Activation Of Peritoneal Mast Cells Research Articles

Activation of P2X7 Receptors in Peritoneal and Meningeal Mast Cells Detected by Uptake of Organic Dyes: Possible Purinergic Triggers of Neuroinflammation in Meninges.

Extracellular ATP activates inflammasome and triggers the release of multiple cytokines in various immune cells, a process primarily mediated by P2X7 receptors. However, the expression and functional properties of P2X7 receptors in native mast cells in tissues such as meninges where migraine pain originates from have not been explored. Here we report a novel model of murine cultured meningeal mast cells and using these, as well as easily accessible peritoneal mast cells, studied the mechanisms of ATP-mediated mast cell activation. We show that ATP induced a time and dose-dependent activation of peritoneal mast cells as analyzed by the uptake of organic dye YO-PRO1 as well as 4,6-diamidino-2-phenylindole (DAPI). Both YO-PRO1 and DAPI uptake in mast cells was mediated by the P2X7 subtype of ATP receptors as demonstrated by the inhibitory effect of P2X7 antagonist A839977. Consistent with this, significant YO-PRO1 uptake was promoted by the P2X7 agonist 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP). Extracellular ATP-induced degranulation of native and cultured meningeal mast cells was shown with Toluidine Blue staining. Taken together, these data demonstrate the important contribution of P2X7 receptors to ATP-driven activation of mast cells, suggesting these purinergic mechanisms as potential triggers of neuroinflammation and pain sensitization in migraine.

Mast cells mediate neutrophil recruitment during atherosclerotic plaque progression.

Activated mast cells have been identified in the intima and perivascular tissue of human atherosclerotic plaques. As mast cells have been described to release a number of chemokines that mediate leukocyte fluxes, we propose that activated mast cells may play a pivotal role in leukocyte recruitment during atherosclerotic plaque progression. Systemic IgE-mediated mast cell activation in apoE(-/-)μMT mice resulted in an increase in atherosclerotic lesion size as compared to control mice, and interestingly, the number of neutrophils was highly increased in these lesions. In addition, peritoneal mast cell activation led to a massive neutrophil influx into the peritoneal cavity in C57Bl6 mice, whereas neutrophil numbers in mast cell deficient Kit(W(-sh)/W(-sh)) mice were not affected. Within the newly recruited neutrophil population, increased levels of CXCR2(+) and CXCR4(+) neutrophils were observed after mast cell activation. Indeed, mast cells were seen to contain and release CXCL1 and CXCL12, the ligands for CXCR2 and CXCR4. Intriguingly, peritoneal mast cell activation in combination with anti-CXCR2 receptor antagonist resulted in decreased neutrophil recruitment, thus establishing a prominent role for the CXCL1/CXCR2 axis in mast cell-mediated neutrophil recruitment. Our data suggest that chemokines, and in particular CXCL1, released from activated mast cells induce neutrophil recruitment to the site of inflammation, thereby aggravating the ongoing inflammatory response and thus affecting plaque progression and destabilization.

Herbal Formula-3 inhibits food allergy in rats by stabilizing mast cells through modulating calcium mobilization

The prevalence of food allergy has increased dramatically during the last three decades, but currently there was no effective therapy except avoidance of allergen. This study aimed to investigate the effect of a modified Chinese herbal formula, Formula-3, on mast cell degranulation and the anti-allergic activity in both animal and cell models. With OVA-sensitized food allergic model in Brown-Norway rats, we checked tissue injury in the small intestines by H&E staining. The Th2 cytokine levels and IgE production in serum or supernatant of the intestinal mucosa homogenates were analyzed by ELISA. Meanwhile, rat peritoneal mast cell activation and degranulation were examined by Toluidine Blue Stain and the release of histamine was measured. Furthermore, the regulation of Formula-3 on Ca(2+) mobilization was investigated by probing intracellular Ca(2+) with fluo-4 fluorescence. The direct effect of Formula-3 on mast cell stabilization was also studied in RBL-2H3 cell line. In vivo Formula-3 administration significantly reduced tissue damage in the small intestines of rat and suppressed Th2 cytokine secretion and IgE production. We demonstrated that Formula-3 treatment significantly suppressed FcεR1-mediated mast cell degranulation no matter in OVA-challenged allergic rats or IgE-sensitized RBL-2H3 cell line. Furthermore, Formula-3 significantly decreased Ca(2+) influx through store-operated calcium channels (SOCs) evoked by dinitrophenyl-BSA or thapsigargin in mast cells. Taken together, our data indicate that Formula-3 stabilizes mast cells by suppressing FcεR1-induced Ca(2+) mobilization mainly through inhibiting Ca(2+) entry via SOCs, thus exerting a protective effect against OVA-sensitized food allergy.

Novel aspects of leukocyte trafficking

The inflammatory response to the Toll‐like receptor (TLR) 4 ligand lipopolysaccharide (LPS) involves a sequence of intravascular leukocyte capture, rolling and adhesion, followed by their emigration into surrounding tissue which is accompanied by plasma protein extravasation (PPE). Typically, such events are observed hours after LPS injection, but we have chosen to investigate the earlier stages of leukocyte trafficking during murine experimental endotoxaemia.Using intravital microscopy of the mesentery, we observed a rapid increase in neutrophil emigration and PPE after i.p. LPS injection, reaching a plateau after 2h. The same response was seen with the TLR2 ligand zymosan, but not the non‐specific neutrophil activator ammonium thioglycolate, implicating TLR‐specific mechanisms in early leukocyte trafficking. LPS‐induced trafficking was associated with activation of peritoneal mast cells, shown by selective uptake of the dye ruthenium red from the superfusion buffer, and was inhibited by pre‐treatment with cromolyn sodium.To conclude, our data suggest that mast cells play a role in LPS‐induced peritoneal inflammation in terms of leukocyte emigration. Further work is underway to determine the sequential mechanisms of cell types and mediators involved in these hyperacute stages of LPS‐induced inflammation.Work supported by the BBSRC.

Soluble Mediators Released from PI-IBS Patients’ Colon Induced Alteration of Mast Cell: Involvement of Reactive Oxygen Species

Growing evidence suggests that patients with post-infectious irritable bowel syndrome (PI-IBS) have increased mast cell activation, and that mucosal soluble mediators are involved in the pathophysiology of visceral hyperalgesia. In addition, previous findings show that reactive oxygen species (ROS) and protease-activated receptors (PARs) are mediators of persistent hyperalgesia. This article aims to investigate: (1) the ability of soluble factors from colonic biopsies to active peritoneal mast cells (PMCs) in vitro; (2) whether the effects of PMCs degranulation induced by soluble mediators are related to PARs activation; and (3) the ability of phenyl N-tert-butylnitrone (PBN), a ROS scavenger, to modify these alterations. Supernatant (SUP) from colonic biopsies was collected and applied to PMCs for 12 h. Activation of PMCs was evaluated. The expression of PAR(2) in PMCs was examined by RT-PCR and double-immunofluorescence staining. PBN (10 mM) treatment was administered, then previous alterations were observed again. Stimulation with SUP of PI-IBS led to an increase in activation of PMCs. PAR(2)mRNA expression was significantly increased in PMCs induced by SUP of PI-IBS compared to healthy subjects. After being treated by PBN, the SUP-induced enhancement of PMCs activities could be weakened, and PAR(2)mRNA expression was significantly decreased. A similar result of immunoreactivity for PAR(2) was observed in PMCs. The study shows that ROS scavenger reverses the SUP of PI-IBS-induced enhancement of PMCs activities, and that these effects may be related to activation of PAR(2). These findings might pave the way to new therapeutic targets in PI-IBS.

Mast Cell Activation In Vivo Impairs the Macrophage Reverse Cholesterol Transport Pathway in the Mouse

Chymase released by activated mast cells degrades high-density lipoproteins. We evaluated whether local activation of mast cells would attenuate cholesterol efflux from neighboring macrophage foam cells, thereby disrupting the entire in vivo pathway of macrophage-specific reverse cholesterol transport (RCT). C57Bl/6J mice received intraperitoneal injections of the mast cell-degranulating compound 48/80 to induce peritoneal mast cell activation, human apolipoprotein A-I (apoA-I) to stimulate RCT, and [(3)H]cholesterol-labeled J774 macrophages for measurement of the rate of RCT. After 3 hours, (3)H-radioactivity was measured in the intestinal lumen contents. Activation of mast cells in the peritoneal cavity depleted human apoA-I pre-β-migrating species, impairing the ability of the peritoneal fluid to efficiently promote cholesterol efflux from cultured macrophages. Moreover, intact but not chymase-treated (proteolyzed) apoA-I accelerated the transfer of macrophage-derived (3)H- radioactivity to the intestinal contents. Importantly, stimulation of RCT by human apoA-I was fully blocked by 48/80 in mast cell-competent wild-type C57Bl/6J mice but not in mast cell-deficient W-sash c-kit mutant mice. The ability of intraperitoneally administered phospholipid vesicles to promote RCT in wild-type mice was not blocked by 48/80, supporting the notion that mast cell-dependent proteolysis of the intraperitoneally administered apoA-I was responsible for RCT inhibition. Overall, our results suggest that tissue-specific activation of mast cells with ensuing release of chymase is able to proteolytically inactivate apoA-I in the microenvironment of the activated mast cells, thus locally impairing the initiation of macrophage RCT in vivo.

Duodenase activates rat peritoneal mast cells via protease-activated receptors of type 1

It was found that duodenase, a serine protease from the bovine duodenum, activates rat peritoneal mast cells (PMC) in vitro presumably via protease-activated receptors (PARs). Like thrombin (a serine protease from the blood coagulation system) and the PAR1 agonist peptide (PAR1-AP), duodenase was shown to accelerate the secretion of beta-hexosaminidase (a marker of cell degranulation) by PMC in a dose-dependent manner. The blockage of the proteolytic activity of duodenase toward the substrate Tos-Gly-Pro-Lys-pNA by the soybean Bauman-Birk protease inhibitor substantially reduced (by 40%) the ability of duodenase to stimulate the secretory activity of PMC. Pretreatment of PMC with duodenase decreased the beta-hexosaminidase secretion induced by thrombin and PAR1-AP by 35 and 41.7%, respectively, and abolished the antiinflammatory effect of activated protein C. At the same time, pretreatment of PMC with duodenase did not affect the secretion of beta-hexosaminidase induced by compound 48/80, a nonspecific degranulator of mast cells. Duodenase, unlike PAR1-AP (30-100 microM), in a broad concentration range (10-100 nM) did not induce aggregation of human platelets, but suppressed the platelet aggregation elicited by PAR1-AP.

Intravesical Suplatast Tosilate (IPD-1151T) Inhibits Experimental Bladder Inflammation

Interstitial cystitis is a painful bladder disease characterized by urgency, frequency and variable inflammation but there is no curative therapy. Suplatast tosilate (IPD-1151T) is an immunoregulatory compound that decreases interstitial cystitis symptoms but to our knowledge its mechanism of action is unknown. We investigated the effect of intravesical IPD-1151T on mediator release from bladder explants in experimental cystitis. A catheter was inserted into the bladder of female mice. After urine was emptied normal saline, carbachol (100 nM) or lipopolysaccharide (10 mg/ml) was introduced with or without 10-minute pretreatment with IPD-1151T. Urine was removed after 45 minutes for histamine and tumor necrosis factor-alpha assays. The bladder was removed after 4 hours, minced into 1 mm2 pieces and cultured with or without triggers overnight for mediator release. The effect of IPD-1151T was also tested on rat skin vascular permeability as well as on purified rat peritoneal mast cells and human cord blood derived mast cells. Carbachol significantly increased histamine release in urine (61.3% in 8 preparations, p<0.05) but not in explant medium. IPD-1151T inhibited this effect by 77%. Lipopolysaccharide induced a 350% urine histamine increase in 9 preparations (p<0.05) and a 300% tumor necrosis factor-alpha increase in explant medium. IPD-1151T inhibited the lipopolysaccharide induced medium tumor necrosis factor-alpha increase by 95% in 5 preparations (p<0.05). IPD-1151T did not inhibit rat skin vascular permeability or purified rat peritoneal mast cell activation by compound 48/80 or human cord blood derived mast cells by anti-IgE. IPD-1151T inhibits bladder release of histamine and tumor necrosis factor-alpha through a mechanism that does not appear to involve direct mast cell inhibition. These findings may justify a beneficial effect of IPD-1151T in interstitial cystitis.

Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca(2+)-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca(2+) in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca(2+). Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca(2+) with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca(2+) from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.

Inhibitory Effects of Morus alba on Compound 48/80-Induced Anaphylactic Reactions and Anti-Chicken Gamma Globulin IgE- Mediated Mast Cell Activation

We investigated the effects of hot-water extract from the root bark of Morus alba (HEMA) on anaphylactic reactions. Using in vitro and in vivo experiments, we examined whether HEMA could inhibit compound 48/80-induced systemic anaphylactic shock and anti-chicken gamma globulin (CGG) IgE-mediated rat peritoneal mast cell activation. HEMA significantly inhibited systemic anaphylaxis induced by the compound 48/80 in mice. HEMA also significantly inhibited the passive cutaneous anaphylaxis activated by anti-CGG IgE. HEMA had no cytotoxicity on rat peritoneal mast cells (RPMC). Moreover, HEMA dose-dependently inhibited mast cell degranulation, histamine release and calcium uptake into RPMC induced by the compound 48/80 or anti-CGG IgE. When HEMA was added, the level of intracellular cAMP in RPMC showed a transient and significant increase (5-fold) compared with that of control cells. HEMA also inhibited significantly the compound 48/80-induced cAMP reduction in RPMC. These results suggested that HEMA inhibits the compound 48/80- or anti-CGG IgE-induced mast cell activation and its inhibitory effects on mast cell activations were favorably comparable to disodium cromoglycate. And HEMA is a candidate for effective therapeutic tools of allergic diseases.

Atrial natriuretic peptide induces rat peritoneal mast cell activation by cGMP-independent and calcium uptake-dependent mechanism.

Atrial natriuretic peptide (ANP), a 28 amino acid basic polypeptide, is known to induce histamine release from human and rat mast cells in vitro and cause a wheel formation in rat skin. However, cellular events associated with histamine release are not clearly understood. In this study, we have examined the calcium flux and cGMP formation associated with histamine release in the ANP-treated mast cells. ANP, in vitro, induced mast cell degranulation and histamine release in a dose-dependent manner. ANP also induced an enhanced calcium uptake into cells and increased the cellular level of cGMP in mast cells. A high level of calcium in the media caused an inhibition of ANP-dependent histamine release but enhanced the level of intracellular cGMP of mast cells. ANP inducing a dose-dependent increase in vascular permeability of rat skin was confirmed by the extravasation of the circulating Evans blue. The results indicate ANP induced the histamine release and an increase in vascular permeability through mast cell degranulation in cGMP-independent and calcium uptake-dependent manner.

Effect of Avène Spring Water on the Activation of Rat Mast Cell by Substance P or Antigen

The biological activity of Avène water from two different springs (‘Sainte Odile’ and ‘Val d’Orb’) was studied in vitro on rat peritoneal mast cell activation. A dilution-dependent inhibition of both histamine and prostaglandin D₂ antigen-induced release was observed when cells were preincubated with both Avène spring waters. They also inhibited histamine release triggered by substance P. The ability of Avène water to inhibit mast cell activation in vitro may be related with its antiallergic and anti-inflammatory properties and its use in hydrotherapy.

Impaired mast cell-dependent natural immunity in complement C3-deficient mice.

The complement system is widely regarded as essential for normal inflammation, not least because of its ability to activate mast cells. However, recent studies have called into question the importance of complement in several examples of mast cell-dependent inflammatory responses. To investigate the role of complement in mast cell-dependent natural immunity, we examined the responses of complement-deficient mice to caecal ligation and puncture, a model of acute septic peritonitis that is dependent on mast cells and tumour necrosis factor-alpha (TNF-alpha). We found that C4- or C3-deficient mice were much more sensitive to caecal ligation and puncture than wild-type (WT) controls (100% versus 20% in 24-h mortality, respectively). C3-deficient mice also exhibited reductions in peritoneal mast cell degranulation, production of TNF-alpha, neutrophil infiltration and clearance of bacteria. Treating the C3-deficient mice with purified C3 protein enhanced activation of peritoneal mast cells, TNF-alpha production, neutrophil recruitment, opsonophagocytosis of bacteria and resistance to caecal ligation and puncture, confirming that the defects were complement-dependent. These results provide formal evidence that complement activation is essential for the full expression of innate immunity in this mast cell-dependent model of bacterial infection.

Phospholipid dynamics and function in cell membrane of blood cells. With reference to mode of action of certain amphiphilic drugs

An asymmetric modification of the cell membrane lipid bilayer of erythrocytes by means of exogenous phospholipase (PL) treatment or of phospholipid addition induced an echinocyte- or stomatocyte-type shape change of the cells. Such membrane shape change was demonstrated to be due to asymmetric swelling or shrinkage of the lipid bilayer halves, from the simultaneous determination of the change or intra-bilayer transfer of the phospholipid molecules. In the collagen-induced activation of blood platelets, the importance of PLA2 activation as the initial reaction of the signaling system was demonstrated, and the regulatory effects of the membrane fluidity change on the enzyme activation were also shown. In the antigen-induced activation of peritoneal mast cells, a pathway through the PLD activation appeared to be more important. Phosphatidic acid produced from the membrane phospholipids through the PLD activation was shown to act as an intracellular mediator to stimulate the PLA2 activation. Various amphiphilic drugs, including benzylisoquinoline alkaloids such as cepharanthine, were shown to inhibit the platelet activation and the mechanism was studied. The anti-platelet activity appeared to be mainly due to inhibition of the PLA2 activation.

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 mast cell-mediated cytotoxicity by IFN-alpha/beta and -gamma.

Although IFN enhance the cytotoxic activity of NK cells, K cells, and monocytes, IFN-alpha/beta and IFN-gamma did not stimulate the cytotoxic activity of rat peritoneal mast cells (PMC), but had an inhibitory effect. Preincubation for 2 h with 100 and 200 U/ml of IFN-gamma and IFN-alpha/beta, respectively, inhibited PMC cytotoxicity against WEHI-164 target cells. Lower concentrations of IFN-gamma (12.5 U/ml) and IFN-alpha/beta (25 U/ml) inhibited cytotoxicity of PMC after 8 h preincubation. The inhibitory effect of IFN was concentration and time dependent. In contrast to cytotoxicity, the release of histamine by PMC was not stimulated by the target cells WEHI-164 and there was no correlation between histamine release and cytotoxic activity of PMC. Specific antibody to subclasses of IFN prevented the inhibition of PMC cytotoxic activity, but preincubation with antibodies to the alternate subclass of IFN did not affect the observed inhibition. Moreover, the presence of both subclasses of IFN showed an additive inhibition of PMC cytotoxicity. The cytotoxic activity of PMC can be completely inhibited by the addition of anti-TNF during the assay. At high concentrations (400 U/ml), IFN inhibited the release of TNF from PMC. In the presence of RNA or protein synthesis inhibitors, IFN did not inhibit cytotoxicity of PMC further. We postulate that IFN may alter gene expression in mast cells in a manner that down-regulates their functions.

Activation of rat peritoneal mast cells in coculture with sympathetic neurons alters neuronal physiology

Rat peritoneal mast cells (PMC) were activated with anti-IgE or 48 80 while in coculture with neurons dissociated from mouse superior cervical ganglia. At 18–24 h of coculture, microelectrode recordings from neurons were made 15 min after activation of PMC with anti-IgE antibody or the secretagogue compound 48 80 . These recordings showed that PMC activation caused depolarization of the neurons and decreased their membrane resistance. Both methods of activation resulted in similar changes in these neuronal characteristics. The resting potential decreased by an average of 15% from the control value of −36.0 ± 0.6 mV ( SEM) ( n = 121). The membrane resistance decreased by an average of 60% from the control value of 34.2 ± 2.0 MΩ ( n = 75). Application of PMC secretion products directly onto neuronal somata caused reversible changes similar to those caused by activation of cocultured PMC. A decrease in membrane resistance that was observed in neurons cocultured with PMC for 18–24 h without stimulation was due to a proportion of PMC undergoing spontaneous activation. These changes in neuronal physiology are attributed to the action of substances released from the mast cells. This coculture model offers the means to study the cellular interactions involved in neuroimmune communication.

Effect of Nedocromil Sodium on the Compound Exocytosis of Mast Cells

The secretory activity of peritoneal mast cells was studied in vitro after challenge with compound 48/80 of cells obtained from normal rats and with anti-rat IgE of cells obtained from rats hyperimmunised with Nippostrongylus brasiliensis. Peritoneal mast cells were isolated from 2 groups of rats aged 2 to 3 months and 5 to 6 months. After purification, the cells were preincubated with or without nedocromil sodium for 5 minutes followed by addition of secretagogue and incubation for an additional period of 5 minutes (compound 48/80) or 15 minutes (anti-IgE). The secretory response was analysed by measurement of the release of endogenous histamine and serotonin (5-hydroxytryptamine) into the medium using high performance liquid chromatography, and by the assay of the heparin content of individual mast cells by cytofluorometry, using the fluorescent dye berberine as a quantitative marker. Concentrations of secretagogues which gave 30 to 40% amine release in controls without inhibitor were selected. In agreement with previous reports, the degree of release of histamine was slightly higher than that of serotonin, but nedocromil sodium suppressed the release of both amines in a parallel manner, without any differential effect, after stimulation with both compound 48/80 and anti-IgE. The effect of nedocromil sodium was concentration-dependent, with the lowest effective concentration in the range of 10(-8) to 10(-7) mol/L, and reduced release to about 40% of control values. Mast cells from young rats responded with a significantly higher amine release than mast cells from old rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor enhances antigen and other secretagogue-induced histamine release from rat peritoneal mast cells in the absence of phosphatidylserine

The effects of 2.5S nerve growth factor (NGF) and epidermal growth factor (EGF), isolated from mouse submaxillary glands, on histamine release from rat peritoneal mast cells (PMCs) were studied. In the absence of phosphatidylserine, NGF (1 ng/ml to 1 μg/ml) did not cause histamine release from PMCs isolated from normal rats and those infected with the nematode Nippostrongylus brasiliensis. However, when PMCs (>97% pure) were preincubated with NGF and then challenged with worm antigen (Ag), there was a marked enhancement of histamine release (approximately twofold with a maximum effect at 10 ng/ml of NGF [3.8 × 10 −10 mol/L]) compared with the release induced by Ag alone. EGF (1 ng/ml to 1 μg/ml) neither produced histamine release from PMCs in the presence of phosphatidylserine nor enhanced Ag-induced histamine release. This suggests that NGF acts directly on PMCs by activation of cell-surface receptors. The early kinetics of Ag-induced histamine release were altered by NGF that increased the initial rate at 15 seconds but did not prolong the overall duration of histamine release. Simultaneous addition of Ag and NGF did not cause enhanced histamine release; thus, some preincubation time with NGF (5 minutes or less) was required for the activation of PMCs. Moreover, after PMCs were activated by NGF, that state persisted for 1 hour, even when unbound NGF was removed by washing, and thereafter subsided gradually. Further studies revealed that NGF enhanced histamine release induced by concanavalin A, compound 48 80 , and ionophore A23187. These results suggest that NGF might be an important molecule in inflammatory responses through the regulation of mediator release from mast cells.

Rat mast cell activation and inactivation: Differences when various ligands are used to induce secretion

The relationship between rat peritoneal mast cell activation and inactivation (desensitization) was studied for a variety of stimuli acting via IgE and IgG receptors on the cell surface. Anti-IgE, antigen (ovalbumin), anti-IgG1, anti-IgG2a and dimers, trimers and higher oligomers of IgE were used to induce histamine release from rat mast cells. All produced similar characteristics of cell activation, with a rapid rate of histamine release from the cells, release being 90% complete within 5 minutes and with calculated doubling times between 21.6 +/- 3.6 s (+/- SEM) for ovalbumin and 93.0 +/- 18.6 s (+/- SEM) for anti-IgG1. The characteristics of inactivation, however, varied with the releasing agent used. Of all the stimuli used only ovalbumin showed a rapid rate of desensitization (t 1/2 = 330 +/- 34.8 s) which correlated with the cessation of histamine release. The other stimuli showed slow rates of desensitization (t 1/2 between 1068 +/- 40.2 s for dimer and 3576 +/- 660 s for anti-IgE) even though the rate of release was rapid. Thus, although these stimuli are thought to be stimulating the cells by cross-linking of either IgE or IgG receptors, the difference in subsequent response of the cells would indicate that the transduction mechanism bringing about release can distinguish between these various stimuli. Also, it seems that the idea that duration of histamine release is determined by the rate of desensitization may need revising as with most of these stimuli, release has terminated when the cells are still in a fully activated state.