Mast Cell Degranulating Agent Compound Research Articles

Leptin Elicits In Vivo Eosinophil Migration and Activation: Key Role of Mast Cell-Derived PGD2.

Eosinophils are key regulators of adipose tissue homeostasis, thus characterization of adipose tissue-related molecular factors capable of regulating eosinophil activity is of great interest. Leptin is known to directly activate eosinophils in vitro, but leptin ability of inducing in vivo eosinophilic inflammatory response remains elusive. Here, we show that leptin elicits eosinophil influx as well as its activation, characterized by increased lipid body biogenesis and LTC4 synthesis. Such leptin-triggered eosinophilic inflammatory response was shown to be dependent on activation of the mTOR signaling pathway, since it was (i) inhibited by rapamycin pre-treatment and (ii) reduced in PI3K-deficient mice. Local infiltration of activated eosinophils within leptin-driven inflammatory site was preceded by increased levels of classical mast cell-derived molecules, including TNFα, CCL5 (RANTES), and PGD2. Thus, mice were pre-treated with a mast cell degranulating agent compound 48/80 which was capable to impair leptin-induced PGD2 release, as well as eosinophil recruitment and activation. In agreement with an indirect mast cell-driven phenomenon, eosinophil accumulation induced by leptin was abolished in TNFR-1 deficient and also in HQL-79–pretreated mice, but not in mice pretreated with neutralizing antibodies against CCL5, indicating that both typical mast cell-driven signals TNFα and PGD2, but not CCL5, contribute to leptin-induced eosinophil influx. Distinctly, leptin-induced eosinophil lipid body (lipid droplet) assembly and LTC4 synthesis appears to depend on both PGD2 and CCL5, since both HQL-79 and anti-CCL5 treatments were able to inhibit these eosinophil activation markers. Altogether, our data show that leptin triggers eosinophilic inflammation in vivo via an indirect mechanism dependent on activation of resident mast cell secretory activity and mediation by TNFα, CCL5, and specially PGD2.

Mast Cell Degranulation Mediates Compound 48/80-İnduced Meningeal Vasodilatation Underlying Migraine Pain

Objective: The cranial dura mater contains plenty of mast cells and is principally supplied by the middle meningeal artery which has a key role in the generation of headaches. Neurogenic inflammation caused by perivascular nerve activation and dural vasodilation is held responsible for migraine pain. Dural mast cells contribute neurogenic inflammation and migraine via vasoactive and proinflammatory mediators in their secretory granules. In the present study, it was aimed to investigate vasoactive effect of mast cell degranulating agent compound 48/80 induced dural mast cell degranulation on the middle meningeal artery and its anterior and posterior branches. Methods: Isolated skulls obtained from male Wistar rats were divided into 2 halves. The skull cavities with intact the dura mater were applied synthetic interstitial fluid for control group or mast cell degranulating agent compound 48/80 (10 μg/ml) in synthetic interstitial fluid for treated group at 37 o C for 15 min. Diameters of middle meningeal artery and its anterior and posterior branches were measured and mast cells were counted from whole-mount preparations of meningeal dura mater. Results: While compound 48/80 induced massive degranulation of dural mast cells (P<0.01), it did not change the number of mast cells in the dura mater. Moreover, compound 48/80 increased diameter of middle meningeal artery (P<0.01) and its anterior (P<0.05) and posterior (P<0.01) branches, respectively compared to synthetic interstitial fluid treatment. Conclusion: Dural mast cell degranulation causes dilatation of middle meningeal artery which is involved in the pathophysiology of migraine, therefore testing of mast cell stabilizing agents in vivo models of migraine pain may promise hope for the next big things in the treatment of migraine headaches.

Degranulation of eosinophilic granule cells in neurofibromas and gastrointestinal tract in the bicolor damselfish

Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting bicolor damselfish ( Stegastes partitus Poey) on Florida reefs. Previous studies have demonstrated high densities of eosinophilic granule containing cells (EGC), the proposed equivalent of mast cells in fishes, in neurofibromas and malignant peripheral nerve sheath tumors (mpnst) in DNF. These lesions are similar to those in the disease neurofibromatosis type 1 (NF1) in humans, which contain large numbers of mast cells. In the present study, experiments were conducted to measure the response of EGC in these tumors as well as in the submucosa of the digestive tract to the mast cell degranulating agent compound 48/80. Degranulation of these cells was visible by light microscopy and characterized by conspicuous swelling of granules and often by the presence of free granules adjacent to the EGC. Degranulation occurred by release of intact granules (diacytosis), as reported in other fishes, rather than by fusion of granules with the cell membrane (exocytosis) as reported in mast cells in mammals. Baseline levels of EGC exhibiting degranulation ranged from 20–26% in the submucosa to 30% in tumors. Within 1–2 h of exposure to compound 48/80, significant increases in average levels of degranulation were observed, to 67% in the gut and 72% in tumors. Degranulation was significantly more extensive in the tumors than in the gut. The outermost edges of the tumors contained significantly higher densities of EGC but these cells exhibited lower rates of degranulation than those in the inner regions of tumors. These observations support the hypothesis that the EGC present in neurofibromas and mpnst in DNF are equivalent to the mast cell component in neurofibromas in NF1.

Suppression of adenosine A(3) receptor-mediated hypotension and mast cell degranulation in the rat by dexamethasone.

Dexamethasone increases the expression of adenosine A(3) receptors and augments degranulation in response to their activation in the rat basophilic leukemia cell line, RBL-2H3. We have studied the effects of dexamethasone on mast cell activation induced by A(3) receptor stimulation in vivo. Administration of the A(3) receptor agonist APNEA [N(6)-2-(4 aminophenyl)ethyladenosine; 10-30 microg kg(-1) i.v.] to anesthetized Sprague-Dawley rats induced falls in blood pressure. Pretreatment with dexamethasone (1 mg kg(-1), i.p., -24 h) blocked the hypotensive response to APNEA but not those induced by the A(1) receptor agonist N(6)-cyclopentyladenosine, the A(2A) receptor agonist 2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine, or the mast cell degranulating agent compound 48/80 (100-300 microg kg(-1), i.v.). APNEA (10 and 30 microg kg(-1), i.v.) and compound 48/80 (100 and 300 microg kg(-1), i.v.) increased plasma histamine concentrations dose dependently. Pretreatment with dexamethasone significantly inhibited the increases induced by the lower doses of each compound. APNEA induced degranulation of mast cells in thymus but not in skin or skeletal muscle, whereas compound 48/80 induced degranulation in each tissue. Pretreatment with dexamethasone inhibited APNEA-induced degranulation of mast cells in the thymus and slightly, yet significantly, reduced degranulation induced by compound 48/80. Thus, in contrast to the findings in RBL-2H3 cells in vitro, in the whole animal, dexamethasone down-regulates the response of the mast cell to A(3) receptor activation. The qualitatively similar effects on compound 48/80 suggest that dexamethasone suppresses mast cell responsiveness by modulating site(s) downstream from the adenosine A(3) receptor, possibly at the level of the G(i) family of trimeric GTP-binding proteins.

Interstitial fluid pressure surrounding rat mesenteric venules during changes in fluid filtration.

The interstitial fluid pressure (P(isf)) has been measured in the exposed superfused mesenteries of anaesthetised rats using the micropipette servo-null technique. When mesenteries were superfused with Ringer-Locke solutions, P(isf) was close to atmospheric pressure with mean +/- S.E.M. values of -0.46 +/- 0.14 cmH(2)O (n = 22). Superfusing with paraffin oil did not alter P(isf) significantly, but P(isf) could be lowered considerably by removing fluid from the upper surface of the mesentery. Measurements of P(isf) were also made in the tissues immediately outside mesenteric venules as the pressure inside these vessels and the filtration of fluid through their walls was varied. No significant changes in perivascular P(isf) could be detected even though the intravascular pressure varied from 20 to 70 cmH(2)O. Addition of histamine or the mast cell degranulating agent compound 48/80 to the superfusate had no significant effect on P(isf). The findings are relevant to experiments on the permeability of single perfused mesenteric microvessels. They strengthen the assumption, which is made in these studies, that P(isf) is close to atmospheric pressure and does not change significantly with changes in the filtration and reabsorption of fluid through the vessel walls. Experimental Physiology (2001) 86.1, 33-38.

Mast cell tryptase in dermal neurogenic inflammation.

Mast cell activation has been assumed to play a role in dermal neurogenic inflammation: C fibre-derived neuropeptides activating mast cells and releasing histamine, which in turn would activate C fibres. To test this hypothesis mast cell tryptase (MCT) was measured inside the axon reflex flare area. Axon reflexes were elicited by histamine or compound 48/80, a polyanionic mast cell-degranulating substance. The time course of plasma extravasation and release of histamine and MCT from dermal mast cells in neurogenic inflammation was measured in vivo by intradermal microdialysis in humans. Single hollow plasmapheresis fibres (pore cutoff size: 3000 kDa) were inserted intracutaneously at the volar forearm and perfused with Ringer's solution (4 microL/min) with one microdialysis fibre located at the planned stimulation site and a second inside the axon reflex area. Neurogenic inflammation was induced by intraprobe delivery of either histamine or the mast cell-degranulating agent compound 48/80. Mediator release was measured at the stimulation sites and inside the arising axon reflex flare area. Mast cell degranulation induced marked plasma protein extravasation (PPE 0.25 +/- 0.04-1.31 +/- 0.6 mg/mL; pre- and post-stimulation, mean +/- sem, n = 7) and release of histamine (2.0 +/- 0.9-38.7 +/- 1.4 ng/mL) and MCT (9.84 +/- 2.4-92.2 +/- 21.6 ng/mL). Interestingly, in addition to increasing PPE (0.33 +/- 0. 11-1.85 +/- 0.9 mg/mL), histamine also induced a slight but significant increase in MCT (11.3 +/- 3.0-12.4 +/- 2.3 ng/mL). No evidence for mast cell activation was observed inside the axon reflex areas, where PPE (0.34 +/- 0.03-0.25 +/- 0.02 mg/mL), histamine (1.64 +/- 0.5-1.46 +/- 0.4 ng/mL) and MCT concentration (11.6 +/- 3.1-7.6 +/- 1.7 ng/mL) gradually decreased. It is concluded that dermal neurogenic inflammation does not degranulate mast cells.

Nitric Oxide Inhibits Numerous Features of Mast Cell–Induced Inflammation

We previously reported that mast cell degranulation causes histamine and P-selectin-dependent leukocyte rolling and platelet-activating factor (PAF)- and CD18-associated leukocyte adhesion, whereas others have reported serotonin-induced edema formation. The purpose of the present study was to determine whether nitric oxide (NO) could inhibit the mast cell-induced multistep recruitment of leukocytes and the associated microvascular dysfunction in single inflamed venules. Intravital fluorescence microscopy was used to demonstrate increased leukocyte rolling and adhesion and increased albumin extravasation in single 25- to 40-microns venules that were treated with the mast cell-degranulating agent compound 48/80 (CMP 48/80). The mast cell-induced histamine-dependent rolling and PAF-dependent adhesion were completely inhibited by the addition of the NO donor spermine NO. However, spermine NO did not directly inhibit histamine-induced leukocyte rolling and only partly affected PAF-induced leukocyte adhesion. Compound 48/80-activated mast cells evoked a significant increase in PAF-dependent neutrophil adhesion in vitro. Spermine-NO prevented the mast cell-dependent neutrophil adhesion but failed to affect direct adhesion with PAF. The mast cell-induced albumin leakage was also inhibited by the NO donor. Taken together, these results suggest that exogenous NO can modulate leukocyte recruitment and microvascular permeability alterations elicited by mast cell activation and raises the possibility that the use of NO donors may be a reasonable therapeutic approach to reducing mast cell-dependent inflammation.

Microvascular leakage induced by substance P in rat urinary bladder: involvement of cyclo-oxygenase metabolites of arachidonic acid.

1. Intravenous administration of substance P (SP) or of the NK1 selective agonist [beta-Ala4, Sar9, Met (O2)11] SP-(4-11) increased vascular permeability in the urinary bladder of urethane-anaesthetized rats, providing evidence for an NK1 receptor-mediated inflammatory response. 2. BW 755C, a dual inhibitor of arachidonate cyclo-oxygenase and lipoxygenase, significantly reduced the plasma extravasation induced by SP, but did not modify the effect of [beta-Ala4, Sar9, Met (O2)11] SP-(4-11). 3. SP-induced microvascular leakage was also inhibited by systemic pretreatment with indomethacin or with the prostaglandin receptor antagonist SC-19220, while it was unaffected by the selective 5-lipoxygenase inhibitor BW A4C or the leukotriene antagonist FPL 55712. 4. Pretreatment of rats with the mast cell degranulating agent compound 48/80 significantly attenuated the inflammatory effect of SP. Indomethacin administration to 48/80-pretreated animals failed to produce further inhibition. 5. These findings indicate that intravascular SP promotes plasma exudation in rat urinary bladder through an NK1-mediated effect on venular permeability and the release of cyclo-oxygenase metabolites of arachidonic acid. The latter effect largely derives from the interaction of the neuropeptide with mast cells.

Antigen-induced Release of Airway Epithelium-derived Inhibitory Factor

The ability of guinea pig trachea to produce a vasoactive epithelium-derived inhibitory factor (EpDIF) in response to mast cell-derived mediators was assessed in a coaxial bioassay system. The mast cell degranulating agent compound 48/80 (10 micrograms/ml) and histamine caused reductions in phenylephrine-induced tone in endothelium-denuded rat aorta preparations mounted coaxially within epithelium-intact guinea pig tracheal tube tissue. Relaxation responses to histamine and to compound 48/80 (10 micrograms/ml) were markedly reduced in the presence of mepyramine (50 microM) or when the epithelium was removed from coaxially mounted guinea pig trachea, indicating that they were mediated via the release of EpDIF. Coaxial bioassay assemblies were also prepared using EpDIF donor tracheal tissue obtained from guinea pigs actively sensitized to ovalbumin. Subsequent challenge with ovalbumin (10(-7) to 10(-1) mg/ml) produced concentration-dependent relaxation mediated by EpDIF. Ovalbumin-induced relaxation responses were not inhibited in the presence of either mepyramine (20 and 100 microM) or SKF 104353-Z2 (10 microM) alone but were significantly reduced when both mepyramine (20 microM) and SKF 104353-Z2 (10 microM) were present. Antigen-induced relaxation was apparently mediated by EpDIF in response to mast cell-derived histamine and leukotrienes. Rat tracheal airway smooth muscle did not relax in response to EpDIF, suggesting selectivity of action on vascular smooth muscle. Vasoactive EpDIF may play a role in protecting against antigen-induced bronchoconstriction by regulating bronchial circulation flow.

Prostaglandin D2 Release by Guinea Pig Skin During In Vitro Anaphylaxis Induced by Antigen and Compound 48/80

The release of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), and histamine induced by antigen and compound 48/80 was studied using an in vitro model of anaphylaxis in guinea pig skin. Abdominal skin from ovalbumin-sensitized guinea pigs was cut into 0.5-1.0 mm-thick slices which were incubated in Tyrode solution at 37 degrees C with or without either ovalbumin or 48/80. Released PGD2 and PGE2 were measured by radioimmunoassay and gas chromatography-mass spectrometry, respectively. Release of PGD2 was detectable at 2 min after challenge (50 micrograms/ml ovalbumin), reaching a maximum at about 15 min. Histamine release was more rapid, achieving 50% of maximum at about 4 min compared to about 7 min for PGD2. In 11 experiments incubation with ovalbumin (50 micrograms/ml for 10 min) induced a significant 6-fold increase in PGD2 compared to unchallenged controls (399 +/- 53 and 67 +/- 19 ng/g dry weight skin, respectively; mean +/- SEM) and a net 47.2% histamine release. In contrast, a smaller (27%) rise in PGE2 was found. Indomethacin (14 microns) completely suppressed evoked PGD2 and PGE2 synthesis without evident effect on histamine release, suggesting that the release of histamine in this model is not dependent on prostaglandin production. The mast cell degranulating agent compound 48/80 (50 micrograms/ml) released significant amounts of PGD2 (340 +/- 86 ng/g skin compared to 89 +/- 30 ng/g for control skin, n = 5) but had no appreciable effect on PGE2. These results show that guinea pig skin can synthesize significant quantities of PGD2 in anaphylactic reactions. Prostaglandin D2 produced in acute allergic reactions in skin in vivo may contribute to the inflammatory reaction, either directly or in synergism with other mediators.

Identification of a high molecular weight alkaline protease in rat heart

We have identified in soluble extracts of rat heart, a 500 000 dalton sulfhydryl-dependent protease which degrades globin and casein to acid-soluble peptides at an alkaline pH optimum. This enzyme was purified more than 1700-fold with respect to the postmicrosomal supernatant. On the basis of various catalytic and biochemical properties the enzyme appears similar to a recently described cytoplasmic protease in rat liver. Protease activity in vitro was stimulated up to 3-fold by physiologic concentrations of ATP and to a lesser extent by some other phosphate-containing compounds. Unlike some alkaline proteases reported in heart tissue, this high molecular weight protease was identified in extracts from isolated cardiac myocytes and in extracts from hearts of rats treated with the mast cell degranulating agent Compound 48 80 . Thus, the identification of the protease in heart does not appear to be accounted for by mast cell contamination.

Mast cells from human respiratory tissue and their in vitro reactivity.

Human respiratory mast cells, which were obtained coincidentally with diagnostic bronchial brush biopsy, were maintained under conditions for short-term tissue culture. Most mast cells were viable and degranulated on exposure to antibody to immunoglobulin E or to the mast cell degranulating agent compound 48-80. The degranulation of human mast cells is characteristically an intracellular process with no extracellular extrusion of granules.