Monoclonal murine anti-DNP IgE:in vitro histamine release of rat mast cells in the presence of reaginic rat and mouse sera

Mast cells from normal rats and animals reinfected withNippostrongylus brasiliensis (N.b.) were sensitizedin vitro with monoclonal anti-DNP mouse IgE, reaginic mouse serum with ovalbumin (OA) specificity and reaginic rat serum against N.b. The sensitized cells were triggered for the release of histamine with DNP-bovine-serum-albumin (DNP-BSA), OA, N.b.-homogenate and guinea-pig antimouse IgE. The histamine release from normal mast cells sensitized with monoclonal mouse IgE was inhibited either with N.b.-reaginic rat serum or OA-reaginic mouse IgE (30g/10⁵ mast cells) completely densensitized mast cells from reinfected rats for specific histamine release in the presence of either N.b.-antigen or DNP-BSA. Anti-mouse IgE which was prepared by monoclonal mouse IgE did not bind to rat mast cells sensitized with rat IgE as was revealed by immunofluorescence experiments. Consequently we observed that anti-mouse IgE failed to trigger the histamine release from mast cells of reinfected rats.     

Histamine modulates mast cell degranulation through an indirect mechanism in a model IgE-mediated reaction

Histamine is released in inflammatory reactions and exerts an immunoregulatory function on cells present in the microenvironment. In this study, we compared the effect of histamine on degranulation of mast cells derived from animals bearing a parasitic infection with those from uninfected animals. Peritoneal mast cells (PMC) were obtained 24 days after infection of Wistar rats with Toxocara canis. The degree of degranulation was assessed either morphologically or by measuring the release of beta-hexosaminidase and TNF-alpha. Non-purified PMC or mast cells immunomagnetically purified with mAb AA4 were used. An increase in degranulation of non-purified mast cells from infected animals was observed after incubation with histamine in vitro or when histamine was injected into the peritoneal cavity. When a purified mast cell population was used, this effect was no longer observed. Supernatants from spleen cells stimulated with histamine induced degranulation of purified mast cells, and again, this was potentiated with PMC from infected animals. However, when supernatants from peritoneal macrophages similarly stimulated were used, a reduction in the degranulation of PMC from infected animals was observed. Our results suggest that histamine may act as a regulator of mast cell degranulation, thus modulating inflammatory responses due to infection with certain parasites.     

IgE receptors, IgE content and secretory response of mast cells in athymic rats.

Athymic, nude (Lewis rnu/rnu) and normal (Lewis +/+) rats were used to study the T-cell dependence of the regulation of IgE receptors and IgE content on mast cells in vivo. We estimated the number of IgE receptors and the IgE content on peritoneal mast cells using a cytofluorometric technique. The secretory capacity of the mast cells was measured in terms of histamine release as a function of anti-IgE concentration by incubation with antibodies in vitro. Two groups of rats, aged 6 and 13 weeks, were examined. The mast cells of the rats belonging to the older age group (both normal and athymic) had a higher total protein (equivalent to dry mass or size) and mediator content (heparin, histamine and 5-hydroxytryptamine) in accordance with previous findings. Mast cells of the athymic rats of both age groups contained similar levels of IgE receptors and IgE and did not differ in these respects from those of the normal rats. Of the IgE receptors available for binding, about 80% were occupied by IgE in mast cells of normal and of athymic rats in both age groups. The anti-IgE-induced histamine release of the mast cells was, however, significantly lower in athymic rats compared to the normal controls. A change in housing from barrier-maintained to conventional conditions for 2 weeks enhanced the IgE-receptor and IgE content, as well as the releasability of histamine of the mast cells of both athymic and normal rats. The basal level of IgE occupancy of the receptors on mast cells was therefore not impaired in the athymic rats, as might be inferred from previous findings of a T-cell dependence of the IgE immune response. The results further indicate that T-lymphocyte-derived cytokines appear not to be required for either the expression of IgE receptors or for their ability to acquire IgE on mast cells, but such factors seem to enhance the release of histamine following the cross-linkage of the IgE receptor on mast cells in normal conditions.     

Ventricular histamine concentrations and mast cell counts in the rat heart during acute ischaemia

The ventricular histamine concentrations and mast cell counts of naive and disodium cromoglycatetreated rats subjected to acute left coronary artery ligation under pentobarbitone anaesthesia were examined. In naive animals, there was a significant increase in the right ventricular histamine level at 2 min following left coronary artery ligation. Left ventricular histamine concentrations tended to decrease, and were significantly lower than those of the right ventricle at 5 min. However, there were no significant changes in mast cell counts of the right or left ventricles after left coronary artery ligation. Treatment with disodium cromoglycate did not significantly alter the ventricular mast cell counts, interfere with the changes in ventricular histamine concentrations, or the occurrence of early ventricular arrhythmias and haemodynamic changes in response to acute left coronary artery ligation. It is suggested that the increase in the right and decrease in the left ventricular histamine concentrations during acute myocardial ischaemia involves mainly the non-mast cell stores, instead of mast cell sources, of cardiac histamine.     

The role of mast cell activation in cholestatic pruritus

Jaundiced patients experience intense pruritus, the pathophysiology of which is unclear. In this study, blood histamine concentrations, skin mast cell counts and intracellular histamine concentrations in peritoneal mast cells were examined in an experimental model of biliary obstruction. Three weeks after bile duct ligation (BDL), total blood histamine concentrations were significantly elevated compared with those from control animals (p<0.0001). Skin mast cell counts were increased (p<0.05) and peritoneal mast cell histamine content decreased (p<0.05) in jaundiced animals. These results demonstrate that mast cells degranulate in biliary obstruction with consequent release of histamine into the systemic circulation. This may contribute to cholestatic pruritus. These data may have significant pharmacological implications in patients with obstructive jaundice.     

Increased tissue survival in experimental skin flaps in mast cell‐deficient rats

Aim: The role of mast cells and their principal mediator, histamine, in surgical skin flap survival was investigated using mast cell‐deficient (Ws/Ws); their congenic littermates wild‐type (+/+), and Wistar rats. Methods: A standardized dorsal skin flap was raised and sutured back into position, and 6 days later the percentage of flap survival was assessed. Moreover, endogenous histamine concentration in the dorsal skin during the surgical preparation was determined using in vivo microdialysis technique together with high performance liquid chromatography‐fluorometry. Accumulation of skin flap myeloperoxidase (MPO) (reflecting leucocyte recruitment) was determined spectrophotometrically. Results: The experimental skin flaps in genetically mast cell‐deficient rats exhibited increased tissue survival and showed little accumulation of MPO and rather low and stable level of histamine output in comparison with skin flaps in the wild‐type (+/+) littermates or normal Wistar rats. Antihistamine treatment inhibited but did not prevent leucocyte recruitment in the skin flaps post‐surgery in +/+ and Wistar rats. Conclusion: It is suggested that mast cell derived histamine plays an important role in leucocyte recruitment in skin flaps. However, mast cell‐independent factors should be taken into consideration and needs further investigation as even in mast cell‐deficient animals there was some accumulation of leucocytes and tissue necrosis in the skin flaps post‐surgery.     

Increased tissue survival in experimental skin flaps in mast cell-deficient rats

AIM: The role of mast cells and their principal mediator, histamine, in surgical skin flap survival was investigated using mast cell-deficient (Ws/Ws); their congenic littermates wild-type (+/+), and Wistar rats. METHODS: A standardized dorsal skin flap was raised and sutured back into position, and 6 days later the percentage of flap survival was assessed. Moreover, endogenous histamine concentration in the dorsal skin during the surgical preparation was determined using in vivo microdialysis technique together with high performance liquid chromatography-fluorometry. Accumulation of skin flap myeloperoxidase (MPO) (reflecting leucocyte recruitment) was determined spectrophotometrically. RESULTS: The experimental skin flaps in genetically mast cell-deficient rats exhibited increased tissue survival and showed little accumulation of MPO and rather low and stable level of histamine output in comparison with skin flaps in the wild-type (+/+) littermates or normal Wistar rats. Antihistamine treatment inhibited but did not prevent leucocyte recruitment in the skin flaps post-surgery in +/+ and Wistar rats. CONCLUSION: It is suggested that mast cell derived histamine plays an important role in leucocyte recruitment in skin flaps. However, mast cell-independent factors should be taken into consideration and needs further investigation as even in mast cell-deficient animals there was some accumulation of leucocytes and tissue necrosis in the skin flaps post-surgery.     

Effect of sensitization on spontaneous and phosphatidylserine-induced histamine release and on cyclic AMP and GMP levels in isolated rat mast cells.

Sprague Dawley rats were sensitized with 20 microgram or 100 mg egg albumin (using pertussis vaccine as adjuvant). Mast cells isolated from the former group of animals showed a higher degree of histamine release upon challenge in vitro with egg albumin than those from the latter group. Using the lower amount of antigen for immunization mast cells from Hooded Lister rats showed an even higher degree of histamine release induced by antigen. An increased antigen-induced histamine release was associated with an increased spontaneous and phosphatidylserine-induced histamine release. Histamine release induced by phosphatidylserine was found to be specific in so far as it was calcium dependent and theophylline-inhibited. The basal level of cyclic AMP in mast cells was significantly depressed by sensitization. There was a relationship between the cyclic AMP/cyclic GMP ratio and the degree of spontaneous, phosphatidylserine-induced and anaphylactic histamine release. The results suggest that sensitization induces an increased release of histamine not only to the specific antigenic stimulus but also to more unspecific stimuli. Concomitantly there is a fall in the cyclic AMP/cyclic GMP ratio. The relationship between these two phenomena is discussed.     

Rat lymphoid cell--derived histamine and 5-hydroxytryptamine releasing factor.

The in vitro production of histamine releasing factor (HRF) by lymphoid cells of rats, both normal and infected with Nippostrongylus brasiliensis, has been studied. Spleen cells and thymocytes were cultured either alone or in the presence of mitogen (PHA, 10 and 50 micrograms/ml) and the dialysed cell-free supernatants were tested for histamine releasing activity on rat peritoneal and pleural mast cell in vitro. We found that spleen cells and thymocytes of normal rats stimulated with PHA in 24 h cultures generated a factor which released histamine and 5-hydroxytryptamine from mast cells, and this ability was potentiated following N. brasiliensis infection of rats - lymphoid cells donors. Pleural mast cells were more sensitive to the action of HRF than peritoneal cells. Rat HRF had an apparent m.w. of 50,000 to 70,000 daltons as determined by gel chromatography and was a heat stable protein inducing histamine release from homologous mast cells in a very rapid (complete in 1-2 min at 37 degrees C), dose and temperature dependent secretory process.     

Interaction of adriamycin with rat and mouse mast cells: Histamine release and cellular uptake

In the present study, we evaluated in mouse and rat mast cells if adriamycin uptake is related to histamine release. In addition, the uptake of the antineoplastic drug and histamine release were studied in other normal and neoplastic cell lines. The effect of sodium cromoglycate was also examined.Adriamycin induced histamine release from mixed or purified mast cells of the mouse and rat. This exocytotic response was quantitatively related to intracellular concentrations of adriamycin. Significant differences in adriamycin uptake were observed between mast cells and other normal and neoplastic cells. When peritoneal mast cells were treated with sodium cromoglycate, histamine release and adriamycin uptake were significantly reduced. In contrast, incubation of other cells with cromoglycate only slightly reduced the cellular concentration of the antineoplastic drug.     

Immature peritoneal mast cells in neonatal rats express the CTMC phenotype, as well as functional IgE receptors.

The purpose of this study was to investigate the relationship between the differentiation and maturation of mast cells and the expression of IgE receptors on their surface in neonatal animals in vivo. Another aim was to clarify whether connective tissue mast cells (CTMC) undergo a maturation process involving a transdifferentiation from mucosal mast cells (MMC) during this period of time. Mast-cell phenotypes were studied in terms of the profiles of proteinases and proteoglycan. In 1-week-old rats, the mast-cell granules stained with Alcian blue rather than with safranin (AB+/S-) in the Alcian blue/safranin staining sequence, normally regarded as a property of MMC. However, the AB+/S-stained proteoglycan was degradable by nitrous acid and stained with berberine sulphate, thus indicating that it contained heparin rather than chondroitin sulphate. The mast cells expressed rat mast-cell proteinase (RMCP) I rather than RMCP II, which is normally found in MMC. The mast cells of 1-week-old rats expressed functional IgE receptors, by showing a dose-dependent IgE-mediated histamine release of mast cells. About 70% of the IgE receptors on the mast cells were occupied by IgE. In 2- to 3-week-old rats, there was a progressive increase in mast cells stained with both Alcian blue and safranin or with safranin alone, i.e. they gradually changed towards the staining properties of CTMC (AB-/S+). The expression and the degree of IgE occupancy of the receptors increased in 1- to 3-week-old animals. This was paralleled by an increment in cell size and in the content of heparin, histamine and serotonin in the mast cells. The findings thus indicate that the peritoneal mast cells of neonatal rats express the CTMC phenotype and undergo a maturation process at from 1 to 3 weeks of age, without involving a transdifferentiation from MMC. The maturation of the mast cells is accompanied by an increase in the expression of functional IgE receptors on the cell surface. production was detectable as early as in 1-week-old rats.     

Anaphylactic histamine release from peritoneal mast cells of two inbred strains of rats sensitized with mouse IgE

Peritoneal mast cells from rats of BH- and DA-inbred strains were compared for their capacity to passive sensitization in vitro with mouse IgE antibodies. In identical experimental conditions mast cells of BH-rats were good receptors for mouse IgE anti-KLH fraction and released histamine when challenged with specific antigen, whereas mast cells of DA-rats were much less sensitized and released little histamine on the challenge. In contrast, mast cells of DA-rats were more susceptible to the challenge with anti-rat IgE and Concanayalin A, than were cells of BH-rats. This susceptibility correlated with total serum IgE level, which was higher in DA- than in BH-rats. These results show higher concentrations of IgE on mast cells of DA-rats. It is suggested that rat IgE present on normal mast cells may be one of the factors interfering in in vitro sensitization of the cells from DA-rats with mouse IgE antibodies.     

Adoptive transfer of mast cells abolishes the inflammatory refractoriness to allergen in diabetic rats

Mast cells are pivotal secretory cells primarily implicated in allergen-evoked inflammatory responses and are downregulated following experimental chemical diabetes. Here we tested the hypothesis that a decrease in the number and reactivity of mast cells would account for the hyporesponsiveness of diabetic rats to allergen-induced inflammation. We found that the anaphylactic release of histamine from sensitized ileum, trachea and skin tissues was clearly reduced in rats turned diabetic via intravenous administration of alloxan. Likewise, actively and passively sensitized diabetic rats mounted a weaker allergen-evoked pleural mast cell degranulation and protein extravasation, as compared to sensitized nondiabetic animals, which paralleled a marked reduction in the mast cell population in the pleural cavity. The number of mast cells enumerated in the mesentery from diabetic rats was also clearly reduced. The allergen-evoked plasma leakage in diabetic rats was restored by the transfer of mast cells from sensitized nondiabetic rats. Moreover, the adoptive transfer of sensitized mast cells from diabetics to naive animals led to a lower allergen-induced exudation as compared to the response noted after the transfer of sensitized naive mast cells. Purified mast cells from diabetic rats were hyporesponsive to antigen and compound 48/80 stimulation in vitro as attested by histamine release. Thus, our results show that the phenomenon of refractoriness of diabetic animals to allergen challenge appears to be accounted for by a reduction in the number and reactivity of mast cells.     

Lysophosphatidylserine as histamine releaser in mice and rats

Lysophosphatidylserine is a specific inducer of histamine release in isolated mast cells. To determine whether a similar effect is manifestin vivo, the phospholipid was injected (1–5 mg/kg i.v.) into mice and rats. A dosedependent rise in blood histamine was observed in both animals. The several-fold increase in blood histamine occurred in the first minutes and was followed by a slower decline toward normal values. A second dose of lysophosphatidylserine was without effect. Systemic manifestations (depression, hypothermia, hypotension) were associated with the increased blood histamine level. When the tissue histamine stores accessible to lysophosphatidylserine were previously decreased by repeated phospholipid injections, no systemic symptoms occurred. Mobilization of carbohydrate reserves was also manifest during the action of lysophosphatidylserine. Prior treatment with compound 48/80 induced sustained refractoriness to lysophosphatidylserine. Structure-activity relationship demonstrated that the property to induce histamine release was linked to the structure of serine head group. Thus, other natural phospholipids or lysophospholipids were inactive. It is concluded that in analogy with the effect seenin vitro lysophosphatidylserine producesin vivo release of mast cell histamine.     

Mast cell hyperplasia in experimental hypersensitivity pneumonitis.

We investigated the presence of mast cells in a model of experimental hypersensitivity pneumonitis (EPH). Guinea pigs exposed to 8 weekly intratracheal challenges with Micropolyspora faeni exhibited significant increases in the number of mast cells within the lung as compared to controls and animals challenged only 2 or 4 times. The number of cells in M. faeni-challenged animals were increased around bronchi, bronchioles, blood vessels and in alveolar septa. There appeared to be contraction of peribronchial, peribronchiolar and vascular smooth muscle. Ultrastructural examination of lung tissue revealed the presence of degranulating mast cells. Bronchoalveolar lavage histamine levels were increased after 8 but not after 2 or 4 weekly challenges. Serum anti-M. faeni antibody was present in all M. faeni-exposed animals but not in control animals. We conclude that mast cells and histamine levels in bronchoalveolar lavage fluid are increased in a model of EHP caused by repetitive, intratracheal injection of M. faeni particulate antigen.     

The role of mast cell activation in cholestatic pruritus

Jaundiced patients experience intense pruritus, the pathophysiology of which is unclear. In this study, blood histamine concentrations, skin mast cell counts and intracellular histamine concentrations in peritoneal mast cells were examined in an experimental model of biliary obstruction. Three weeks after bile duct ligation (BDL), total blood histamine concentrations were significantly elevated compared with those from control animals (p<0.0001). skin=" mast=" cell=" counts=" were=" increased=">p<0.05) and=" peritoneal=" mast=" cell=" histamine=" content=" decreased=">p<0.05) in=" jaundiced=" animals.=" these=" results=" demonstrate=" that=" mast=" cells=" degranulate=" in=" biliary=" obstruction=" with=" consequent=" release=" of=" histamine=" into=" the=" systemic=" circulation.=" this=" may=" contribute=" to=" cholestatic=" pruritus.=" these=" data=" may=" have=" significant=" pharmacological=" implications=" in=" patients=" with=" obstructive=">     

The intracerebroventricularly administered mast cells degranulator compound 48/80 increases the pituitary-adrenocortical activity in rats

The effect of brain mast cells degranulation by compound 48/80 on the pituitary-adrenocortical activity, measured indirectly through corticosterone secretion, and the involvement of a histaminergic mechanism in that stimulation was investigated in conscious rats. All the drugs were given intracerebroventricularly (icv), histamine antagonists 15 min prior to compound 48/80. Compound 48/80 induced a significant dose- and time-related increase in the serum corticosterone levels. That increase, measured 1 h after adminstration of compound 48/80, was moderately diminished by icv pretreatment of rats with mepyramine and cimetidine, histamine H₁- and H₂-receptor antagonists. Three hours after administration of compound 48/80 mast cells of the thalamus and the hypothalamus were completely degranulated. At the same time the thalamus and the whole brain histamine levels were substantially higher than in the saline-treated control rats.The above results suggest that histamine liberated from the brain mast cells and central histamine receptors play a moderate role in increasing the pituitary-adrenocortical activity by compound 48/80.The study was supported by the Polish Academy of Sciences, grant No. 06.03.     

Migration of neutrophils is dependent on mast cells in nonspecific pleurisy in rats.

To determine the role of mast cells in the recruitment of neutrophils and eosinophils, acute nonspecific pleurisy was induced by injecting isologous serum into normal +/+ and mast cell-deficient Ws/Ws rats. In +/+ rats, neutrophil infiltration peaked 4 h after serum administration, followed by influx of eosinophils after 24-48 h. The levels of neutrophil influx after 4 h as well as the activity of myeloperoxidase (MPO) in pleural lavage-cell extract were significantly lower in Ws/Ws rats than in +/+ rats. In contrast, numbers of eosinophils as well as activity of eosinophil peroxidase (EPO) did not differ significantly between Ws/Ws and +/+ rats. For local reconstitution of mast cells, +/+ rat peritoneal mast cells (PMC) or mesenteric lymph node cells (MLNC) as a control were transferred into the Ws/ Ws pleural cavity. Serum injection into animals with PMC transfer 7 days previously triggered augmented neutrophil influx by approximately 4.7-fold as compared to that in MLNC-transferred animals. Mast cells recovered from the pleural cavity of PMC-transferred rats showed histamine contents equivalent to 20% of that of freshly isolated PMC and retained the reactivity to compound 48/80. These results indicated that dependency of neutrophil recruitment on resident mast cells is greater than that of eosinophils in isologous serum-induced pleurisy.     

The effect of zinc ions on selective and nonselective histamine release in vitro

The effect of different concentrations of zinc ions on the in vitro release of histamine from isolated rat peritoneal mast cells induced by compound 48/80 and Triton X-100, as well as the influence of the in vivo administration of zinc ions on the subsequent in vitro release of histamine from isolated peritoneal mast cells and lung tissue, have been investigated. The results show that intraperitoneal administration of zinc for 20 days significantly inhibited the spontaneous release of histamine from lung tissue as well as that induced by a low dose of compound 48/80. The in vitro addition of zinc ions to isolated mast cells significantly inhibited the release of histamine induced by low doses of compound 48/80 but not by high doses or by Triton X-100. Inhibition of histamine release increased up to 100% with increase in the zinc ions concentration up to 10^?3 M. An inhibition of the histamine release from mast cells and lung tissue from animals which had received in vivo injection of zinc was also observed. The results indicate that the inhibitory action of zinc ions on the release of histamine is connected with an action on the biomembrane rather than on the histamine binding within the mast cells.     

Histamine content and mast cell numbers in tissues of normal and athymic rats

Tissue histamine levels and mast cell numbers were determined in the skin, tongue and jejunum of female rnu/nu and rnu/+ rats aged between 5 and 29 weeks. The tongue and jejunal mucosa of rnu/nu rats had a larger mast cell density and histamine content than rnu/+. There was a marked increase in subepithelial mast cells in the skin of rnu/nu rats compared with their normal littermates, while mast cell numbers in the deep skin layer and the histamine content were similar in the two groups of rat. Subepithelial skin mast cells were smaller, of more variable shape and contained fewer granules than mast cells in the deep dermal layer, and, unlike the latter, did not emit a yellow fluorescence after treatment with o-phthaladehyde. The results indicate that the bulk of the skin histamine is contained in mast cells residing in deep skin layers. They also support the view that the thymus may have a suppressive effect on both mucosal and connective tissue mast cellsin vivo.