Response of bronchial smooth muscle to mast cell degranulation in situ

We studied the response of bronchial smooth muscle to mast cell degranulation with Ascaris suum antigen (AA) and compound 48/80 (48/80) in 26 mongrel dogs in situ. Bronchial smooth muscle response was measured isometrically in situ from a segment of the right middle lobe bronchus; tracheal response was monitored isometrically as a control. After intra-arterial (ia) injection of AA into the bronchial circulation, bronchial contraction preceded tracheal contraction by 19.2 +/- 4.6 s (P less than 0.002). Bronchial contraction to AA (21.7 +/- 3.4 g) was substantially greater than to 48/80 (10.5 +/- 1.8 g, P less than 0.05) corresponding to differences in maximal systemic histamine concentrations (146 +/- 24.1 vs. 1000 +/- 236 ng/ml, P less than 0.01). In 5 dogs, the effect of leukotriene D4 (LTD4) and FPL 55712 was studied. Injection of 10(-8) mol ia LTD4 caused no bronchial contraction. In four dogs, 10(-7) mol FPL 55712 caused no bronchial relaxation after initial precontraction during immune degranulation with AA; intravenous chlorpheniramine (5 mg/kg) caused 69.7 +/- 9.4% relaxation. We demonstrate a model that permits selective immune degranulation of a single major resistance bronchus in situ. We conclude that AA-induced degranulation in dogs caused bronchial contraction predominantly by secretion of preformed mediator.     

Age-induced reprogramming of mast cell degranulation

Mast cell degranulation can initiate an acute inflammatory response and contribute to the progression of chronic diseases. Alteration in the cellular programs that determine the requirement for mast cell degranulation would therefore have the potential to dramatically impact disease severity. Mast cells are exposed to increased levels of PGE2 during inflammation. We show that although PGE2 does not trigger the degranulation of dermal mast cells of young animals, in older mice, PGE2 is a potent mast cell stimulator. Intradermal administration of PGE2 leads to an EP3 receptor-dependent degranulation of mast cells, with the number of degranulated cells approaching levels observed in IgE- and Ag-treated controls. Taken together, these studies suggest that the ability of PGE2 to initiate mast cell degranulation changes in the aging animal. Therefore, elevated PGE2 levels might provide an important pathway by which mast cells are engaged to participate in inflammatory responses in the elderly patient.     

Somatostatin inhibits intestinal mucosal mast cell degranulation in normal conditions and during mast cell hyperplasia

Several studies demonstrate that intestinal mucosal mast cells (IMMC) are modulated by nervous reflexes as well as by intraluminal content. We recently demonstrated that peptones, such as ovalbumin hydrolysate (OVH), induce the release of rat mast cell protease II (RMCP II), indicating IMMC degranulation. The response is due to complex neuroendocrine reflexes. Somatostatin (SS) and its analogues have been used as potential treatments for inflammation in other body systems with contradictory results. The aim of this study was to evaluate if somatostatin could contribute to the reduction of intestinal mucosal mast cell degranulation. Anesthetized rats were prepared for duodenal perfusion and mast cell activation was measured by analysis of RMCP II concentration in the duodenal perfusate. Somatostatin significantly decreased RMCP II concentration in both nonstimulated conditions and after ovalbumin hydrolysate perfusion. However, when somatostatin was given previously to OVH, the peptone still induced a slight increase of RMCP II. Similar effects were observed in animals previously treated with capsaicin. These protocols were repeated in animals infected with Trichinella spiralis, which induces mucosal mast cell hyperplasia. In these cases, somatostatin blocked the effect of OVH, thus, preventing an increase in RMCP II concentration. Fresh frozen tissue sections from the duodenum were processed in an attempt to demonstrate the presence of SS receptors in mast cells using immunofluorescence and Fluo-peptide labeling techniques. Confocal images from duodenum specimens demonstrate the existence of SS receptors in positive cells for RMCP II. Taken together, these results indicate that somatostatin diminishes mast cell activity and in consequence could prevent the intestinal responses to mast cell hyperplasia.     

Role of reactive oxygen species in mast cell degranulation

Mast cells are a heterogeneous multifunctional cellular population that promotes connective tissue homeostasis by slow release of biologically active substances, affecting primarily the permeability of vessels and vascular tone, maintenance of electrolyte and water balance, and composition of the extracellular matrix. Along with this, they can rapidly release inflammatory mediators and chemotactic factors that ensure the mobilization of effector innate immune cells to fight against a variety of pathogens. Furthermore, they play a key role in initiation of allergic reactions. Aggregation of high affinity receptors to IgE (FcεRI) results in rapid degranulation and release of inflammatory mediators. It is known that reactive oxygen species (ROS) participate in intracellular signaling and, in particular, stimulate production of several proinflammatory cytokines that regulate the innate immune response. In this review, we focus on known molecular mechanisms of FcεRI-dependent activation of mast cells and discuss the role of ROS in the regulation of this pathway.     

Gastrin-releasing peptide induces itch-related responses through mast cell degranulation in mice

Gastrin-releasing peptide (GRP), secreted from the central terminals of primary afferents, is involved in the transmission of itch signals in the spinal dorsal horn. Although primary afferents containing GRP are distributed throughout the skin, the role of peripherally released GRP in the itch response is unknown. We investigated whether GRP acts on the skin to induce an itch response in mice. Intradermal injections of GRP(18-27) (1-300 nmol/site) elicited scratching. GRP(18-27)-induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone hydrochloride, the BB(2) bombesin receptor antagonist RC-3095, the H(1) histamine receptor antagonists fexofenadine hydrochloride and chlorpheniramine maleate, and the PAR(2) proteinase-activated receptor antagonist FSLLRY-NH(2). Mast cell deficiency significantly, but not completely, reduced the GRP(18-27)-induced scratching. BB(2) bombesin receptors are present in mast cells in the skin, and intradermal injection of GRP(18-27), not only induced scratching, but also led to mast cell degranulation. GRP(18-27)-induced mast cell degranulation was inhibited by the BB(2) bombesin receptor antagonist RC-3095. These results suggest that peripherally released GRP can induce an itch response, at least partly, through activation of BB(2) receptors present in the mast cells, triggering their degradation and the release of histamine and the serine proteinase, tryptase.     

The initiation of mast cell degranulation: activation at the cell membrane.

The low molecular weight mast cell activator, polymyxin B, has been covalently bound to an insoluble matrix of Sepharose 4B. It has been demonstrated that mast cells in preparations of rat peritoneal cells bind to Sepharose 4B-polymyxin B beads but not to control beads. The bound cells are stimulated to degranulate by this interaction at the cell membrane with the resultant release of biogenic amines.     

Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation

Molecular and Cellular Biochemistry - Calcium ion (Ca2+) uptake into the mitochondrial matrix influences ATP production, Ca2+ homeostasis, and apoptosis regulation. Ca2+ uptake across the...     

Norepinephrine-induced cardiac hypertrophy and fibrosis are not due to mast cell degranulation

The norepinephrine (NE)-induced hypertrophy of the left ventricle (LV) in the rat is preceded by increased interleukin (IL)-6 expression and associated with LV fibrosis. We have examined whether the elevated level of IL-6 may be due to mast cell degranulation. Therefore we tested the effect of cromoglycate sodium salt (cromolyn), an inhibitor of mast cell degranulation with anti-inflammatory and membrane-stabilizing activity, on the increased expression of IL-6 mRNA and of mRNAs of proteins involved in the remodelling of the extracellular matrix (ECM) which is induced by NE (0.1 mg/kg·h). After 4 h, the NE-induced increase in IL-6 mRNA expression was not influenced by cromolyn (20 mg/kg·h). Cromolyn-infusion for 3 days did not affect the extent of LV hypertrophy induced by NE, as measured by the LV weight/body weight (LVW/BW) ratio and by atrial natriuretic peptide (ANP) expression. Cromolyn induced a slight depression of the NE-induced elevation of the matrix metalloproteinase (MMP)-2. However, it did not affect the NE-induced elevated levels of mRNAs of collagen I and III and the tissue inhibitor of matrix metalloproteinase (TIMP)-2. Since cromolyn did not reduce the NE-effects in rat hearts in vivo we conclude that mast cell degranulation seems not to be involved in them.     

Nerve growth factor induces mast cell degranulation without changing intracellular calcium levels

Nerve growth factor (NGF) induces degranulation of rat peritoneal mast cells (RPMC) in a dose-dependent manner, providing direct evidence for its action on non-neuronal tissues. Activation of RPMC by NGF depends on lysophosphatidylserine and extracellular calcium. NGF-mediated RPMC degranulation is not coupled to a transient increase in intracellular free calcium ([Ca2+]i). It is suggested that NGF has a unique mode of action independent of [Ca2+]i and presumably also without involving protein kinase C activation as indicated by the effects of phorbol esters and NGF on antigen-evoked [Ca2+]i rise.     

Endothelin-1 induces mucosal mast cell degranulation in the rat small intestine

The enhanced production of endothelial cell-derived vasoactive mediators and the activation of mast cells (MCs) have been implicated in the pathogenesis of mucosal damage during ischemia and reperfusion injuries. The first objective of our study was to define the in vivo relation between endothelin-1 (ET-1) and the MC system. Secondly, we determined whether pretreatment with ET receptor antagonists would attenuate MC responses to exogenous ET-1. In the first series of experiments, increasing doses of ET-1 (0. 1, 1 and 3 nmol/kg i.v.) were administered to anesthetized rats. In the second series, the animals were pretreated with equimolar doses of the ET-A receptor antagonist BQ-610 or ETR-P1/fl peptide, and the ET-B receptor antagonist IRL-1038. Intestinal perfusion changes and macrohemodynamics were recorded, and the proportion of degranulated MCs was determined in ileal biopsies. The average mucosal thickness was recorded with an image analysis system. ET-1 induced dose-dependent alterations in the hemodynamic and morphological parameters and caused pronounced mucosal injury, with a significant reduction in villus height. The ratio of degranulated MCs was similar in all ET-treated groups (77%, 82% and 86%) to that observed in animals subjected to 15-min ischemia and 60-min reperfusion (85% degranulation). Pretreatment with BQ-610 and ETR-P1/fl peptide attenuated the ET-1 induced alterations in the hemodynamic parameters and decreased structural injury to the mucosa. ET-induced MC degranulation was significantly inhibited by the ET-A receptor antagonists, but not by IRL-1038. These results indicate that elevated levels of circulating ET-1 might induce intestinal mucosal tissue injury and MC degranulation via activation of ET-A receptors, and raise the possibility that ET-A receptor antagonist administration could exert a potentially beneficial effect through a mechanism other than the blockade of vasoconstriction in pathologies associated with an increased ET-1 release.     

Alterations in mast cell degranulation and ovarian histamine in the proestrous hamster

Mast cells in the ovary of cyclic hamsters were observed exclusively in the hilum and in the vicinity of blood vessels that enter and exit the ovary. Ovaries were collected on proestrus from hamsters at 0900 h preluteinizing hormone (LH) surge, 1500 h (peak LH surge), and 2100 h (post-LH surge) and processed for routine histologic staining with toluidine blue. A significant increase in the percentage of extensively degranulating mast cells was observed coincident with the gonadotropin surge (0900 h: 5.39 +/- 0.97%; 1500 h: 20.39 +/- 2.76%). At the peak of the LH surge the ovarian histamine concentration was also significantly higher than those before and after the surge (1500 h: 5.13 +/- 0.94 ng/mg ovary; 0900 h and 2100 h: 2.84 +/- 0.35 and 3.02 +/- 0.48 ng/mg, respectively). The results indicate that a major source of ovarian histamine may be mast cells residing in the ovarian hilum and surrounding the ovarian blood vessels that enter and exit the ovary. In addition, the gonadotropin surge on the day of proestrus may be a trigger for release of mast cell histamine.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation

Introduction

Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.

Methods

HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.

Results

Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G₀/G_i receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A₄ attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.

Conclusions

Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G₀/G_i receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.     

Phosphorylation of SNAP-23 by IkappaB kinase 2 regulates mast cell degranulation

Mast cells are known to play a pivotal role in allergic diseases. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) leads to degranulation and allergic inflammation; however, the regulatory mechanisms of IgE-dependent exocytosis remain unknown. We show here that IkappaB kinase (IKK) 2 in mast cells plays critical roles in IgE-mediated anaphylaxis in vivo, and IgE-mediated degranulation in vitro, in an NF-kB-independent manner. Upon FcvarepsilonRI stimulation, IKK2 phosphorylates SNAP-23, the target membrane soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE), and ectopic expression of a phospho-mimetic mutant of SNAP-23 partially rescued the impaired IgE-mediated degranulation in IKK2-deficient mast cells. These results suggest that IKK2 phosphorylation of SNAP-23 leads to degranulation and anaphylactic reactions. While this reaction is NF-kB-independent, we additionally show that IKK2 also regulates late-phase allergic reactions promoted by the release of proinflammatory cytokines in an NF-kB-dependent manner. The findings suggest that IKK2 is a central player in allergic reactions.     

Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation

Regulation of mast cell degranulation is dependent on the subtle interplay of cellular signaling proteins. The Src homology 2 (SH2) domain-containing inositol-5'-phosphatase (SHIP), which acts as the gatekeeper of degranulation, binds via both its SH2 domain and its phosphorylated NPXY motifs to the adapter protein Shc via the latter's phosphorylated tyrosines and phosphotyrosine-binding domain, respectively. This theoretically leaves Shc's SH2 domain available to bind proteins, which might be part of the SHIP/Shc complex. In a search for such proteins, protein kinase C-delta (PKC-delta) was found to coprecipitate in mast cells with Shc and to interact with Shc's SH2 domain following antigen or pervanadate stimulation. Phosphorylation of PKC-delta's Y(332), most likely by Lyn, was found to be responsible for PKC-delta's binding to Shc's SH2 domain. Using PKC-delta(-/-) bone marrow-derived mast cells (BMMCs), we found that the antigen-induced tyrosine phosphorylation of Shc was similar to that in wild-type (WT) BMMCs while that of SHIP was significantly increased. Moreover, increased translocation of PKC-delta to the membrane, as well as phosphorylation at T505, was observed in SHIP(-/-) BMMCs, demonstrating that while PKC-delta regulates SHIP phosphorylation, SHIP regulates PKC-delta localization and activation. Interestingly, stimulation of PKC-delta(-/-) BMMCs with suboptimal doses of antigen yielded a more sustained calcium mobilization and a significantly higher level of degranulation than that of WT cells. Altogether, our data suggest that PKC-delta is a negative regulator of antigen-induced mast cell degranulation.     

Recycling of mast cells following degranulation in vitro: An ultrastructural study

Mature mast cells, isolated from the rat peritoneal cavity, were placed into suspension culture, either as resting cells or after degranulation by exposure to compound $\text{48}\text{80}$, and were maintained for up to 63 hr. No mitotic cells were observed, and cell number was conserved. The culture conditions did not cause spontaneous degranulation and cell survival was better than 80%. However, with time in culture, an increasing percentage of cells acquired a vesiculated appearance, characterized by a Golgi area with distended cisternae, the accumulation of lysosomal or autophagic-like vesicles, and enlarged, irregular or fused secretory granules. In the degranulated group, about one-fourth of the cells recovered the morphological appearance of resting cells by 63 hr, indicating that they are capable of ‘recycling’. A cell type with a unique morphology, characterized by a large central vacuole containing secretory product, an eccentric nucleus, and mature secretory granules at the cell periphery appeared in the stimulated group after 22 hr of culture. It may be a possible intermediate stage in the mast cell regranulation process, based on its occurrence exclusively in the stimulated group, the correlation between its distribution and the recovery of mast cells to the resting state, and the morphological resemblance of its granule contents to stages in granule maturation in differentiating embryonic mast cells.     

Exogenous dendritic cell homing to draining lymph nodes can be boosted by mast cell degranulation

Dendritic cells (DCs), as potent antigen presenting cells, are increasingly used for immunotherapeutic approaches, predominantly in oncology. Low efficiency of injected Ag-pulsed DC homing to draining lymph nodes (DLNs) is one of the factors that affect the efficacy of therapy. As Langerhans cell emigration was enhanced after skin mast cell degranulation, we investigated the effect of local mast cell activation on exogenous bone marrow-derived DCs (BM-DCs) homing to DLNs. Product of activated MC/9 mast cells enhanced chemotaxis of BM-DCs to CCL21 in vitro. Intradermal injection of compound 48/80 (c48/80) induced local skin mast cell obvious degranulation and boosted exogenous BM-DC homing to DLNs. Both Ag-specific lymphocyte proliferation and TH1/TH2 cytokine production increased after HBsAg-pulsed BM-DC was injected into c48/80 pretreated mice. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy.