Histamine synthesis is required for granule maturation in murine mast cells

Mast cells are the major sources of histamine, which is released in response to immunological stimulations. The synthesis of histamine is catalyzed by histidine decarboxylase (HDC). Previous studies have shown that Hdc^?/? mast cells exhibit aberrant granule morphology with severely decreased granule content. Here, we investigated whether the histamine synthesized in mast cells regulates the granule maturation of murine mast cells. Several genes, including those encoding granule proteases and enzymes involved in heparin biosynthesis, were downregulated in Hdc^?/? peritoneal mast cells. Impaired granule maturation was also found in Hdc^?/? BM‐derived cultured mast cells when they were cocultured with fibroblasts in the presence of c‐kit ligand. Exogenous application of histamine and several H₄ receptor agonists restored the granule maturation of Hdc^?/? cultured mast cells. However, the maturation of granules was largely normal in Hrh4^?/? peritoneal mast cells. Depletion of cellular histamine with tetrabenazine, an inhibitor of vesicular monoamine transporter‐2, did not affect granule maturation. In vivo experiments with mast cell deficient Kit^W/Kit^W‐v mice indicated that the expression of the Hdc gene in mast cells is required for granule maturation. These results suggest that histamine promotes granule maturation in mast cells and acts as an proinflammatory mediator.     

Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the ‘state of equilibrium’ at the cell surface caused by the superfusion (Uvnäs et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 μm and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 μm. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.     

TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism

Histamine is a bioactive amine that exerts immunomodulatory functions, including many allergic symptoms. It is preformed and stored in mast cells and basophils but recent evidence suggests that other cell types produce histamine in an inducible fashion. During infection, it has been suggested that neutrophils may produce histamine. We also observed that histamine is released in a neutrophil-mediated LPS-induced model of acute lung injury. Therefore, we sought to examine whether innate signals promote histamine production by neutrophils. Bone marrow-derived neutrophils stimulated with a range of TLR agonists secreted histamine in response to LPS or R837, suggesting TLR4 or TLR7 are important. LPS-driven histamine was enhanced by coculture with GM-CSF and led to a transient release of histamine that peaked at 8 h post stimulation. This was dependent upon de novo synthesis of histamine, since cells derived from histidine decarboxylase (HDC) deficient mice were unable to produce histamine but did generate reactive oxygen species upon stimulation. Using pharmacological inhibitors, we show that histamine production requires PI3 kinase, which has been shown to regulate other neutrophil functions, including activation and selective granule release. However, unlike mast cells, HDC deficiency did not alter the granule structure of neutrophils, suggesting that histamine does not participate in granule integrity in these cells. Consequently, our findings establish that neutrophils generate histamine in response to a select panel of innate immune triggers and that this might contribute to acute lung injury responses.     

Histamine and Heparin Release from Isolated Rat Mast Cells Exposed to Compound 48/80

The relationship between the release of histamine and heparin from rat mast cells exposed to compound 48/80 in vitro has been studied in order to elucidate the mechanism of action of this histamine releasing agent. Heparin is almost exclusively localized to the granule fraction of mast cells and therefore its release can be used as an index of granule release. The ratio percentage histamine release/percentage heparin release was found to be 1.4–1.7 and 1.6–2.0 following exposure of mast cells to 2.5 and 0.25 μg/ml respectively of compound 48/80. These “found” ratios are artificially high due to the fact that it is impossible to effect a complete separation of the released granules from the mast cells. Bearing this in mind, these results are consistent with the hypothesis (Uvnäs and Thon 1966) that the release of histamine-containing granules is a primary event in the release of histamine induced by this agent. How ever, they do not exclude the possibility that part of the histamine release can take place without granule release. Studies of the specific activities of the ³⁵S-labelled heparin released and retained by mast cells taken from rats at different intervals after Na₂³⁵SO₄ injection and exposed to compound 48/80 suggest that the most mature granules are released first on treatment of mast cells with this substance.     

Surface histamine receptors in rat peritoneal mast cells. Separation of receptor-bearing cells via binding to a histamineprotein conjugate,Cell-ectTM

The Seragen Cell-ect Total Histamine kit^TM was used to separate histamine receptor-bearing rat peritoneal mast cells from cells lacking these receptors. It was found that approximately 75% of the peritoneal mast cell population carried cell surface histamine receptors.The results further suggest that the mast cell histamine receptors present are mainly of an H₁-type, as judged by the capacity of a specific H₁-antagonist to reduce the histamine receptor-dependent cell adhesion. Moreover, an H₁-agonist is less efficient in this respect and an H₂-antagonist does not affect the cell adhesion at all. A possible functional role for these receptors, however, remains to be clarified.     

Turnover of histamine in mucosal and connective tissue mast cells of the rat

Mast cells constitute a heterogenous cell system. The specific type of mucosal mast cell (MMC) of the gut differs with respect to a number of properties from the classical connective tissue mast cell (CTMC) found in, e.g. skin, tongue and the peritoneal cavity. This report summarizes recent findings concerning turnover rates of amines and heparin in the two cell types. The elimination rate of radiactively prelabelled histamine, 5-hydroxytryptamine (5-HT) and heparin from peritoneal CTMC was compared with the elimination of radiolabelled histamine from tongue, where histamine is stored in CTMC and duodenum where it is stored in MMC. The elimination of histamine from peritoneal CTMC was slow (t _1/2=23 days) and did not differ significantly from that of 5-HT (t _1/2=25 days) and heparin (t _1/2=35 days) suggesting a low degree of secretory activity in the normal rat. The elimination rate of histamine from the tongue was also very slow. The specific radioactivity of histamine in duodenum was decreasing more rapidly. This was explained by a dilution of the radioactivity since the histamine content increased during the experimental period, and also by MMC death. The results are compatible with the assumption that CTMC and MMC are secretory cells, but with low activity until recruited by adequate, immunological or other stimuli.     

No Role for Zinc in the Storage of Histamine in Rat Peritoneal Mast Cells

The histamine in the granules of mast cells has been proposed to be stored as a heparin-zinc-histamine complex. Due to its chelating action zinc should increase (double) the histamine binding capacity of heparin. We have determined the zinc content of isolated rat peritoneal mast cells to 2.4–4.1 nmol/10⁶ cells and of their granules to 13.0–21.4 nmol/mg dry weight. The corresponding amounts of histamine were 150 and 680 nmol respectively. The zinc content found is far (20 times) too low to allow for an adequate binding of histamine in a heparin-zinc-histamine complex. In vitro the granules take up zinc in the same manner as they take up other cations and zinc competes with histamine for granule storage sites. Consequently, Hi-uptake is reduced and not enhanced in the presence of zinc in the suspension medium. In summary no evidence was found for a storage function of zinc in a heparin-zinc-histamine complex.     

On the Cation Exchanger Properties of Rat Mast Cell Granules and their Storage of Histamine

Mast cell granules free of a surrounding membrane were isolated from water-lysed rat peritoneal and thoracic mast cells by differential centrifugation. The granules were depleted of their histamine by suspension in 10 mM sodium phosphate buffer and the sodium-charged granules then converted into the “hydrogen form” by repeated washing in slightly acid deionized water. The cation exchanger properties of the mast cell granules were investigated by testing the applicability of the Rothmund-Kornfeld equation for cation exchangers to the binding of Na⁺ and Hi⁺ ions to granule sites. The results lend further support to the view that the mast cell granule acts as a cation exchanger with the exchanger function localized to protein carboxyls in the protein-heparin complex of the granule matrix.     

The Release of Histamine,Heparin and Granule Protein from Rat Mast Cells Treated with Compound 48/80 in vitro

Fillion , G. M. B., S. A. Slorach and B. Uvnäs . The release of histamine, heparin and granule protein from rat mast cells treated with compound 48/80 i n vitro. Acta physiol. scand. 1970. 78. 547–560. The quantitative relationship between the release of histamine, heparin and granule protein from rat mast cells exposed to compound 48/80 in vitro has been investigated in order to clarify the mechanism of action of this releaser. A correlation was found between the three release curves. The ratio of heparin to granule protein was similar in extruded granules and granules remaining in the cell after compound 48/80 treatment. From these results we conclude that the principal mechanism of histamine release induced by this agent involves an initial extrusion of histamine-containing granules, followed by an exchange of histamine in the granules for cations in the extracellular medium.     

Plasma extravasation induced by dietary supplemented histamine in histamine-free mice

Histidine decarboxylase (HDC) synthesizes endogenous histamine from histidine in mammals. To evaluate the role of histamine in skin allergic reaction, we used HDC gene knockout mice lacking histamine. No plasma extravasation reaction was observed in HDC-/- mice after passive cutaneous anaphylaxis (PCA) test. Compound 48/80, a mast cell granule depletor, produced plasma extravasation inHDC+/+ mice but no extravasation in HDC-/- mice. Interestingly, orally administered histamine was distributed in the skin in HDC-/- mice and in these histamine-supplemented mice the plasma extravasation reaction was observed after the injection of compound 48/80 and the PCA test. Cultured bone marrow-derived mast cells of HDC-/- mice took up histamine from the histamine-supplemented medium into the secretory granules. The absorbed histamine was released in response to the same antigen and antibody combination used as in PCA test. In contrast to the immediate-type response, the delayed-type hypersensitive response, observed as a thickening of the ear skin after trinitrochlorobenzene challenge (following sensitization), showed no differences between HDC+/+ and HDC-/- mice. Therefore, among the allergic skin reactions, histamine is revealed to be an important mediator especially for the plasma extravasation in an immediate-type allergy model.     

Ultrastructural autoradiographic analysis of RNA in isolated human lung mast cells during secretion and recovery from secretion

BACKGROUND: Previous work has implicated isolated, control human lung mast cell granules in RNA metabolism using multiple methods of high-magnification imaging based on different mechanistic principles. These methods have demonstrated ribosomes, RNA, U1snRNP and uridine in, around and attached to secretory granules. METHODS: Here, we have extended these studies using ultrastructural autoradiography of radiolabeled uridine incorporation in degranulating and recovering mast cells. RESULTS: We found that control cells incorporated uridine into granules, with values that decreased dramatically in conjunction with stimulated histamine secretion and granule extrusion, and that granule stores of tritiated uridine increased together with the reconstitution of secretory granules in recovering mast cells. CONCLUSION: These findings support a possible new role for secretory granules in RNA metabolism in mast cell biology.     

Differentiation of second messenger systems in mast cell activation

Pretreatment of rat peritoneal mast cells with either Staurosporine or an analog K-252a, lead to a dose-related inhibition of histamine release when stimulated with Anti-IgE (IC₅₀: Staurosporine=110 nM; K-252a=100 nM). In contrast, the two PKC inhibitors (1–1000 nM) failed to inhibit histamine release induced by compound 48/80 (0.5–1 g/ml). Exposure of Anti-Asc-IgE sensitized mouse bone marrow derived mast cells to Asc-BSA lead to the release of both histamine (510 ng±12.6 ng/10⁶ cells) and immunoreactive Leukotriene C₄ (27.0±12.6 ng/10⁶ cells) LTC₄ release was inhibited by Staurosporine and K-252a with an IC₅₀ of 75 nM for both compounds. Pretreatment of rat peritoneal mast cells with PMA 100 nM lead to a small but significant release of histamine (18.3±3.6%). Pretreatment of these cells with K-252a or Staurosporine lead to a dose related inhibition of histamine release with an ED₅₀ of 10 nM for Staurosporine and 60 nM for K-252a. Treatment of rat peritoneal mast cells with the calcium ionophore A23187 lead to a significant release of histamine which was not inhibited by either of the two kinase inhibitors (0.1–1000 nM). The two kinase inhibitors also inhibited mouse bone marrow derived mast cell proliferation in response to IL-3 with IC₅₀ of 80 nM for Staurosporine and 270 nM for K-252a.     

The function of CCR3 on mouse bone marrow‐derived mast cells in vitro

The mechanisms governing the population of tissues by mast cells are not fully understood, but several studies using human mast cells have suggested that expression of the chemokine receptor CCR3 and migration to its ligands may be important. In CCR3‐deficient mice, a change in mast cell tissue distribution in the airways following allergen challenge was reported compared with wild‐type mice. In addition, there is evidence that CCR3 is important in mast cell maturation in mouse. In this study, bone marrow‐derived mast cells (BMMCs) were cultured and CCR3 expression and the migratory response to CCR3 ligands were characterized. In addition, BMMCs were cultured from wild‐type and CCR3‐deficient mice and their phenotype and migratory responses were compared. CCR3 messenger RNA was detectable in BMMCs, but this was not significantly increased after activation by immunoglobulin E (IgE). CCR3 protein was not detected on BMMCs during maturation and expression could not be enhanced after IgE activation. Resting and IgE‐activated immature and mature BMMCs did not migrate in response to the CCR3 ligands eotaxin‐1 and eotaxin‐2. Comparing wild‐type and CCR3‐deficient BMMCs, there were no differences in mast cell phenotype or ability to migrate to the mast cell chemoattractants leukotriene B₄ and stem cell factor. The results of this study show that CCR3 may not mediate mast cell migration in mouse BMMCs in vitro. These observations need to be considered in relation to the findings of CCR3 deficiency on mast cells in vivo.     

Anaphylactic histamine release and membrane fluidity of mast cells from young and aged rats

The effect of aging on mast cell secretory function was studied in young (8 weeks old) and aged (72 weeks old) female BD₆ rats using passive cutaneous anaphylaxis (PCA) and the histamine release testin vitro. Young rats were more susceptible to PCA and their peritoneal mast cells released more histamine in anin vitro anaphylactic reaction. Measurement of electron spin resonance (ESR) using methyl-5-doxylstearate and methyl-16-doxylstearate as spin labels demonstrated that cell membranes of mast cells of young rats showed higher fluidity in the resting state and a more pronounced increase in membrane fluidity after anaphylactic stimulation than mast cell membranes of aged rats. The study of lipid composition of mast cell membranes showed that the ratio of cholesterol (C) to phospholipids (P) was lower in mast cells of young rats than in mast cells of aged rats. This suggests that a decrease in membrane fluidity, associated with an increase in C/P ratio may be one of the factors responsible for decreased histamine releasability of mast cells from aged rats.     

Degranulation and Histamine Release,Two Consecutive Steps in the Response of Rat Mast Cells to Compound 48/80

Isolated rat peritoneal mast cells were exposed to compound 48/80 in A isotonic salt solution and in B isotonic sucrose solution. In both cases degranulation and histamine release occurred. In A all the released histamine appeared dissolved in the suspension medium, the discharged granules having lost their histamine. In B the discharged granules were show to retain histamine. These granules released their histamine when suspended in a NaC1-containing medium. Depleted granules could be refilled with histamine by suspension in a histamine-containing medium and again depleted of the amine by exposure to sodium chloride. The proposal is made that the histamine release induced by compound 40/80 is a two stage process, a primary energy-requiring transport of histamine-containing granules to the outside of the mast cell and a secondary nonenergy-requiring physico-chemical process, an extracellular cation exchange in the shed granules between histamine and cations, mainly sodium in the tissue fluid.     

Anaphylactic histamine release and membrane fluidity of mast cells from young and aged rats

The effect of aging on mast cell secretory function was studied in young (8 weeks old) and aged (72 weeks old) female BD₆ rats using passive cutaneous anaphylaxis (PCA) and the histamine release testin vitro. Young rats were more susceptible to PCA and their peritoneal mast cells released more histamine in anin vitro anaphylactic reaction. Measurement of electron spin resonance (ESR) using methyl-5-doxylstearate and methyl-16-doxylstearate as spin labels demonstrated that cell membranes of mast cells of young rats showed higher fluidity in the resting state and a more pronounced increase in membrane fluidity after anaphylactic stimulation than mast cell membranes of aged rats. The study of lipid composition of mast cell membranes showed that the ratio of cholesterol (C) to phospholipids (P) was lower in mast cells of young rats than in mast cells of aged rats. This suggests that a decrease in membrane fluidity, associated with an increase in C/P ratio may be one of the factors responsible for decreased histamine releasability of mast cells from aged rats.

     

Oxatomide protects against degranulation of rat peritoneal mast cells during in vitro challenge with antigen or compound 48/80. Ultrastructural aspects

The ultrastructure of isolated rat peritoneal mast cells was evaluated after in vitro degranulation and treatment with oxatomide, a new anti-allergic compound.In a first series of experiments, mast cells of rats infected withTrichinella spiralis larvae were incubated withTrichinella larvae somatic antigen to produce histamine release. The release was visualized in the electron microscope by exocytosis of the peripheral amine-containing granules, which resulted from fusion between several perigranular membranes and fusion of these membranes with the plasma membrane.A more drastic degranulation was provoked in a second series by incubation of unsensitized mast cells in the presence of the amine liberator compound 48/80. This treatment led to a complete extrusion of the granules in most of the cells, while in a smaller number of cells, only large vacuoles containing remnants of several granules were seen. The plasma membrane of these cells however was intact and there were no signs of exocytosis.The effect of oxatomide against mast cell degranulation was dose-dependent and comparable for the two types of histamine release. After incubation with high doses (10^–4 M, 5.10^–5 M) granule liberation was rarely observed in antigen-challenged and compound 48/80-challenged cells. Protection was apparently situated at the level of the plasma membrane which seemed to be unable to fuse with the perigranular membranes while fusion of perigranular membranes of individual granules was still possible. None of the tested concentrations of oxatomide induced spontaneous degranulation. High doses, however, led in a number of cells to some ultrastructural alterations such as partial disappearance of plasmalemmal folds, slight cytoplasmic oedema and the appearance of intranuclear microtubules. The latter were also seen in oxatomide-treated challenged mast cells.     

Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells.

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the 'state of equilibrium' at the cell surface caused by the superfusion (Uvn?s et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 microM and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 microM. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.     

Expression of non‐mast cell histidine decarboxylase in tumor‐associated microvessels in human esophageal squamous cell carcinomas

Histamine is produced by mast cells and many other types of cells. The role of histamine released from mast cells in promoting tumor angiogenesis has been intensively studied; however, the role of non‐mast cell histamine in regulating tumor angiogenesis has been largely ignored. In this study, tissue specimen sections from 43 patients with esophageal squamous cell carcinoma (ESCC) and normal esophageal biopsies from 17 heath individuals obtained from a high incidence area of north China were used to assess changes in microvessel density (MVD) and non‐mast cell L‐histidine decarboxylase (HDC) (the only rate‐limiting enzyme that catalyzes the formation of histamine from L‐histidine) expression in the tumor microenvironment by immunohistochemistry (IHC). In addition, the cellular characterization of non‐mast cell HDC‐positive cells in microvessels was examined by double IHC combined with HDC/CD34 and HDC/PCNA antibodies. These IHC analyses revealed a significantly increased HDC‐positive MVD in ESCC as compared with normal controls, which accounted for ～61% of CD34‐labeled general MVD in ESCC. Furthermore, IHC in serial sections and double IHC showed that most of these HDC‐positive cells were CD34‐positive endothelial cells in microvessels with an increased proliferative capacity. Thus, our results suggest that non‐mast cell histamine expressed in endothelial cells of microvessels could be an additional cellular source and might play a role in regulating angiogenesis in ESCC.     

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.