Ultrastructural cytochemical, immunocytochemical and in situ hybridization methods with polyuridine probes detect mRNA in human mast cell granules

Mature human mast cells are classical secretory cells that are filled with secretory-storage granules but are poorly endowed with visible free or membrane-bound cytoplasmic ribosomes. We recently reported close associations of ribosomes and various components essential to RNA metabolism in and close to human mast cell granules using multiple ultrastructural imaging methods. In view of these findings and an increased awareness of RNA sorting and localization to specific subcellular sites and organelles, we used human mast cells purified from non-tumour portions of lung samples resected at surgery for carcinoma and ultrastructural methods to investigate this further. Poly(U) probes were used to detect direct en grid binding, and radiolabelled as well as non-radiolabelled poly(U) probes were used in in situ hybridization protocols to detect poly(A)-positive pre-mRNA and mRNA in nuclear, cytoplasmic and granular compartments of mature human mast cells. Negative controls verified specificity of label; expected nuclear and cytoplasmic locations of poly(A)-positive RNA served as positive controls for each sample. These findings lend support to the hypothesis that site-specific synthesis in secretory-storage granules may occur in secretory cells.     

RNA is closely associated with human mast cell lipid bodies

Both novel and multiple ultrastructural studies based on different principles show relationships of cytoplasmic lipid bodies and ribonucleic acid (RNA) of potential importance to RNA metabolism in human mast cells. The methods include general ultrastructural morphological observations, imaging of RNA with an EDTA regressive stain, imaging of the incorporation of radio labeled uridine by ultrastructural autoradiography, postembedding immunogold labeling of uridine, ribosomes and small nuclear ribonuclear proteins and ultrastructural in situ hybridization detection of poly(A)-positive messenger RNA. Altogether these studies implicate human mast cell lipid bodies in RNA metabolism and are analogous to earlier similar studies which showed that human mast cell granules also curtain RNA.     

Mast cells of lymph hearts during ontogenesis of frogs Rana temporaria

Electron microscopic observations of the lymph hearts of tadpoles and yearling frogs of Rana temporaria showed that mast cells (MCs) were present not only between muscle fibers (population of resident MCs), but in the cavities of lymph heart (population of circulating MCs), too. There were some differences in the ultrastructure of the resident MCs at each studied stage of larval development. The first recognizable MCs were revealed in the lymph hearts at premetamorphosis (stages 39-41). MCs presented as mononuclear relatively small and slightly elongated cells with a few immature secretory granules and numerous free ribosomes, polysomes and short cisternae of rough endoplasmic reticulum (RER) in the cytoplasm. Chromatin of their nuclei was poorly condensed; the Golgi apparatus was moderately developed. At pro-metamorphosis (stages 44-45), we revealed MCs at different levels of their differentiation. Some MCs demonstrated an active process of granulogenesis in their cytoplasm. Among densely packed cytoplasmic organelles, immature secretory granules were closely associated with cisternae of RER and free ribosomes. Other MCs appeared as more differentiated cells. They were characterized by a predominantly heterochromatic nuclei and cytoplasm filled with polymorphic and heterogeneous granules. MCs also showed a reduction in the number of free ribosomes and cisternae of RER in the cytoplasm. On the contrary, the Golgi apparatus was well developed. Stacks of Golgi cisternae, detaching vacuoles, and progranules occupied the perinuclear region. The majority of the outlines above ultrastructural features of differentiated MCs were typical for MCs of yearling frogs. At metamorphic climax (stages 52-53), MCs often tightly contacted with macrophages. We did not reveal apoptotic MCs. However, some MCs exhibited morphological features typical for programmed necrosis-like death, which was characterized by mitochondria swelling, dilatation of cisternae of RER and nuclear envelope, plasma membrane rupture and subsequent loss of intracellular contents. Electron microscopical immunocytochemistry revealed the localization of atrial natriuretic peptide (ANP), substance S (SP) and heat shock protein (Hsp70) in the secretory granules of the resident and circulating MCs at different stages of tadpole development and in yearling frogs.     

Morphologic mast cell cycles

During ultrastructural studies of the early kinetics of anaphylactic degranulation and early and late recovery intervals from these release reactions induced in isolated, partially purified, cultured human lung mast cells stimulated to release histamine and granules by exposure to anti-IgE, we noted the ability of mast cells to undergo morphologic cycles. Following release of most cytoplasmic granules and shedding of large amounts of membranes and cellular processes, small, immature mast cells, nearly devoid of granules, were seen. These lymphocyte-like cells had condensed nuclear chromatin and large nucleoli. Blast transformation of these small cells and expansion of synthetic machinery-laden cytoplasm resulted in large, immature mast cells with small numbers of small, immature progranules and large numbers of lipid bodies in their cytoplasm. This cycle of mast cell morphologic change has considerable similarities to, as well as some differences from, lymphocyte morphologic cycles.     

Ultrastructure of the APUD-like cells of the frog thymus gland

Within the thymus gland of the European common frog, Rana temporaria, cells with endocrine- like appearance have been found. At the ultrastructural level the most characteristic feature of their cytoplasm is the presence of secretory granules. Some cells possess irregular electron lucent granules with an eccentrically located dense core while others possess smaller electron dense granules. The cytoplasm contains also cisterns of rough endoplasmic reticulum, free ribosomes, and small mitochondria. The cells possess irregular nuclei with the pronounced nucleoli. These endocrine-like cells are connected by desmosomes with neighbouring non-granulated epithelial cells. Ultrastructural features of the cells described here resemble those seen in polypeptide hormone-secreting cells belonging to the family of cells of the APUD (Amine Precursor Uptake and Decarboxylation) series.     

Ribonuclease-gold labels proteoglycan-containing cytoplasmic granules and ribonucleic acid-containing organelles--a survey

An enzyme-affinity-gold method to detect RNA in routinely prepared ultrastructural samples is based on the affinity of the gold-coupled enzyme, ribonuclease, for its substrate, RNA. High concentrations of a known inhibitor of RNase, heparin, are uniquely located in human mast cell granules. Specific labeling for the presence of heparin in these structures was determined using the RNase-gold (R-G) reagent based on the RNase inhibitor property of heparin. This property was used to probe for the presence of proteoglycans (PG) known to be present in a wide variety of ultrastructural samples, none of which contain heparin. In addition to known subcellular sites of RNA, the R-G reagent was shown to bind to PG-rich cytoplasmic granules in a wide variety of leukocytes and secretory cells of epithelial, endocrine, and neuroendocrine origin. This newly recognized property was used to image the changing distribution of labeled PGs during cellular maturation, secretion, and recovery from secretion of secretory cells in vivo, ex vivo, in vitro and in isolated, biochemically defined guinea pig basophil granule preparations.     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

In situ characterization of mast cells in the frog Rana esculenta

The number, distribution, and ultrastructural characteristics of mast cells were assessed in the tongue, heart, and kidney of the frog Rana esculenta. The density of tongue mast cells (253±45 mast cells/mm²) was significantly higher than that of the heart (5.3±0.4/mm²) and kidney (15.3±1.4 /mm²). A striking feature of this study was the remarkable association of frog mast cells to nerves. The ultrastructural study of the mast cell/nerve association demonstrated that mast cells were closely apposed to or even embedded in nerves. Mast cells were also physically associated with melanocytes even in the heart. Mast cells were Alcian blue⁺/safranin⁺ in the tongue and in the peritoneum, whereas in the heart and in the kidney they were Alcian blue^–/safranin⁺. The mast cells in the lamina propria of the gastrointestinal tract were Alcian blue⁺/safranin^–. The cytoplasm of frog mast cells was packed with numerous heterogeneous, membrane-bound granules. The ultrastructure of these cytoplasmic granules was unique, being totally unlike any other previously described granules in other animal species as well as in man. The histamine content/frog mast cell (≈0.1 pg/cell) was approximately 30 times lower than that of human mast cells isolated from different tissues (≈3 pg/cell). A monoclonal anti-histamine antibody was used to confirm the ultrastructural localization of histamine in secretory granules in frog mast cells.     

In situ hybridization and immuno-electron microscope analyses of the Us11 gene of herpes simplex virus type 1 during transient expression

The distribution of Us11 RNA and of its encoded protein have been investigated at the ultrastructural level in HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1. In these transfected cells, Us11 protein accumulates at sites identical to those of lytically infected cells, i.e., in nucleoli and in regions of the cytoplasm that contain ribosomes. Us11 RNA and polyadenylated RNA are scattered over the ribosomerich areas of the cytoplasm. They also accumulate in the nucleoplasm on clustered ribonucleoprotein (RNP) fibrils but also in clusters of interchromatin granules, some of them contiguous to nucleoli. However they are never found in nucleoli. These data reveal the involvement of interchromatin granules in some steps of Us11 mRNA maturation and/or transport.     

Seasonal ultrastructural variations in pinealocytes of the woodchuck,Marmota monax

The ultrastructure of the pinealocyte in the woodchuck, Marmota monax, was studied during the four seasons of the year. Fall cells have a fairly uniform cytoplasmic density, organelles consistent with synthetic and/or secretory activity and rather extensive pericapillary and intercellular spaces. Many winter pinealocytes are nearly devoid of ribosomes and granular endoplasmic reticulum but contain lipid droplets associated with mitochondria. Pericapillary and intercellular spaces are minimal. Spring glands have the greatest variation in cytoplasmic density with intercellular and pericapillary spaces similar to that seen in fall glands. Cells containing electron dense cytoplasm have Golgi zone associated, secretory granules, free ribosomes, short sections of granular endoplasmic reticulum and dense bodies. Cells with a more electron lucent cytoplasm are similar to the most frequently observed summer pinealocytes which have numerous Golgi zones but few associated secretory granules. Microtubules are prominent in the cytoplasm of these cells, the plasma membranes are smooth and intercellular and pericapillary spaces are minimal. A yearly rhythm or cyclic activity of the pinealocyte is suggested.     

The effect of aging on the rat submandibular gland: an ultrastructural, cytochemical and biochemical study

The effect of aging on the rat submandibular gland was studied by using ultrastructural, ultrastructural cytochemical and biochemical techniques. There was an age-related clumping of the nucleolar-associated and peripheral chromatin in many of the acinar cells and a decrease in the number of cisternae of rough endoplasmic reticulum. Many aged acinar cells were binucleated. There was also an age-related increase in pigment granules throughout the gland. These membrane bound granules consisted of a lipid droplet and an associated dense cap which had a granular matrix and pigment droplets. The lead capture method for acid phosphatase activity demonstrated that activity was associated with the granular matrix of the dense cap. These results were correlated with the age-related increase in acid phosphatase activity as determined by colorimetric procedures. There was an age-related increase in the number of cells characterized by small secretory granules. These cells were found as part of the intercalated ducts or at the junction of the duct with the acini. Oncocytes were also found as part of the parenchyma of the aged submandibular gland. These cells were characterized by the pleomorphic mitochondria that fill their cytoplasm. Occasionally, cells that possessed extraordinary numbers of mitochondria and small secretory granules were also observed. The determinations of total DNA and RNA revealed and age-related decrease in RNA while there was no significant change in the concentration of DNA.     

Accumulation of adrenocorticotropin secretory granules in the midbody of telophase AtT20 cells: evidence that secretory granules move anterogradely along microtubules

During the cell cycle the distribution of the ACTH-containing secretory granules in AtT20 cells, as revealed by immunofluorescence labeling and electron microscopy of thin sections, undergoes a cycle of changes. In interphase cells the granules are concentrated in the Golgi region, where they form, and also at the tips of projections from the cells, where they accumulate. These projections contain many microtubules extending to their tips. During metaphase and anaphase the granules are randomly distributed in the cytoplasm of the rounded-up mitotic cells. On entry into telophase there is a rapid and striking redistribution of the granules, which accumulate in large numbers in the midbody as it develops during cytokinesis. This accumulation of secretory granules in the midbody is dependent upon the presence of microtubules. The changing pattern of distribution of the secretory granules during the cell cycle fulfills the predictions of a model envisaging first that secretory granules associate with and move along interphase microtubules in a net anterograde direction away from the centrioles, and secondly that they do not associate with microtubules of the mitotic spindle during metaphase and anaphase.     

Accumulation of low density lipoproteins in stimulated rat serosal mast cells during recovery from degranulation

Stimulation of rat serosal mast cells in vitro with compound 48/80, a degranulating agent, resulted in an immediate increase in binding of low density lipoproteins (LDL) to the stimulated mast cells. The increase in binding was dose-dependent and closely followed the increase in histamine release, i.e., the exocytosis of mast cell granules. It could be demonstrated that the LDL were bound to exocytosed secretory granules which remained cell-associated. During the recovery period the granule-bound LDL were internalized by the mast cells along with the granules. A single stimulation of mast cells rendered their cytoplasm to be filled with granular material showing positive staining for both apoB and neutral lipid. This change was accompanied by a 30-fold increase in the cellular content of cholesteryl esters. Thus, rat serosal mast cells possess a specific mechanism for uptake of LDL that is activated by stimuli that lead to degranulation, the result being massive uptake of LDL by stimulated mast cells during recovery from degranulation.     

In situ detection of the mast cell proteases chymase and tryptase in human lung tissue using light and electron microscopy

Scroll-rich, "mucosal" mast cells are the predominant human lung mast cell type. It has been proposed that these mast cells store tryptase but are mostly chymase deficient. We present a detailed immunolocalisation study of chymase and tryptase in lung specimens of eight patients. Using monoclonal antibody B7 in a conventional tissue processing method for light microscopy, chymase-positive mast cells were much fewer than tryptase-positive ones. However, they approached the number of tryptase-positive cells when optimised processing was used. Two different monoclonal antibodies, B7 and CC1, were used to visualise chymase in purified lung mast cells of two patients using ultrastructural immunogold labelling. Immunoabsorption controls demonstrated a reactivity of B7 with both tryptase and chymase, but indicated specificity of CC1 for chymase. On the ultrastructural level, all of more than 1,400 lung mast cells evaluated labelled for chymase. Reactivity was seen in cytoplasmic granules, cytoplasm and vesicles, but not elsewhere. Tryptase labelling using monoclonal antibody G3 was also present in all mast cells detected, and was retained in altered granules (=activated mast cells), where B7 labelling was sparse. The average labelling density was approximately sixfold higher than for chymase. In summary, chymase may be more abundant in human lung mast cells than hitherto thought.     

P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA

The germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA.     

Ultrastructure of the parathyroid gland of the japanese lizard in the spring and summer season

The ultrastructure of the parathyroid glands of adult Japanese lizards (Takydromus tachydromoides) in the spring and summer season was examined. The parenchyma of the gland consists of chief cells arranged in cords or solid masses. Many chief cells contain numerous free ribosomes and mitochondria, well-developed Golgi complexes, a few lysosome-like bodies, some multivesicular bodies and relatively numerous lipid droplets. The endoplasmic reticulum is mainly smooth-surfaced. Cisternae of the rough endoplasmic reticulum are distributed randomly in the cytoplasm. Small coated vesicles of 700-800 Å in diameter are found occasionally in the cytoplasm, especially in the Golgi region. The chief cells contain occasional secretory granules of 150-300 nm in diameter that are distributed randomly in the cytoplasm and lie close to the plasma membrane. Electron dense material similar to the contents of the secretory granules is observed in the enlarged intercellular space. These findings suggest that the secretory granules may be discharged into the intercellular space by an eruptocrine type of secretion. Coated vesicles (invaginations) connected to the plasma membrane and smooth vesicles arranged in a row near the plasma membrane are observed. It is suggested that such coated vesicles may take up extracellular proteins. The accumulation of microfilaments is sometimes recognized. Morphological evidence of synthetic and secretory activities in the chief cells suggests active parathyroid function in the Japanese lizard during the spring and summer season.     

Translation and granule localization of mouse mast cell protease-5. Immunodetection with specific antipeptide Ig.

An antibody to mouse mast cell protease-5 (MMCP-5) was obtained by immunizing a rabbit with a 17-residue synthetic peptide corresponding to the unique amino acid sequence at residues 146 to 162 in this serine protease. After affinity purification, anti-MMCP-5(146-162) Ig reacted in SDS-PAGE immunoblots to recombinant MMCP-5 and to the native MMCP-5 protein present in the lysates of mouse serosal mast cells and the MC5 line of Kirsten sarcoma virus-immortalized mouse mast cells. Immunocytochemical staining localized MMCP-5 to the cytoplasmic granules of serosal mast cells and Kirsten sarcoma virus-immortalized mouse mast cells. Because mouse bone marrow-derived mast cells express abundant amounts of MMCP-5 mRNA, anti-MMCP-5(146-162) Ig was used to study the translation and granule accumulation of this protease when progenitor cells differentiate into these immature mouse mast cells. Maximal expression of MMCP-5 mRNA occurred after bone marrow cells had been cultured for 2 wk in IL-3-rich WEHI-3 cell conditioned medium, and MMCP-5 protein was detected in these cells. However, electron-microscopic analysis with gold-labeled antibody revealed that the amount of MMCP-5 in the individual granules of bone marrow-derived mast cells varied. The highest concentration of MMCP-5 was found in the most electron-dense secretory granules of the cells. These studies demonstrate the ultrastructural localization of the earliest transcribed mouse mast cell chymase, MMCP-5, and its granule accumulation during the differentiation of mouse bone marrow progenitor cells into immature mouse mast cells.     

Electron microscopic demonstration of calcitonin in human medullary carcinoma of thyroid by the immuno gold staining method

In a human medullary carcinoma of thyroid gland containing calcitonin in light microscopic demonstration by the avidin biotin complex (ABC) method characteristic secretory granules were found electron microscopically in the cytoplasm of the tumour cells. They consisted in so-called type I granules (270 +/- 25 nm) and type II granules (135 +/- 17 nm). By the immuno gold staining (IGS) method the content of many secretory granules measuring 85-270 nm (152 +/- 18 nm) in diameter could be identified as calcitonin. These granules seemed to be predominantly of type II because of their nearly corresponding size and feature. The type I granules were less frequent in number and they showed no or little immunoreactivity. The results indicate that the IGS-method is practicable to demonstrate the ultrastructural localization of calcitonin and to identify clearly the nature of intracytoplasmic granules in electron microscopy.