Remodeling of neural, glial, and tracheal cell populations in the developing Manduca wing

In the rat larynx, plasma exudation and edema formation were studied by light and electron microscopy after i.v. injections of the mast cell activator compound 48/80, substance P, and capsaicin. The morphological effects of substance P and capsaicin on connective tissue mast cells in vivo were also examined. Of the drugs tested, only compound 48/80 degranulated the connective tissue mast cells. All drugs induced a subepithelial plasma exudation in the subglottic region, with edema in the lamina propria and widened intraepithelial intercellular spaces, though the tight junction regions seemed intact. In the epiglottis, 10 min after compound 48/80 injection, there was edema in the lamina propria on the lingual side, with an intact and tight epithelial lining. No morphological sign of edema was found in the epiglottis after injection of substance P or capsaicin. The pronounced effect found in the epiglottic region after compound 48/80 injection was due to the release of mediators such as histamine and 5-hydroxytryptamine from the connective tissue mast cells. This study supports the belief that substance P in vivo mediates an increased vascular permeability by a direct effect on the blood vessels – a mechanism distinct from mast cell degranulation.     

Substance P induces granulocyte infiltration through degranulation of mast cells

Substance P, a potent vasodilatory neuropeptide, is released from peripheral nerve endings of sensory neurons by various stimuli. Although in vitro incubation of rat and human mast cells with substance P causes their degranulation, it is not known whether inflammatory changes induced by substance P are mediated by degranulation of mast cells. We investigated this point by using genetically mast cell-deficient WBB6F1-W/Wv and WCB6F1-Sl/Sld mice. The s.c. injection of substance P induced degranulation of mast cells in the skin of WBB6F1-+/+ mice, and then a marked eosinophil infiltration around the degranulated mast cells. However, WBB6F1-W/Wv and WCB6F1-Sl/Sld mice showed little or no eosinophil infiltration in the skin after the injection of substance P. When the mast cell deficiency of WBB6F1-W/Wv mice was rescued either systemically by bone marrow transplantation or locally by injection of cultured mast cells, injection of substance P induced the infiltration of eosinophils, suggesting that substance P-induced eosinophil infiltration was mediated through degranulation of mast cells.     

Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event

Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized 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.     

Functional and biochemical characterization of rat bone marrow derived mast cells

The functional and biochemical characterization of rat bone marrow derived mast cells (RBMMC) confirms both species-related differences between rat and mouse bone marrow-derived mast cells (MBMMC) as well as mast cell heterogeneity in a single species. Such RBMMC have the staining characteristics of mucosal mast cells and contain the mucosal mast cell protease. The RBMMC release the preformed granule mediator beta-hexosaminidase both in response to immunologic stimulation with 200 ng Ag (net release 15.8 +/- 3.8%) and in response to 1 microM calcium ionophore A23187 (net release 21.8 +/- 6.8%). However, compound 48/80, substance P, and somatostatin did not induce mast cell degranulation. In experiments with optimal beta-hexosaminidase release, the RBMMC generated similar quantities of the newly formed arachidonic acid metabolites leukotriene C4 and PGD2 when stimulated with either Ag or calcium ionophore A23187. The RBMMC incorporate [35S]sulfate into proteoglycans consisting of 90% chondroitin sulfates and 10% heparin. The chondroitin sulfates were comprised of chondroitin 4 sulfate and chondroitin sulfate diB sulfated disaccharides in a ratio of 4/1. Although we show that RBMMC and MBMMC share a low histamine content, functional IgE receptors and unresponsiveness to cromolyn and selective secretagogues (compound 48/80, substance P, and somatostatin), we also provide evidence that RBMMC differ from MBMMC in their profile of newly generated mediators, preformed granule proteoglycan, and lack of proliferative response to mouse IL-3.     

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

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

Strain differences in histamine release from mouse peritoneal mast cells induced by compound 48/80 or A23187

Strain differences among BALB/c, BDF1, CDF1, C3 H/He, C57 BL/6, DBA/2, ddy and ICR mice were investigated with respect to the ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80, a Ca2+ dependent histamine releaser, and the Ca2+ ionophore A23187. The ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80 were found to be high in BALB/c, ddY and ICR mice, but low in BDF1, CDF1, C3 H/He, C57 BL/6 and DBA/2 mice. Those induced by Ca2+ ionophore A23187 were high in BALB/c, BDF1, CDF1, C3 H/He, DBA2, ddy and ICR mice but low in C57 BL/6 mice. These results indicate that differences in histamine release from mouse peritoneal mast cells are strain dependent.     

Effect of ion composition on the changes in membrane potential induced with several stimuli in rat mast cells

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na⁺/K⁺ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity-stimulated depolarization is inhibited by removing sodium and potassium. © 1994 Wiley-Liss, Inc.     

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

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

Degranulation of mast cells due to compound 48/80 induces concentration-dependent intestinal contraction in rainbow trout (Oncorhynchus mykiss Walbaum) ex vivo

Rainbow trout (Oncorhynchus mykiss) intestinal strips (n = 10) were mounted in an isolated organ bath and the effect of incremental doses of compound 48/80 was recorded. Compound 48/80 induced concentration-related contraction in all the examined strips following a sigmoidal dose-response curve fit. Values for maximal contraction (E(max) , g?cm(-2)), negative logarithm of the EC(50) (pD(2)), and hill slope were, respectively (mean±standard error), 12.88 ± 0.51, 1.88 ± 0.05, 1.49 ± 0.27. The histological modification induced on mast cells (MCs) due to compound 48/80 was characterized by mean of gray-levels and texture analysis. Significant differences were observed between gray-levels values (Linear mixed model, P<0.01), contrast, and entropy (Linear mixed model, P<0.05) of MCs from compound 48/80-treated strips compared with MCs from untreated strips. Moreover, maximal intestinal contraction (due to compound 48/80) correlates positively and significantly (Pearson and Spearman correlations, P<0.05) with degranulation intensity determined by means of gray-levels analysis. Four antisera were tested on intestinal sections and no MCs positive to serotonin, substance P, met-enkephalin, and bombesin were found. This study demonstrates that compound 48/80 induces the degranulation of trout intestinal MCs ex vivo, and that the aforementioned degranulation promotes a concentration-dependent intestinal contraction.     

Novel phenotype in beagle dogs characterized by skin response to compound 48/80 focusing on skin mast cell degranulation

Beagle dogs have long been employed in toxicology studies and as skin disease models. Compared with other experimental animal species, they are known to be susceptible to skin responses, such as rashes, from exposure to various chemical compounds. Here, a unique dog phenotype was identified that showed no skin response to compound 48/80, a mast cell degranulating agent. Although the skin responses to intradermal injection of polyoxyethylene castor oil derivative (HCO-60, a nonionic detergent), histamine dihydrochloride, concanavalin A (IgE receptor-mediated stimuli), or calcium ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 were comparable in wild-type (WT) dogs and these nonresponder (NR) dogs, only the response to compound 48/80 was entirely absent from NR dogs. The skin mast cell density and histamine content per mast cell were histologically comparable between WT and NR dogs. By checking for skin responses to compound 48/80, NR dogs were found to exist at the proportion of 17–20% among four animal breeders. From retrospective analysis of in-house breeding histories, the NR phenotype appears to conform to the Mendelian pattern of recessive inheritance. The standard skin response in WT dogs developed at 2–4 months of age. In conclusion, this unique phenotype, typified by insensitivity in the compound 48/80-induced degranulation pathway in mast cells, has been widely retained by recessive inheritance in beagle dogs among general experimental animal breeders. The knowledge concerning this phenotype could lead to better utilization of dogs in studies and aid in model development.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Human skin mast cells: their dispersion, purification, and secretory characterization

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.     

Immunologic mast cell-mediated responses and histamine release are attenuated by heparin.

Heparin has been shown to act as a competitive inhibitor of inositol 1,4,5-triphosphate (InsP3) receptors in various cell types. Because InsP3 is one of the second messengers involved in stimulus-secretion coupling in mast cells, it is possible that heparin may inhibit mast cell-mediated reactions. Therefore, in allergic sheep, we tested this hypothesis in two mast cell-mediated reactions induced by immunologic and nonimmunologic stimuli: immediate cutaneous reaction (ICR) and acute bronchoconstrictor response (ABR). In 12 sheep allergic to Ascaris suum antigen, the surface area of the skin wheal was determined 20 min after intradermal injection (0.05 ml) of increasing concentrations of specific antigen, compound 48/80, and histamine, without and after pretreatment with heparin (100, 300, or 1,000 U/kg i.v.). Antigen, compound 48/80, and histamine produced concentration-dependent increases in ICR. Heparin "partially" inhibited the ICR to antigen and compound 48/80 in a dose-dependent manner without modifying the ICR to histamine. The heparin preservative benzyl alcohol was ineffective. In 11 additional sheep, specific lung resistance was measured before and after inhalation challenges with antigen, compound 48/80, and histamine without and with aerosol heparin pretreatment (1,000 U/kg). Heparin blocked the antigen- and compound 48/80-induced bronchoconstriction without modifying the airway effects of histamine. In isolated human uterine mast cells, heparin inhibited the anti-immunoglobulin E- but not the calcium ionophore- (A23187) induced histamine release. These data suggest that heparin inhibits the ICR and ABR induced by stimuli that produce immunologic and nonimmunologic mast cell degranulation without attenuating the effects of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of histamine liberation in mast cells from the lung and the intestine of the dog by isoproterenol]

Enzymatically isolated dog lung and gut mast cells were stimulated with compound 48/80, ionophore A23187, concanavalin A and FNa-Ca. Cell response elicited by A23187, concanavalin A or 48/80 is almost completely inhibited by isoproterenol. Concanavalin A induced histamine release on gut mast cells is high, indicating an elevated degree of sensitization of these cells. Results point to the existence of beta adrenergic inhibitory activity on dog lung and gut mast cells.     

Release of 14-kDa group-II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process.

Group II phospholipase A2 was detected in appreciable amounts in rat peritoneal mast cells. The effect of several inhibitors specific to 14-kDa group-II phospholipase A2, including two proteinaceous inhibitors and a product of microorganisms with a low molecular mass, on mast-cell activation was examined. When rat peritoneal mast cells were sensitized with IgE and then challenged with antigen, the specific phospholipase-A2 inhibitors suppressed histamine release in a concentration-dependent manner. By contrast, these inhibitors showed no effect on prostaglandin generation under the same conditions. Histamine release from rat peritoneal mast cells subjected to non-immunochemical stimuli, such as concanavalin A, the Ca2+ ionophore A23187, compound 48/80 and substance P was also suppressed. When rat peritoneal mast cells were treated with 14-kDa-group-II-phospholipase-A2-specific inhibitors, washed and stimulated, histamine release was not affected appreciably. Similar suppressive effects of the inhibitors on histamine release were observed with mouse cultured bone-marrow-derived mast cells. When bone-marrow-derived mast cells were activated, they secreted both a soluble and an ecto-enzyme form of 14-kDa group-II phospholipase A2, although appearance of the enzyme associated with the external surface of cells was observed transiently. An appreciable amount of membrane phospholipids was degraded during activation of mast cells, which was decreased by treatment with 14-kDa-group-II-phospholipase-A2 inhibitor. These observations suggest that degranulation and eicosanoid generation in mast cells are regulated independently by discrete phospholipases A2 and that the 14-kDa group-II phospholipase A2 released from mast cells during activation may play an essential role in the progression of the degranulation process.     

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

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

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

Summary A mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/W ^vmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [³H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [³H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.Supported by a Grant-in-Aid for Scientific Research, No. 366, from the Japanese Ministry of Health and WelfareThe authors are indebted to Drs. Motomu Minamiyama and Yukio Hirata for valuable advices, and to Miss Mitsuko Inoue for technical assistance     

Immunomodulatory activity of boswellic acids of Boswellia serrata Roxb

Extract of gum resin of B. serrata containing 60% acetyl 11-keto beta boswellic acid (AKBA) along with other constituents such as 11-keto beta-boswellic acid (KBA), acetyl beta-boswellic acid and beta-boswellic acid has been evaluated for antianaphylactic and mast cell stabilizing activity using passive paw anaphylaxis and compound 48/80 induced degranulation of mast cell methods. The extract inhibited the passive paw anaphylaxis reaction in rats in dose-dependant manner (20, 40 and 80 mg/kg, po). However, the standard dexamethasone (0.27 mg/kg, po) revealed maximum inhibition of edema as compared to the extract. A significant inhibition in the compound 48/80 induced degranulation of mast cells in dose-dependant manner (20, 40 and 80 mg/kg, po) was observed thus showing mast cell stabilizing activity. The standard disodium cromoglycate (50 mg/kg, ip) was found to demonstrate maximum per cent protection against degranulation as compared to the extract containing 60% AKBA. The results suggest promising antianaphylactic and mast cell stabilizing activity of the extract.     

Influence of ethanolic extract of Tephrosia purpurea Linn. on mast cells and erythrocytes membrane integrity

The ethanolic extract of T. purpurea Linn. was studied for its in vitro effect on rat mast cell degranulation and erythrocyte membrane integrity in vitro. The extract in concentration of 25-200 microg/ml showed a dose-dependant inhibition of rat mast cell degranulation induded by compound 48/80 and egg albumin. T. purpurea extract was found to inhibit haemolysis of erythrocytes induced by hypotonic solution but accelerated haemolysis induced by heat at a concentration of 100 microg/ml. The studies reveal that the ethanolic extract of T. purpurea may inhibit degranulation of mast cells by a mechanism other than membrane stabilization.