Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro

Rat serosal heparin-containing mast cells (HP-MC) were maintained in vitro for as long as 30 days when co-cultured with mouse skin-derived 3T3 fibroblasts. In contrast, when the mast cells were cultured alone, on fibronectin-, gelatin-, or dermal-collagen-coated dishes, on acid and heat-killed fibroblasts in the presence or absence of 24 hr fibroblast-conditioned medium, or on a monolayer of mouse serosal macrophages, they failed to adhere to the dishes, released significant amounts of their histamine and lactate dehydrogenase, and stained with trypan blue, indicating a loss of viability. The rat serosal HP-MC cultured with the 3T3 fibroblasts became so adherent to the fibroblasts that the two cell types could be separated from one another only by trypsinization. The cultured HP-MC stained with both alcian blue and safranin and continued to synthesize proteoglycan at a rate comparable to that of freshly isolated cells. The 35S-labeled proteoglycan synthesized by these cultured cells, like that produced by freshly isolated rat serosal HP-MC, was a 750,000 to 1,000,000 m.w. proteoglycan containing only heparin glycosaminoglycans of 50,000 to 100,000 m.w. When HP-MC were cultured for 1 wk with the fibroblasts and were then incubated for 5 min with a 1/20 dilution of rabbit anti-rat IgE, they generated and released an average of 22 +/- 10 ng (mean +/- SD, n = 5) of prostaglandin D2 per 10(6) cells and exocytosed a higher net percentage of their total histamine content (44 +/- 11% [mean +/- SD, n = 8]) than did cells just isolated from the animal (6 +/- 4% [mean +/- SD, n = 4]). As assessed by electron microscopy, many of the cultured HP-MC resembled freshly isolated cells except that some secretory granules had fused with one another in some cells. Morphologically, after activation the cultured HP-MC underwent compound exocytosis like freshly isolated cells. These results demonstrate that the in vivo differentiated rat HP-MC maintain their histology, morphology, immunologic responsiveness, histamine content, and ability to synthesize heparin proteoglycan when co-cultured with living fibroblasts.     

DIFFERENTIATION AND PROLIFERATION OF EMBRYONIC MAST CELLS OF THE RAT

Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are confined to the loose connective tissue in the embryo. The alcian blue-safranin reaction distinguishes between weakly sulfated and strongly sulfated mucopolysaccharides by a shift from alcian blue to safranin staining. Based on this reaction and morphologic characteristics, four stages were identified. Stage I mast cells are lymphocyte-like cells with cytoplasmic granules which invariably stain blue with the alcian blue-safranin reaction. In Stage II cells the majority of granules are alcian blue-positive, but some safranin-positive granules have appeared. Stage III mast cells are distinguished by a majority of safranin-positive cytoplasmic granules; some alcian blue-positive granules still remain. Stage IV cells contain only safranin-positive granules. Thymidine-H³ uptake and identification of mitotic figures indicates that mast cells in Stages I and II comprise a mitotic pool while those in Stages III and IV are mitotically inactive. The pattern of S³⁵O₄ incorporation and the sequence of appearance of histochemically identifiable mast cell constituents corroborates division of the proliferation and differentiation of embryonic mast cells into the four stages described above. The process of formation of mast cell granules is interpreted as reflecting the synthesis and accumulation of a heparin precursor in alcian blue positive granules followed by the synthesis and accumulation of highly N-sulfated heparin along with mast cell chymase and finally histamine in safranin-positive granules.     

Differentiation of mast cells during postnatal development of neonatally estrogen-treated rats

Summary The accumulation of mast cells in the testicular interstitium of neonatally estrogen-treated rats was studied from 15 to 90 days of age. The maturation of these cells was assessed by ultrastructural analysis and their histochemical properties were examined with the sequential alcian blue-safranin staining method. The first identifiable mast cells appeared in the testis at 17–20 days of age, as immature cells with proliferative capacity. The density of mast cells increased up to 45 days of age, showing a slight decrease from 45 to 90 days of age. Before 45 days of age, most mast cells showed alcian blue-stained granules, whereas at 45 days of age, most cells presented a mixture of alcian blue and safranin-stained granules. From this age onward, most cells were stained with safranin. These maturational changes were well-correlated with their ultrastructural features. Mast cells presented few and heterogeneous immature granules up to 45 days of age, and many uniform electron-dense granules at 90 days of age. These results indicate that the testicular interstitium of neonatally estrogen-treated rats provides an adventageous environment for the recruitment, proliferation and maturation of connective tissue mast cells.     

The histological localization of heparin in the northern quahog clam, Mercenaria mercenaria.

The cellular location of heparin in Mercenaria mercenaria, the northern quahog clam, was identified histologically by azure II and alcian blue/safranin staining. A survey of various organs indicates that heparin was found in several morphological cell subpopulations of the labial palp, ctenidia, intestine, siphon, and pallium. Cells containing these compounds were restricted to areas proximal to epithelial surfaces and granules were observed in all of the cell types except those of the intestine. The "mast-like" cells that we report here may play a role analogous to that of mammalian mast cells in inflammatory responses, using heparin proteoglycan as a supportive defense molecule.     

Loss of histochemical identity in mast cells lacking carboxypeptidase A

Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa-/- mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa-/- peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa-/- peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa-/- mice. The Mc-cpa-/- mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.     

Demonstration of the heterogeneity of the mast cell population on the basis of the mucopolysaccharide content.

From the aspect of its mucopolysaccharide content the mast cell population is not homogeneous. The pulmonary and heart muscle mast cells of the rat are alcian blue positive, the mast cells of the thyroid gland, lymph nodes, subcutaneous connective tissue, mesentery and peripheral nerve are safranin positive, whereas among the mast cells of the peritoneal cavity and the thymus there are both alcian blue and saffranin positive forms. The least acid mucopolysaccharides are in the mast cells of the peritoneal fluid, the mesentery and the lungs, whereas the most acid ones are in the mast cells of the lymph nodes, the subcutaneous connective tissue and the thyroid gland. There is a considerable difference between the two last mentioned organs. The mast cells of the subcutaneous connective tissue are end-product cells without amine or precursor turnover, whereas the mast cells of the thyroid gland incorporate and deliver amines, which may participate in the regulation of the host gland.     

Stochastic model for mast cell proliferation in culture of murine peritoneal cells

We recently identified two types of mast cell colonies derived from murine peritoneal cells: type 1 and type 2. Type 1 mast cell colonies consisted of berberine sulfate(+)- safranin(+) connective tissue-type mast cells (CTMC) and were derived from mature CTMC in the heaviest fraction obtained by Percoll density gradient centrifugation. In contrast, type 2 mast cell colonies consisted of alcian blue(+)- berberine sulfate(-)- safranin(-) mucosal mast cells (MMC) and were derived from immature progenitors in low density fractions. We replated a total of 60 type 1 and 60 type 2 mast cell colonies and examined their capability for producing secondary colonies. Although all of the primary colonies yielded secondary colonies, the replating efficiencies of individual colonies varied over a wide range. Cumulative distributions of secondary colonies from both type 1 and type 2 primary colonies could be fitted well by gamma distributions obtained by computer simulation. These findings are in agreement with the stochastic model for CTMC- and MMC proliferation. Cytological analyses of secondary colonies from primary type 1 colonies revealed heterogeneous distributions of alcian blue(+)- safranin(-)- berberine sulfate(-) mast cells, suggesting that transdifferentiation from mature CTMC to safranin(-)- berberine sulfate(-) mast cells is also governed by stochastic mechanisms.     

Some histochemical reactions of mast cells of gerbils,hogs, armadillos and cats

Summary Mast cells of the Mongolian gerbil Meriones unguiculatus, the hog Sus scrofa, the cat Felis catus and the armadillo Pasypus novemcinctus were studied histochemically in relation to various fixation procedures, using azure A at pH 1 and 3, alcian blue at pH 1 and 2.5, diazosafranin at pH 3 and 7.8–8, and the PAS reaction. Fixations were performed in buffered 10% formol and 5% glutaraldehyde, in Kose's fluid, buffered sublimate (B4), lead nitrate and lead acetate formol.With azure A and alcian blue many mast cells were found in the gerbil with the aldehyde fixatives, fewer with the heavy metals. The diazosafranin reaction was present only in the aldehyde material, the PAS reaction was negative.In the hog, mast cells were more numerous after heavy metal fixation, fewer with aldehydes. Azure A stained metachromatically at pH 1 and 3, alcian blue reacted only at pH 1, the PAS reaction was negative, the pH 3 and 8 diazosafranin reactions were positive with all 4 fixations.In the cat, mast cells were moderately numerous with lead acetate formol, rare with formol and absent with glutaraldehyde. They stained with azure A at pH 1 and 3, with alcian blue at pH 1 and 2.5, with diazosafranin at pH 3 and 8 and by the PAS reaction.Armadillo mast cells were more numerous after heavy metal fixations, stained with azure A and alcian blue at pH 1 and 2.5 to 3, and with acid and alkaline diazosafranin.The mast cells of the 4 species vary in their requirements for aldehyde and heavy metal fixation, in their PAS reactivity and in their pH 2.5 alcian blue staining. All are sufficiently sulfated to react to cationic dyes at pH 1, but vary in PAS reactivity, indicating partial or complete sulfation. The presence of 5-hydroxytryptamine is indicated in all four species.Assisted by grant from National Cancer Institute C-04816.     

Staining properties of duodenal mast cells in 48/80-treated rats

Summary Mast cells in the tongue, mesentery and lamina propria of the duodenal mucosa in normal and 48/80-treated rats were observed at different time intervals. The tissues were studied comparatively after staining with toluidine blue, acridine orange or alcian bluesafranin. Under the experimental conditions used, the mast cells in the tongue and mesentery showed constant positive reactions to toluidine blue and acridine orange, both of which failed to demonstrate the presence of mast cells in the lamina propria of the duodenal mucosa. The combined alcian blue-safranin stain elicited a safranin-positive reaction in the mast cells of the tongue and mesentery and an alcian blue reaction in those of the lamina propria of the duodenal mucosa. This alcianophilia of the duodenal mast cells was not affected by compound 48/80. On the other hand, the safranin stain of the tongue and mesentery mast cells was altered to alcian blue by the drug. The results are discussed in the light of recent developments in mast cell research.This work was supported by grant MA-2236 of the Medical Research Council of Canada.     

新生大鼠雌激素注射后睾丸肥大细胞的变化

新生大鼠注射雌二醇后,睾丸肥大细胞于第30天可见到,细胞数量随年龄增长而增多,生后4－6个月,睾丸网附近仍可见大量肥大细胞。睾丸内的肥大细胞比皮肤内的结缔组织肥大细胞（CTMC）小而与小肠粘膜的粘膜肥大细胞（MMC）相近,AB－S染色后基本着蓝色,硫酸小檗碱荧光染色后呈现中等强度黄色荧光,结果提示,新生大鼠雌激素注射后睾丸内肥大细胞的增多可能与免疫过程有关,睾丸内肥大细胞与CTMC和MMC皆有所不同。     

Cells containing IgE in the intestinal mucosa of mice infected with the nematode parasite Trichinella spiralis are predominantly of a mast cell lineage

To determine whether IgE+ cells in the intestinal mucosa of nematode-infected mice were of a mast cell or a lymphocyte lineage, the intestinal mucosae of mast cell-deficient w/wv mice were examined for IgE+ cells after inoculation with Trichinella spiralis muscle-stage larvae. Immunofluorescence staining techniques were used to detect IgE associated with cells in the intestinal mucosa. Comparisons were made among four strains of mice, w/wv (mast cell-deficient), +/+ (normal congenic littermates of w/wv), BALB/c, and SJL, that were either uninfected controls or inoculated with T. spiralis. Tissue sections from the small intestine of T. spiralis-infected BALB/c, SJL, and +/+ mice were fixed in ethanol and were stained with an affinity-purified F(ab')2 rabbit anti-mouse IgE followed by FITC goat anti-rabbit IgG. Large numbers of cells in the intestinal mucosa exhibited bright fluorescence. When other sections of intestines from these mice were processed in Carnoy's fixative and were stained with alcian blue at low pH (a metachromatic stain for mast cells) or alcian blue followed by immunofluorescence staining for IgE, large numbers of mast cells were observed in the intestinal mucosa, and 70 to 90% stained positively for IgE. There was a considerable number of cells in the intestinal mucosa which were IgE+ but which did not stain with alcian blue. Few alcian blue-positive cells and no IgE+ staining cells were present in the intestinal mucosa of control, uninfected +/+, BALB/c, and SJL mice. To determine whether these IgE+ alcian blue-negative cells were of a lymphocyte or a mast cell lineage, the mast cell-deficient w/wv mouse strain was examined after infection with T. spiralis. In contrast to BALB/c, SJL, or +/+ mice, few cells in the intestinal mucosa of T. spiralis-infected w/wv mice stained with alcian blue or were positive for IgE. However, when the IgE response in the MLN of the w/wv mice was compared to the IgE response of BALB/c, SJL, and +/+ mice, numerous IgE+ cells, but no alcian blue-positive cells, were observed in the parenchyma of the MLN from all four strains of T. spiralis-infected mice. In addition, flow microfluorometric analysis of MLN cells stained for surface IgE in suspension showed a comparable proportion of IgE-bearing cells, which were mostly B lymphocytes, among all four strains of T. spiralis-infected mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphometric evaluation of murine pulmonary mast cells in experimental hemorrhagic shock

Respiratory failure resulting frequently in death is one of the complications in the course of post-hemorrhagic changes. A systemic inflammatory reaction plays a significant role in the pathogenesis of this syndrome. Mast cells also contribute to this effect. To broaden our knowledge of the pathogenesis of respiratory insufficiency, we evaluated morphometrically lung mast cells in hemorrhagically shocked rats. Lung sections were stained with alcian blue and safranin, and four separate locations were distinguished: under the lung pleura, around the bronchi and the large vessels, and in the interalveolar septa. A decrease in the area and volume of mast cells and an increase in their circularity index in interalveolar septa and around the bronchi was observed. An enlargement of mast cells around lung vessels was also found. There were no changes in the morphometric parameters of mast cells under pleura. The results suggest an activation and degranulation of mast cells and a role in the inflammatory process causing acute lung injury in hemorrhagic shock.     

Cytofluorometric quantitation of 5-hydroxytryptamine and heparin in individual mast cell granules.

Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.     

Biochemical and phenotypic characterization of human basophilic cells derived from dispersed fetal liver with murine T cell factors

Metachromatically granulated cells were generated from human fetal liver stem cells cultured in heterologous mouse conditioned medium rich in interleukin 3. After 2 to 3 wk of culture with biweekly changes of medium and selection of nonadherent cells, all cells present in five cultures had cytoplasmic granules, and 60 to 95% of the cells stained metachromatically with toluidine blue or with alcian blue but not with the safranin counterstain. Ultrastructurally, many granules contained fibrillar material or electron-dense cores with fibrils and vesicular fragments. In addition, the granules of many cells were filled with electron-dense material, which in some cases had a fine structure consisting of concentric whorls or a reticular pattern. Analysis of high-affinity IgE receptors on the cultured cells by flow cytometry demonstrated a unimodal fluorescence pattern, suggesting that most cells were in the basophil or mast cell lineage. The cultured cells lacked the lymphoid cell surface determinants B1, B4, T3, and T11, the myeloid determinants Mo2 and MY9, the natural killer cell determinant 901, and Ia histocompatibility antigens, but expressed the myeloid determinant MY7. The cells contained 52 ng/10(6) cells of histamine and incorporated [35S]sulfate at an average rate of 31,300 cpm/10(6) cells/4 hr into 175,000 m.w. chondroitin sulfate A proteoglycans. Upon activation with 1 microM calcium ionophore A23187, the cultured cells released 53% of their cell-associated histamine and metabolized arachidonic acid to 15.0 ng/10(6) cells of immunoreactive leukotriene C4 equivalents, 0.5 ng/10(6) cells of leukotriene B4, and 3.1 ng/10(6) cells of prostaglandin D2 (means, n = 3). Thus, stem cells present in human fetal liver give rise, as do stem cells in mouse fetal liver, to metachromatically granulated cells when cultured in the presence of mouse interleukin 3. In both species, the cultured cells bear IgE receptors, lack characteristic lymphoid and most myeloid cell surface determinants, and contain histamine and chondroitin sulfate proteoglycans. The human fetal liver-derived cells are similar in morphology and T cell factor dependence to basophil-like cells derived from umbilical cord blood, but are novel in their capacity to generate leukotrienes and prostaglandin D2.     

Uptake and turnover of dopamine in rat mast cells studied by cytofluorometry and high performance liquid chromatography

Summary Uptake and turnover of dopamine (DA) in rat peritoneal mast cells were studied by a cytofluorometric technique. The main advantage of the method is that it permits the study of the distribution of amine content within populations of cells. Catecholamines and indolamines can be differentiated, but subtler structural differences in this group of compounds cannot be distinguished. We, therefore, combined the cytofluorometric measurements with a liquid chromatographic method based on reversed-phase chromatography followed by amperometric detection in a thin layer flow cell. Intraperitoneally injectedl-DOPA was rapidly decarboxylated to DA, which was accumulated in mast cell granules. The elimination of DA from the mast cells was much faster than previously published 5-hydroxytryptamine and histamine elimination rates. No evidence of intracellular conversion of DA before its elimination was found and simultaneous heparin quantitations gave no evidence of an elimination pathway due to exocytosis of granules. Electron microscopy disclosed no structural changes that could be related to exocytosis during the elimination phase of DA. The rapid elimination together with absence of inhibition of DA-uptake after storage of exogenous 5-hydroxytryptamine suggest that the mechanism of DA storage differs from the mechanism of storage of endogenous mast cell amines.     

The effect of polymyxin B and some mast-cell constituents on mucosal mast cells in the duodenum of the rat

Summary Mucosal mast cells in the rat duodenum show no morphological signs of exocytosis of granules and do not release histamine after treatment with polymyxin B in doses large enough to cause almost complete degranulation of connective-tissue mast cells of tongue, skin, and mesentery with concomitant release of 60% of the tissue histamine. Administration of polymyxin B in gradually increasing doses over a period of 5ds resulted in a statistically significant increase in mucosal mast cells and a comparable increase in duodenal histamine content, whereas the connective-tissue mast cells in the other tissues examined became fewer in number, the remaining cells showing profound morphological changes, and tissue histamine levels, were reduced to 40% of the controls. A similar increase in mucosal mast cells has been observed after treatment with another mast-cell secretagogue, compound 48/80. This suggests that the increase in mucosal mast cells may be an indirect effect of these compounds, related to their activation of other mast cells and mediated by material(s) secreted by the connective-tissue mast cells. Possible mediators such as heparin, histamine, and 5-hydroxytryptamine injected for 5 ds in doses large enough to account for the amount released from the degranulated mast cells had no effect on the morphology or numbers of mast cells in any of the tissues examined.Supported by grants from the Swedish Medical Research Council, Project no 2235     

Concomitant Staining of Mast and Parietal Cells in Human Gastric Mucosa

A simple technique for concomitant staining of mast and parietal cells in the same section is described. Mast cells were stained by alcian blue or astra blue in methanol-formalinacetic acid fixed biopsies of gastric mucosa. Parietal cells were visualized by Dolichos biflorus lectin binding.     

Mast and enterochromaffin-like cells of the gastric mucosa]

An attempt has been made to reveal simultaneously both mast and ECL cells in the fundic mucosa of some laboratory animals and man. In Bouin fluid fixed specimens, toluidine blue pH 5.0 and alcian blue pH 1.0 failed to reveal mucosal mast cells in rats and mice only. In those animals mucosal mast cells became demonstrable in Carnoy fluid fixed tissues after staining with alcian blue pH 1.0. A double staining technique has been applied using Grimelius silver method followed by staining either with toluidine blue after acid hydrolysis or with alcian blue. Both mast and ECL cells became visible showing here and there their close arrangement. The latter might be a point for some functional relations between both cell types.     

鸡中枢淋巴器官肥大细胞的组织化学与形态学

对哺乳动物的,特别是啮齿动物和人类肥大细胞已有了比较深入的研究, 但关于家禽肥大细胞的研究很少.本研究旨在阐明鸡中枢淋巴器官中肥大细胞的组织化学与形态学特征.本研究证实Carnoy 氏液是鸡肥大细胞的优良的固定液,而中性缓冲福尔马林(NBF) 却阻断了大多数肥大细胞的着染力.甲苯胺蓝和阿尔新蓝是鸡肥大细胞的良好的染料,但阿尔新蓝能使更多的肥大细胞着染,虽然其也可使杯状细胞着染.作者的一种新的染色法, 长时间阿尔新蓝染色(LAB-S)可用于NBF固定的组织中肥大细胞的染色,因为其着染的细胞数与Carnoy 氏液固定甲苯胺蓝染色的细胞数无显著差异(P<0.001).在胸腺髓质中见有大量的肥大细胞,而胸腺皮质仅可见个别肥大细胞位于血管周围及小叶间结缔组织中.腔上囊的皮质与髓质中很少见有肥大细胞.肥大细胞有血管周围分布的倾向,但一个有趣的发现是血管内偶尔也有个别肥大细胞.电镜下可见肥大细胞的胞浆颗粒内充满无定形的颗粒状基质,但其电子密度有的较高,有的较低.少数胞浆颗粒内有旋涡状及网状亚微结构.但未见有人类肥大细胞胞浆颗粒内特征性的晶格状和卷轴状的亚微结构,也未见到在绵羊肥大细胞中描述过的特殊亚微结构.     

Orthochromatic, Species-Limited Staining of Mast Cells With Night Blue

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.