Local histamine release after immunological and non-immunological mast cell degranulation in vivo

Plasma histamine concentration in the circulation has been proposed as an index of mast cell degranulation occurring in vivo but there are problems with this approach in practice. Local elevations in plasma histamine occur in blood draining the site of antigen challenge in forearm skin. We have compared changes in plasma histamine concentration with time following intradermal injection of antigen, codeine or histamine to produce matched wheal and flare responses in 4 atopic subjects. Less histamine appears to be released after non-immunological challenge.     

The effect of capsaicin application on mast cells in normal human skin

Peptides released from sensory nerves during an axon reflex are thought to cause mast cell degranulation, histamine (Hi) release and Hi-induced vasodilatation leading to the flare of the triple response. Capsaicin stimulates peptide release from sensory neurones and causes flare in vivo but does not cause Hi release from mast cells in vitro. The effects of capsaicin on mast cell degranulation in human skin in vivo has been studied by histological examination of skin biopsies after topical capsicin (1%) treatment of stratum corneum-denuded forearm in four volunteers. The results show a significant reduction in the visible numbers of mast cells and the appearance of degranulated mast cells ghosts in the skin six hours after capsaicin application. Since capsaicin itself does not release Hi from mast cells, these data suggest that capsaicin-induced release of peptides from neurones could cause mast cell degranulation.     

Histamine is released from skin by substance P but does not act as the final vasodilator in the axon reflex.

We have explored in man the hypothesis that histamine released from dermal mast cells by neurotransmitters from afferent nerves contributes to vasodilatation of the axon reflex. The ability of substance P to release histamine from human skin in vivo, and the effects of a histamine H1-receptor antagonist on capsaicin-induced axon reflex flares were studied. Intradermal injections of substance P (50 pmol) produced a weal and flare response which was associated with increased histamine concentration in blood draining the site (mean plasma histamine concentration before injection 0.17 +/- 0.02 ng ml-1 (+/- s.e.mean), concentration one minute after injection 1.26 +/- 0.28 ng ml-1, n = 6). Terfenadine, an H1-receptor antagonist, had no effect on the flare response to intradermal injection of capsaicin at a dose which inhibited by more than 60% the flare response to exogenous histamine and to histamine released from dermal mast cells by substance P. Substance P releases histamine from human skin in vivo. However, whatever the nature of the neurotransmitter released from afferent nerves during the axon reflex, it does not produce vasodilatation through release of histamine from dermal mast cells. Histamine may still contribute to the flare by initiation of the reflex.     

Activation of connective tissue-type and mucosal-type mast cells in compound 48/80-induced airway response

The pathology of non-immunological airway contraction is not well understood. To define the activation of different phenotypes of mast cells, a rat non-immunological asthmatic model was prepared. Airway contraction in rats was measured by an unrestrained whole-body plethysmographic system following a 10-min inhalation challenge with a 5% solution of compound 48/80. Histamine, leukotrein C(4) (LTC(4)) and tumor necrosis factor (TNF)-alpha levels in bronchoalveolar lavage fluid, as well as tissue histamine content were quantified. Mast cells and eosinophils were detected by histology. Both the early and late phase of airway responses were induced by inhalation of compound 48/80. Histamine and TNF-alpha levels increased significantly 30 min after challenge, but no increases were detected at either 8 or 24 h after challenge. A high LTC(4) level was detected in 30 min and 8 h after challenge. Tissue histamine content decreased at 30 min after challenge and returned to the unstimulated level by 8 h. Connective tissue mast cells in rat trachea showed a degranulation response. Along with the increase in numbers of mucosal mast cells, rat mast cell protease II at both mRNA and protein levels in the trachea epithelial layer was also increased significantly at 30 min after challenge. We conclude that compound 48/80 inhalation causes both the early and late phase of airway contraction in rats. Mast cell degranulation is responsible for the early phase of airway response, which subsequently triggers the late phase of airway response.     

Evidence that mast cell degranulation, histamine and tumour necrosis factor alpha release occur in LPS-induced plasma leakage in rat skin.

1. In the present study we investigated the role of mast cells during inflammation in rat skin. As the release of several pro-inflammatory mediators, such as histamine and tumour necrosis factor alpha (TNFalpha), occurs following mast cell activation we studied whether mast cell degranulation and the release of both histamine (H) and TNFalpha occurred in a model of lipopolysaccharide (LPS)-induced plasma leakage in rat skin. 2. Plasma leakage in the rat skin was measured over a period of 2 h as the local accumulation of intravenous injection of 125I-human serum albumin (125I-HSA) in response to intradermal injection of LPS. LPS (10 microg site-1) produced an increase of plasma leakage (50.1+/-2.3 microl site-1) as compared to saline (9.0+/-3.2 microl site-1). Histological analysis of rat tissue showed that LPS induced a remarkable mast cell degranulation (59.8+/-2.1%) as compared to saline (13.5+/-2.2%). 3. Ketotifen (10-9 - 10-7 mol site-1), a well-known mast cell-membrane stabilizer, produced a dose-related inhibition of LPS-induced plasma leakage by 36+/-3.5%, 47+/-4.0%, 60+/-3.3% respectively. In addition, ketotifen (10-7 mol site-1) inhibited mast cell degranulation by 59. 2+/-2.7%. 4. Chlorpheniramine maleate (CPM) (10-9 - 10-7 mol site-1), an H1 histamine receptor antagonist only partially inhibited LPS-induced plasma leakage in rat skin (38+/-1.1% at the highest dose). Furthermore, CPM (10-7 mol site-1) did not prevent mast cell degranulation. 5. A polyclonal antibody against TNFalpha (1:500, 1:100, 1:50 v v-1 dilution), locally injected, decreased LPS-induced plasma leakage in the skin by 15+/-2.0%, 24+/-2.1% and 50+/-3.0% respectively. 6. Taken together these results suggest that LPS-induced plasma leakage in rat skin is mediated, at least in part, by mast cell degranulation and by the release of histamine and TNFalpha from these cells.     

In vitro anti-allergic activity of the fungal metabolite pyridovericin

Mast cells play a critical role during the development of an allergic response. Upon activation by an antigen and IgE, via FcεRI receptors, mast cells release histamine and other mediators that initiate and propagate immediate hypersensitivity reactions. Mast cells also secrete cytokines that regulate the immune responses. In this way, inhibitors of mast cell activity could work as promising therapeutics for allergic disorders. In the present work, we investigated the capacity of pyridovericin, a natural product isolated from the entomopathogenic fungus Beauveria bassiana, to inhibit mast cell degranulation and cytokine secretion. It was found that pyridovericin strongly decreased the release of β-hexosaminidase, a marker for mast cell degranulation, when mast cells were stimulated by both FcεRI-dependent and independent pathways. In addition, pyridovericin strongly abrogated secretion of interleukin-4. Pyridovericin-mediated suppression of stimulated increase in intracellular Ca^2 + levels, a crucial signal for mounting of both degranulation and cytokine production responses, was ascribed as one of the inhibition targets of pyridovericin. Those initial studies identify pyridovericin as a potential new candidate for the development of new anti-allergic drugs.     

Apomorphine-induced inhibition of histamine release in rat peritoneal mast cells.

The apomorphine-induced inhibition of histamine release in rat peritoneal mast cells was studied by means of secretagogues stimulating different pathways of mast cell activation. Apomorphine inhibited the mast cell response to all releasing agents (lysophosphatidylserine plus nerve growth factor, compound 48/80, substance P, ATP, tetradecanoylphorbolacetate, melittin). The IC50 ranged from 4 microM to 24 microM at concentrations of secretagogues releasing 30-50% of mast cell histamine. However, the potency of the drug decreased at higher secretagogue concentrations. Mast cells, pretreated with apomorphine and washed, released little histamine upon stimulation. The secretory response could be partially restored on increasing the concentration of secretagogues. The results suggest that apomorphine affects a regulatory step controlling the terminal sequence of mast cell secretory activity. As indicated by the reduced potency of the drug, the control by the apomorphine-sensitive reaction loses efficiency under conditions of massive histamine release.     

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.     

Lipopolysaccharide from Porphyromonas gingivalis stimulates rat mast cells to cysteinyl leukotriene generation and upregulates Toll-like receptor -2 and -4 expression

Mast cells are found in all tissues of the oral cavity and it is suggested that they take part in the development of oral inflammation. As Porhyromonas gingivalis is widely recognized as a major pathogen in the development and progression of gingivitis and periodontitis, the aim of our study is to determine the effect of P. gingivalis lipopolysaccharide (LPS) on mast cell degranulation, cysteinyl leukotriene (cysLT) generation, and migration, as well as Toll-like receptor (TLR)-2 and -4 expression. Experiments were carried out in vitro on rat peritoneal mast cells. LPS-induced mast cell histamine release was estimated by a spectrofluorometric method and cysLT generation by ELISA test. Mast cell migration in response to this antigen was examined according to Boyden's modified method and TLR expression was determined by flow cytometry. We found that P. gingivalis LPS did not induce mast cell degranulation and histamine release. However, activation of mast cells with this bacterial antigen resulted in generation and release of significant amounts of cysLTs. We also documented that LPS from P. gingivalis did not stimulate mast cell migration, even in the presence of laminin, whereas it strongly upregulated TLR2 and TLR4 expression on mast cells. Observations that P. gingivalis LPS activates mast cells to generate and release proinflammatory mediators such as cysLTs and modulates TLR2 and TLR4 expression indicates that these cells might be involved in the emergency of inflammatory processes evolved in response to P gingivalis infection.     

The influence of ranitidine, alone and in combination with clemastine, on histamine-mediated cutaneous weal and flare reactions in human skin.

Significant, (P less than 0.05) inhibition of histamine induced cutaneous weal and flare reactions by ranitidine at 10(-5) M concentration has been demonstrated in a double blind, in vivo study; the results support the evidence for cutaneous H2-histamine receptors provided by previous studies using cimetidine and metiamide. The degree of inhibition of histamine mediated cutaneous reactions achieved by clemastine was increased by the administration of clemastine and ranitidine together, for ranitidine concentrations 10(-5) M (P less than 0.001) and 10(-6) M (P less than 0.05). The magnitude of the inhibitory effect of ranitidine, both administered alone and in combination with clemastine, suggests that in the production of histamine induced cutaneous weal and flare reactions, the contribution afforded by stimulation of cutaneous H2-histamine receptors is only small.     

Single-walled carbon nanotube exposure induces membrane rearrangement and suppression of receptor-mediated signalling pathways in model mast cells

Carbon nanotubes (CNT) are environmental challenges to the respiratory and gastrointestinal mucosa, and to the dermal immune system. Mast cells (MC) are pro-inflammatory immunocytes that reside at these interfaces with the environment. Mast cells are sources of pro-inflammatory mediators (histamine, serotonin, matrix-active proteases, eicosanoids, prostanoids, cytokines and chemokines), which are released in a calcium-dependent manner following immunological challenge or physico-chemical stimulation. Since C-60 fullerenes, which share geometry with CNT, are suppressive of mast cell-driven inflammatory responses, we explored the effects of unmodified SWCNT aggregates on mast cell signaling pathways, phenotype and pro-inflammatory function. We noted SWCNT suppression of antigen-induced signalling pathways and pro-inflammatory degranulation responses. Mast cells recognize unmodified SWCNT by remodeling the plasma membrane, disaggregating the cortical actin cytoskeleton and relocalizing clathrin. Clathrin was also identified as a component of an affinity-purified ‘interactome’ isolated from MC using an SWCNT affinity matrix for mast cell lysates. Together, these data are consistent with the ability of SWCNT to suppress mast cell pro-inflammatory function via a novel recognition mechanism.     

The effects of astemizole on histamine-induced weal and flare

The effect of astemizole on the weal and flare response to intradermal histamine has been studied in normal volunteers after single dosing, and in patients with urticaria and pruritus after chronic dosing with the drug. Single doses of astemizole (40 mg) in normal volunteers significantly reduced histamine weal and flare at 24 and 48 h (p less than 0.001). Before treatment, there was a significantly greater response to intradermal histamine in patients with urticaria than pruritus (p less than 0.05). Chronic dosing with astemizole produced significant reduction in histamine-induced weal and flare in both patient groups which did not show evidence of tachyphalaxis over the period of the study. The effect of astemizole on histamine-induced weal and flare was accompanied by a significant increase in the rate of weal and flare disappearance in both patients (weal p less than 0.002, flare p less than 0.05) and volunteers (weal p less than 0.02, flare p less than 0.005); but there was no change in the rate of weal formation. The effects of astemizole on weal and flare kinetics may be a function of its high level of H1 antagonist activity.     

Mast cell degranulation in rat mesenteric venule: effects of L-NAME, methylene blue and ketotifen.

Mast cells are present in proximity to the microvessels, and on stimulation with inhibition of NO synthesis, are a rich source of numerous inflammatory mediators. A microcirculatory study was undertaken to clarify whether nitric oxide (NO) and activation of guanylate cyclase is involved in degranulation of perivascular mast cells in the rat mesenteric venule, and whether oral administration of ketotifen suppress the degranulation. Intravital microscopy was used to monitor the rates of adherence and extravasation of leukocytes in single unbranched venules with diameters between 25 and 35 microm of rat mesentery. Leukocyte rolling velocity, red blood cell velocity, vessel diameter and blood pressure were also measured. Mast cell degranulation was quantified within 30 microm from the venule. NG-nitro-L-arginine methyl ester (L-NAME) at an intravenous dose of 30 mg kg-1 increased the number of degranulated cells, while its enantiomer, D-NAME at the same dose had no effect. Superfusion with methylene blue (MB), an inhibitor of soluble guanylate cyclase, at 50 microm elicited similar degranulation of the mast cells. The degranulation was associated with increased adhesion of leukocytes to the endothelium and the slowed rolling. Pretreatment with ketotifen at an oral dose of 1 mg kg-1 inhibited mast cell degranulation in responses to either L-NAME or MB. It is conceivable that guanylate cyclase for NO production pathway in endothelial and/or mast cells is involved in the mast cell degranulation process, and the process or subsequent action of NO may be preserved by ketotifen, eliciting down-modulation of mast cell activation.     

Targeting cardiac mast cells: pharmacological modulation of the local renin-angiotensin system

Enhanced production of angiotensin II and excessive release of norepinephrine in the ischemic heart are major causes of arrhythmias and sudden cardiac death. Mast cell-dependent mechanisms are pivotal in the local formation of angiotensin II and modulation of norepinephrine release in cardiac pathophysiology. Cardiac mast cells increase in number in myocardial ischemia and are located in close proximity to sympathetic neurons expressing angiotensin AT1- and histamine H3-receptors. Once activated, cardiac mast cells release a host of potent pro-inflammatory and pro-fibrotic cytokines, chemokines, preformed mediators (e.g., histamine) and proteases (e.g., renin). In myocardial ischemia, angiotensin II (formed locally from mast cell-derived renin) and histamine (also released from local mast cells) respectively activate AT1- and H3-receptors on sympathetic nerve endings. Stimulation of angiotensin AT1-receptors is arrhythmogenic whereas H3-receptor activation is cardioprotective. It is likely that in ischemia/reperfusion the balance may be tipped toward the deleterious effects of mast cell renin, as demonstrated in mast cell-deficient mice, lacking mast cell renin and histamine in the heart. In these mice, no ventricular fibrillation occurs at reperfusion following ischemia, as opposed to wild-type hearts which all fibrillate. Preventing mast cell degranulation in the heart and inhibiting the activation of a local renin-angiotensin system, hence abolishing its detrimental effects on cardiac rhythmicity, appears to be more significant than the loss of histamine-induced cardioprotection. This suggests that therapeutic targets in the treatment of myocardial ischemia, and potentially congestive heart failure and hypertension, should include prevention of mast cell degranulation, mast cell renin inhibition, local ACE inhibition, ANG II antagonism and H3-receptor activation.     

Inhibitory effects of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on allergic inflammatory responses in rat basophilic leukemia cells

Mast cells play crucial roles in the initiation of allergic inflammatory responses by releasing various mediators such as histamines, cytokines, and leukotrienes. In addition, signaling cascade pathways, such as the mitogen-activated protein kinase (MAPK) pathway, contribute to the regulation of mast cell degranulation. Accordingly, different research strategies have been pursued to develop anti-inflammatory and anti-allergic drugs by regulating these signaling pathways. The development of new drugs that inhibit mast cell degranulation may help in the treatment of allergies. In this study, we investigated the effects of coumarin derivatives on mast cell degranulation. The effect of coumarin derivatives on degranulation in rat basophilic leukemia (RBL)-2H3 cells was determined by a β-hexosaminidase assay and histamine assay. A coumarin derivative 1 (C1), 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate, inhibited degranulation in a dose-dependent manner and demonstrated maximum therapeutic effect when used at 25 μM. Additionally, these compounds inhibited the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. Taken together, these results indicate that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibits mast cell degranulation by suppressing the activation of the ERK pathway and this inhibitory effect suggests potential therapeutic strategies towards the prevention of allergic disorders.     

Nedocromil sodium inhibits histamine-induced itch and flare in human skin

This study was designed to test the hypothesis that nedocromil sodium inhibits sensory nerve function to reduce flare and itch in human skin. Nedocromil sodium (2%) or water (control) was introduced into the volar forearm skin of eight non-atopic volunteers by iontophoresis (8 mC) and histamine (20 microl of 1 microM and 300 nM) injected intradermally 10 min later at the same site. Itch was assessed on a visual analogue scale every 20 s for 5 min. Weal and flare areas and mean blood flux within the flare were assessed by scanning laser Doppler imaging at 10 min. The results showed that nedocromil sodium reduced itch scores, totalled over 5 min, by approximately 74.0% (P<0.005) and flare areas by approximately 65% (P<0.03). Neither weal areas nor blood flux within were reduced. These data demonstrate that nedocromil sodium is effective in reducing neurogenic itch and flare in the skin. We suggest that its mechanism of action is modulation of sensory neurone activation or conduction in the skin.     

Non cytotoxic guinea-pig mesenteric mast cell stimulation by protamine

Protamine stimulates guinea-mesenteric mast cells in a concentration-dependent manner, both histamine release and mast cell degranulation being correlated. Mast cell stimulation is blocked by 2,4-DNP (0.03 mM), low (0 degrees C) and high (45 degrees C) temperature. The inhibitory effect by 2,4-DNP is reversed by glucose (5.0 mM), while incubation at 37 degrees reverses that by low and high temperature. Lack of calcium from the incubation medium does not influence mast cell stimulation by protamine. However calcium chelation with EDTA (2.0 mM) or EGTA (2.0 mM) blocks mast cell stimulation. Addition of calcium (0.9 mM) reverses this inhibition. These observations indicate that guinea-pig mast cell stimulation by protamine is a nonlytic, energy and calcium dependent process, similar to anaphylaxis, but different from that of other basic compounds which induce mast cell lysis.     

The role of histamine in the acute inflammatory responses to intradermal platelet activating factor.

1. The role of histamine in PAF-induced acute inflammatory responses (flare and weal) in the skin has been evaluated in a series of three separate studies. 2. Terfenadine, a potent H1-selective histamine antagonist virtually abolished the flare response and significantly inhibited the weal response. 3. Histamine depletion in the skin using compound 48/80 resulted in similar effects on the flare and weal response. Two consecutive daily injections of compound 48/80 were found to deplete comprehensively skin sites of histamine and the ability of skin to respond to PAF was completely restored within 2 weeks of compound 48/80 treatment. 4. Intradermally injected PAF was associated with acute rises in plasma histamine in blood drawn from a draining vein with peak concentrations occurring within 5 min of injection. 5. No difference in PAF-induced flare and weal response was found between atopic and non-atopic subjects and this was reflected in the peak plasma histamine results. A significantly higher baseline plasma histamine was found in the atopic group, however, when compared with the non atopic group. 6. It is concluded that histamine has an important role in the acute inflammatory responses to intradermally injected PAF, although there does appear to be a significant direct vascular component in the PAF-induced weal response.     

Patients with chronic bronchitis differ in their mast cell subtypes as compared with normal subjects

Mast cells were isolated by enzymatic digestion from lung tissue obtained from patients with chronic obstructive lung disease and from normal subjects. Two mast cell subtypes could be demonstrated in human lung tissue. Mast cell subtypes were differentiated in formalin-sensitive and formalin-insensitive mast cells. It appeared that compared with normal individuals, patients suffering from chronic bronchitis had increased numbers of mast cells of the formalin-sensitive type, whereas patients with emphysema had reduced numbers, but the same ratio, of both mast cell subtypes.     

Prostaglandin D2 release from human skin mast cells in response to ionophore A23187

1. Cells were dispersed from human foreskin by proteolytic digestion and enriched or depleted in mast cell content by density gradient flotation on discontinuous gradients of Percoll. 2. Cells were harvested at six interfaces on the density gradient. Mast cell purity ranged from 0.6-85.0%, compared to 5.5% in the unfractionated cells. 3. Challenge of the cells with the calcium ionophore A23187 resulted in release of both histamine and prostaglandin D2 (PGD2). In fractions depleted of mast cells, histamine release and net PGD2 generation were low, but increasing amounts of these mediators were released as mast cell purity was increased up to 59%. 4. Overall, there was a significant correlation between the net generation of PGD2 and histamine (r = 0.9234, P less than 0.001) and also between PGD2 release and mast cell number (r = 0.7475, P less than 0.001). 5. These data provide the first direct evidence of the capacity of the human cutaneous mast cell to synthesize and release PGD2.