ear-swelling responses derived from compound 48/80 by 29.75%. A PCA reaction was inhibited by 17.9%. In an in vitro model, FKME (1 mg/ml) inhibited histamine release from the RPMCs by 13.8% and TNF-α, IL-6, and IL-8 production from HMC-1 cells by 71.16% (P < 0.001), 86.72% (P < 0.001), and 44.6%, respectively. However, FKME had no cytotoxic effects on cell viability. In conclusion, FKME inhibited not only systemic anaphylaxis and ear swelling induced by compound 48/80 but also inhibited a PCA reaction induced by anti-DNP IgE in vivo. Treatment with FKME showed significant inhibitory effects on histamine, TNF-α, IL-6, and IL-8 release from mast cells.     

Role of histamine in the inhibitory effects of phycocyanin in experimental models of allergic inflammatory response

It has recently been reported that phycocyanin, a biliprotein found in the blue-green microalgae Spirulina, exerts anti-inflammatory effects in some animal models of inflammation. Taking into account these findings, we decided to elucidate whether phycocyanin might exert also inhibitory effects in the induced allergic inflammatory response and on histamine release from isolated rat mast cells. In in vivo experiments, phycocyanin (100, 200 and 300mg/kg post-orally (p.o.)) was administered 1 h before the challenge with 1 microg of ovalbumin (OA) in the ear of mice previously sensitized with OA. One hour later, myeloperoxidase activity and ear edema were assessed. Phycocyanin significantly reduced both parameters. In separate experiments, phycocyanin (100 and 200 mg/kg p.o.) also reduced the blue spot area induced by intradermal injections of histamine, and the histamine releaser compound 48/80 in rat skin. In concordance with the former results, phycocyanin also significantly reduced histamine release induced by compound 48/80 from isolated peritoneal rat mast cells. The inhibitory effects of phycocyanin were dose dependent. Taken together, our results suggest that inhibition of allergic inflammatory response by phycocyanin is mediated, at least in part, by inhibition of histamine release from mast cells.     

Antiallergic effects of Vitis amurensis on mast cell-mediated allergy model

In this study, we investigated the effect of the methanol extract of fruits of Vitis amurensis Rupr. (Vitaceae; MEVA) on the mast cell-mediated allergy model and studied the possible mechanism of action. Mast cell-mediated allergic disease is involved in many diseases, such as asthma and sinusitis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. MEVA inhibited compound 48/80-induced systemic reactions and serum histamine release in a dose-dependent manner in mice. MEVA decreased immunoglobulin E (IgE)-mediated local allergic reactions, passive cutaneous anaphylaxis. MEVA dose-dependently reduced histamine release from mast cells activated by compound 48/80 or IgE. The inhibitory effect of MEVA on histamine release was mediated by the modulation of intracellular calcium. In addition, MEVA attenuated the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated secretion of tumor necrosis factor-alpha, interleukin-6 (IL-6), and IL-8 in human mast cells. The inhibitory effect of MEVA on these proinflammatory cytokines was p38 mitogen-activated protein kinase and nuclear factor-kappaB (NF-kappaB) dependent. Our findings provide evidence that MEVA inhibits mast cell-derived, immediate-type allergic reactions and involvement of proinflammatory cytokines, p38 MAPK, and NF-kappaB in these effects.     

Mast cell degranulation mediates compound 48/80-induced hyperalgesia in mice

Mast cells mediate allergies, hypersensitivities, host defense, and venom neutralization. An area of recent interest is the contribution of mast cells to inflammatory pain. Here we found that specific, local activation of mast cells produced plantar hyperalgesia in mice. Basic secretagogue compound 48/80 induced plantar mast cell degranulation accompanied by thermal hyperalgesia, tissue edema, and neutrophil influx in the hindpaws of ND4 Swiss mice. Blocking mast cell degranulation, neutrophil extravasation, and histamine signaling abrogated these responses. Compound 48/80 also produced edema, pain, and neutrophil influx in WT C57BL/6 but not in genetically mast cell-deficient C57BL/6-Kit(W-sh)(/)(W-sh) mice. These responses were restored following plantar reconstitution with bone marrow-derived cultured mast cells.