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.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Adriamycin Binds to the Matrix of Secretory Granules during Mast Cell Exocytosis

The antineoplastic drug adriamycin induces exocytosis in rat peritoneal mast cells followed by a significant uptake of the drug into the secretory granules. The drug is fluorescent, allowing visualization of its accumulation and binding to mast cell granules by fluorescence microscopy. At the same time, the well known inorganic dye ruthenium red was used as a probe because of its great affinity for heparin in the mast cell secretory granules as visualized by bright field microscopy. Competition between adriamycin and ruthenium red for binding to the negatively charged matrix of granules was demonstrated. Biochemical studies were also performed to confirm microscopic observations. Adriamycin may be of interest for studying mast cell secretion; it is not only a strong fluorescent dye for mast cell granules that are in communication with the extracellular space, but it also induces mast cell exocytosis.     

Abnormal mast cell granules in the beige (Chédiak-Higashi syndrome) mouse.

Mast cells of beige (C57BL/6J) (bg-j/bg-j) mice were examined histochemically and ultrastructurally. Mast cell granules in the beige mice were markedly enlarged and irregular in shape. Granule contents stained uniformly with acidified toluidine blue, but with ruthenium red and Alcian Blue-safranin, two components were evident. The rims of the abnormal granules stained with ruthenium red and with Alcian Blue; the centers of the granules were clear with ruthenium red and stained with safranin. Mast cell granules thus represent another abnormal organelle in the Chédiak-Higashi syndrome.     

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.     

Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation

Summary Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1–0.01 m) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized ³H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.     

A Neoplastic Connective Tissue Mast Cell Capable of Continuous Growth In Tissue Culture

A neoplastic connective tissue mast cell from a dog mast cell sarcoma has been grown in tissue culture for 50 passages over a period of 2 years. The cells were grown as monolayer cultures in glass bottles, using Eagle's basal medium fortified with calf serum. The cultures were contaminated with an Alkaligenes sp. for 10 months but finally were sterilized bacteriologically by treatment with specific antiserum combined with antibiotics. The cells grow in a fibroblastic pattern, and contain mitochondria, mast cell granules, and lipid granules or droplets. The mast cell granules stain basophilic with Giemsa's stain and metachromatically with azure A or toluidine blue. They also stain with Sudan black B and with periodic acid-Schiff stain. The interphase nuclei are vesicular, contain from 1 to 20 nucleoli, and frequently show bizarre outlines. Multinucleate cells are often seen, as are mitotic figures. Extracellular fibrous material occurs in all cultures and apparently originates from the cell surface. This material does not have the structure of connective tissue fibers and has not been identified. The cells develop an increased number of metachromatic granules when grown in medium containing heparin and an increased number of sudanophilic granules when grown in medium containing stearic acid. Only small amounts of histamine were present in the tumor from which this cell line was derived and in the cells grown in tissue culture.     

Comparison of histochemical staining techniques for detecting mast cells in oral lesions

ABSTRACT

Mast cells are large cells with granular cytoplasm that participate in wound healing, angiogenesis and defense against pathogens. They also contribute to inflammation by initiating innate and acquired immunity. The granules of these cells exhibit characteristic staining properties. We investigated toluidine blue, astra blue, Alcian blue-pyronin Y and May-Grunwald Giemsa stains for mast cells in various oral lesions and assessed the efficacy of each for identifying mast cells. Sections were obtained from 10 each of diagnosed cases of inflammatory fibrous hyperplasia, periapical cyst, mild dysplasia, oral submucous fibrosis and oral squamous cell carcinoma and stained using the stains listed above. Mast cells were assessed for their presence, contrast of the mast cell in the connective tissue background and number. We found that May-Grunwald Giemsa stain was the best for identification of mast cells, although toluidine blue staining is less time-consuming. Overall we obtained better results using May-Grunwald Giemsa and toluidine blue for staining mast cells.     

An histochemical approach to characterization of anionic constituents in mast cell secretory granules

We used cationized colloidal gold in order to investigate the distribution of anionic sites in different secretory granules of rat and mouse mast cells. The localization of the anionic sites was performed by post-embedding labeling of thin sections of rat peritoneal cells or mouse skin tissue, fixed in Karnovsky's fixative and OsO₄ and embedded in Araldite or LR white, respectively. In all cases anionic sites were demonstrated with a high density variation depending on cell type. In all mast cell secretory granules we have observed the highest density (ca. 500–900 gold particles/m²), while in other peritoneal cell granules it was about 10 times less (ca. 40–80 gold particles/m²). Pretreatment of the LR white sections with heparinase I and III resulted in a reduction of 97% and 72%, respectively, in the binding of the gold particles to the granules, indicating that the majority of the gold binding reactivity is due to heparin. Correlation of section profile area with labeling density revealed that the smaller granules were significantly more labeled when compared to the larger profiles. On the basis of these observations it seems that a post-translational change (mainly sulfation of heparin) of secretory content influences the granule anionic charge and thus may affect the intragranule buffer capacity.     

Electron microscopic studies on the pars intermedia in normal and in mice with hereditary nephrogenic diabetes insipidus

Summary The ultrastructure of the pars intermedia (PI) of the normal VII +/+ and hereditary nephrogenic diabetes inspidus DI Os/+ mice has been studied with particular reference to the morphology of the glandular cells and their innervation. Four types of cells were observed in both the genotypes of mice, 1) the light glandular cell, 2) the dark cell, 3) a type of cell similar to ependymal cells and 4) a small percentage of typical ACTH cells, observed mostly on the PI border of the cleft and rarely in the centre of PI. The predominant light glandular cells contain mainly two types of membrane bound granules: 1) electron dense core granules, which measure 1500–2500 Å and 2) electron lucent vesicles, which measure 3000–4000 Å in diameter. Granules of intermediate size with various density are also present in both types of mice. The electron dense core granules are predominant in DI Os/+ mice, whereas, electron lucent vesicles are predominant in the normal VII +/+ mice. Similar uniform size membrane bound electron dense granules have been observed in ACTH cells of PI and pars distalis. From earlier experimental evidences and the present observations, it is concluded that the dense core granules in PI may be synthesizing ACTH or ACTH-like substance. It is also discussed that these dense core granules may further mature and give rise to MSH in the form of electron lucent vesicles. If it is so, PI light glandular cells may have dual functions, of producing MSH and ACTH. One of the functions of ependymal-like cells, may be the transport of PI secretion.Three types of nerve endings are observed throughout the PI, making synaptic contact with the predominant cell type. The innervation is more in DI Os/+ mice than in normal mice. The classification of these nerves is according to Bargmann and co-workers 1) peptidergic neurosecretory fibers, contain mainly membrane bound dense core granules, measuring 1200 to 1800 Å, and are the classic neurosecretory granules; 2) adrenergic fibers, measuring 700–900 Å; 3) cholinergic fibers, measuring 300–400 Å. Adrenergic and cholinergic fibers are more towards the hypophysial cleft. The increased innervation, the synaptic contact, the extremely hypertrophied PI and the greater activity of its light glandular cells in the DI Os/+ mice show the PI is under the influence of the nervous system.This study was supported by MRC of Canada Grant No. MA-3759.     

Mast cell heterogeneity between two different species of Hoplias sp. (Characiformes: Erythrinidae): response to fixatives, anatomical distribution, histochemical contents and ultrastructural features

Mast cells from two erythrinid species: Hoplias malabaricus and Hoplias lacerdae, were studied in several tissues throughout the body using light and electron microscopy. Mast cells were found in all organs studied, but were especially abundant in the gastrointestinal tract, and were always in association with connective tissue. These cells showed different characteristics between the two species studied, like varied morphology, anatomical distribution, density, basophilic/eosinophilic staining and heparin content. In H. malabaricus, the tissues fixed with Helly's solution contained mast cells that were basophilic, metachromatic and had heparin in their cytoplasmic granules, while the tissues fixed with Karnovsky's solution contained eosinophilic and orthochromatic mast cells in which heparin was not detected. In H. lacerdae, the use of both fixatives resulted in mast cells that were eosinophilic, orthochromatic, with no identifiable heparin content. Exclusively in H. malabaricus oesophagus, the mast cells were additionally seen among the epithelial cells. The ultrastructural studies performed in hindgut fixed with Karnovsky's solution revealed that the cytoplasmic granules seen in H. lacerdae mast cells were better preserved than in H. malabaricus mast cells. The latter had electron-lucent granules that were often merged, forming channels. The present study demonstrated that mast cells from two species belonging to the same genus or even mast cells from the same species but under different fixatives can present heterogeneous characteristics, possibly due to their functional properties or to their sensitivity to fixatives.     

Electron microscopic observations on synapse-like contacts between pituicytes and different types of nerve fibers in the anuran pars nervosa

Summary Pituicytes of Rana pipiens could be classified into two types, pale and dense, according to their relative densities of cytoplasm and the populations of free ribosomes and cell organelles. An intermediate type of pituicyte was also recognized.Lipid droplet such as are typical in the cytoplasm of mammalian pituicytes, are not in the cytoplasm of either types of frog pituicyte. Both types have long cytoplasmic processes which run among the nerve fibers, and some of them end at the pericapillary space.Nerve endings making synapse-like contacts with the cell bodies or the processes of the pituicyte are frequent. According to the structures and sizes of granules and vesicles in the nerve endings, these endings are classified into one of three types: 1) A, which appears to be a peptidergic neuronal ending containing dense granules 1,200–2,000 Å in diameter and small clear vesicles 300–400 Å in diameter; 2) B, which appear to be monoaminergic endings containing cored vesicles 600–1,000 Å in diameter and small clear vesicles 300–500 Å in diameter; 3) C, which appear to be cholinergic endings containing only small clear vesicles. Type C endings are relatively rare. In the synaptic area the axonal membranes appose those of the pituicytes across a gap of about 200 Å and numerous presynaptic vesicles are clustered or accumulated near the presynaptic membranes.The author wish to express his hearty thanks Professor Dr. A. Gorbman, Zoology Department, University of Washington, Seattle, U.S.A. and Professor Dr. H. Fujita for their helpful advices and criticisms. The frog tissues were obtained and fixed in Professor A. Gorbman's laboratory supported by U.S.P.H.S. grant NS 04887.     

Electron microscopic autoradiography on the localization of serotonin in the frog median eminence

Summary For the identification of the nerve fiber containing serotonin (5-hydroxytryptamine) in the frog median eminence, an electron microscopic autoradiography was performed with 5-hydroxytryptophane-³H which is the precursor of serotonin. At 1 and 24 hours after the intraperitoneal injection, most silver grains were located over the nerve fibers and endings, and a few were also found over the glia cell and the perivascular space. A large number of silver grains were located over the type 3 nerve endings (Nakai, 1971) containing small dense granules about 600–1000 Å in diameter 1 and 24 hours after the injection. Some silver grains were localized over the nerve endings containing intermediate-size dense granules 1100–1700 Å in diameter. Silver grains were also frequently observed over the nerve fibers in the inner layer of frog median eminence. There is no significant difference in the pattern of distribution of silver grains between tissues of 1 hour and 24 hours after the injection.The authors wish to thank Prof. H. Fujita for his advice and criticism.     

Mast Cell Proteoglycans

Mast cells are versatile effector cells of the immune system, contributing to both innate and adaptive immunity toward pathogens but also having profound detrimental activities in the context of inflammatory disease. A hallmark morphological feature of mast cells is their large content of cytoplasmic secretory granules, filled with numerous secretory compounds, including highly negatively charged heparin or chondroitin sulfate proteoglycans of serglycin type. These anionic proteoglycans provide the basis for the strong metachromatic staining properties of mast cells seen when applying various cationic dyes. Functionally, the mast cell proteoglycans have been shown to have an essential role in promoting the storage of other granule-contained compounds, including bioactive monoamines and different mast cell-specific proteases. Moreover, granule proteoglycans have been shown to regulate the enzymatic activities of mast cell proteases and to promote apoptosis. Here, the current knowledge of mast cell proteoglycans is reviewed.     

Structure of the chromosomal material in inactive nuclei of chicken red blood cells

Electron microscope sections have been cut of chicken red blood cell nuclei by a novel procedure that produces zones of material below 100 Å in thickness. After fixing and staining, electron dense structures are observed that have dimensions closely correlating with those determined for nucleosomes. These structures appear as wedge-shaped or as circular objects with a diameter of approximately 90–100 Å. Five to eight of these objects are arranged in a spiral around a central core giving rise to a 250–300 Å structure which may represent a superbead or a cross section of a solenoidal fibre.     

Ribonuclease–gold Labels Chondroitin Sulphate in Guinea Pig Basophil Granules

Basophilic leucocytes are metachromatic granule-containing secretory granulocytes that contain a mixture of granular proteoglycans devoid of heparin. In guinea pigs, isolated basophilic leucocyte granules primarily contain chondroitin sulphate. We have recently demonstrated that an enzyme-affinity– gold technique to image RNA, using the reagent RNase gold, also binds specifically to heparin in human mast cell granules. Such binding is based on the known property of heparin as a competitive inhibitor of RNase. Using similar methods, we show here that RNase–gold binds to the chondroitin sulphate in the secretory granules of guinea pig basophils, thus broadening the applicability of this post-embedding affinity–gold method to studies that require imaging of chondroitin sulphate in routinely prepared electron microscopical samples.     

Mast Cells Produce a Unique Chondroitin Sulfate Epitope

The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.     

Distribution, Histochemical and Enzyme Histochemical Characterization of Mast Cells in Dogs

This study describes the distribution, proteoglycan properties and protease activity of mast cells from 15 different dog organs. In beagles and mixed breed dogs, staining with Alcian Blue-Safranin O revealed mast cells in all the organs examined. However, their numbers varied and they demonstrated unique localization patterns within some of these organs. Berberine sulphate fluorescence-positive mast cells were observed in the submucosa, muscularis and serosa of the intestines, as well as the tongue and liver (within the connective tissue). Mast cells within the intestinal mucosa were negative for, or demonstrated weak, berberine sulphate staining. Heterogeneity of mast cells in terms of the proteoglycans contained within their granules was further confirmed by determination of critical electrolyte concentrations (CECs). The CECs of mast cells within the connective tissue of several organs, including the intestines (submucosal and muscularis-serosal layers) were all greater than 1.0 M. The results from CEC experiments together with berberine staining indicate that heparin was contained within their granules. Relative to the CECs of mast cells in other organs, mast cells in the intestinal mucosa exhibited lower CECs, suggesting that the proteoglycans within their granules were of lower charge density and/or molecular weight. Although mast cells were classified into two groups by proteoglycans within the granules, enzyme histochemical analysis in beagles revealed three subtypes of mast cells: chymase (MC(C)), tryptase (MC(T)) and dual positive (MC(TC)) cells. There was no correlation between the proteoglycan content and enzyme properties of the mast cell granules.     

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.     

Vesicle and granule content of sympathetic ganglion cells during limb regeneration of the newt Triturus

Summary Fine structural observations were made on the vesicle and granule content of ganglion cells in the posterior subclavian ganglion and peripheral nerve fibers of the upper forelimb of the newt Triturus. The populations of vesicles and granules in normal ganglion cells and nerve fibers were compared with those observed after limb transection. In normal neurons, clear vesicles range in size from 250 to 1000 Å in diameter, but are most frequently 400–500 Å. Vesicles with dense contents (granules) also vary greatly in size, but most are 450–550 Å in diameter and correspond to dense-core vesicles. Large granules that contain acid phosphatase activity are thought to be lysosomes. During limb regeneration, in both the ganglion cells and peripheral nerves, the ratio of dense vesicles to clear vesicles increases. There is a large increase in number of dense granules with a diameter over 800 Å, particularly in the peripheral regenerating fibers. This study shows that regenerating neurons differ from normal in their content of vesicular structures, especially large, membrane-bounded granules.This work was supported by grants from the National Science Foundation (GB 7912) and from the National Cancer Institute (TICA-5055), National Institutes of Health, United States Public Health Service.