Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study

Summary The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 m in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled. From these findings, it is suggested that the granules of rat Clara cells consist of two types of granules of distinct origin; one appears to derive from condensing vacuoles of Golgi origin, whereas the other may be formed by membranefusions with small cytoplasmic vesicles of unknown source.     

Streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit azurophilic granule fusion with phagosomes

We recently reported that the human pathogen Streptococcus pyogenes of the M1 serotype survives and replicates intracellularly after being phagocytosed by human neutrophils. These data raised the possibility that the generation of reactive oxygen metabolites by neutrophils, and the release of microbicidal molecules from their azurophilic and specific granules into phagosomes, can be modulated by S. pyogenes bacteria expressing surface-associated M and/or M-like proteins. We now demonstrate, using flow cytometry, immunofluorescence microscopy and transmission electron microscopy, that live wild-type S. pyogenes, after internalization by human neutrophils, inhibits the fusion of azurophilic granules with phagosomes. In contrast, azurophilic granule-content is efficiently delivered to phagosomes containing bacteria not expressing M and/or M-like proteins. Also, when heat-killed wild-type bacteria are used as the phagocytic prey, fusion of azurophilic granules with phagosomes is observed. The inhibition caused by live wild-type S. pyogenes is specific for azurophilic granule-phagosome fusion, because the mobilization of specific granules and the production of reactive oxygen species are induced to a similar extent by all strains tested. In conclusion, our results demonstrate that viable S. pyogenes bacteria expressing M and M-like proteins selectively prevent the fusion of azurophilic granules with phagosomes.     

Accumulation of phosphate-containing granules in the nucleoid area of Pseudomonas aeruginosa

Many electron-dense granules were found in the nucleoid area of Pseudomonas aeruginosa strain K by electron microscopy with the technique of the freeze-substitution method. These granules contained phosphorus and calcium as determined by X-ray microanalysis. The size and the numbers of the granules decreased when the bacteria was cultured in the medium from which phosphate-containing compounds were depleted. From these observations we concluded that the granule was a phosphate-containing granule and possibly a polyphosphate granule. The excellent preservation of the fine structures by the freeze-substitution technique enables us to show very small polyphosphate granules in the nucleoid area of the bacterial cells which cannot be revealed by the conventional chemical fixation method. As we could not see the granules in other bacteria cultured in nutrient medium such as Serratia, Escherichia, Bacillus and Vibrios, the accumulation of the phosphate granules in Ps. aeruginosa might be a unique character of this bacteria and might be related to the growing capability of this bacteria in extremely low nutrient supply.     

Ultrastructural study of periodic lamellar granules in human neutrophils

Granules consisting of periodically arranged membranous lamellae and amorphous electron-opaque material, i.e., periodic lamellar granules, are present in human neutrophils. To date, no extensive ultrastructural studies have been carried out on these granules because of their infrequent presence in neutrophils. The bone marrow of 18 cases of chronic myeloproliferative disorders, including one case of chronic neutrophilic leukemia in which periodic lamellar granules were frequently seen in neutrophils, was investigated by electron microscopy. Periodic lamellar granules were seen in neutrophils in 12 of the 18 cases at varying frequencies. They were preferentially seen in immature neutrophils. The transverse profiles of these granules revealed concentric complete/incomplete rings or periodic parallel straight lines, i.e., various patterns of lamellar arrangement were present. Periodic lamellar granules were positive for myeloperoxidase and lysozyme at the electron-microscopic level. These results suggest that these granules represent a primary neutrophil granule subtype. However, their functional and pathologic significance remains unknown.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Ultrastructure and morphogenesis of ceroid pigment. II. Late changes of lysosomes in Kupffer cells of rat liver after phagocytosis of unsaturated lipids (author's transl)]

Wistar rats were injected intravenously with cod liver oil emulsion. The lipid droplets ere phagocytized by Kupffer cells and stored in lysosomes. The transformation of these lipid-containing lysosomes into ceroid pigment granules was studied electron-microscopically and cytochemically for a period of 12 weeks after the injection. The lipid droplets enclosed in lysosomes show an increasing and continous condensation from the periphery towards the center due to oxidation and polymerization of unsaturated fatty acids. During the first week almost the total amount of the stored lipids is transformed into an amorphous, highly electron-dense material which disintegrates into cloddy and globular fragments during the following time. The fragments are embedded in a fine granular, slightly electron-dense matrix showing a marked activity of acid phosphatase. The lysosomal structures which contain remnants of condensed oxidized and polymerized lipids are the electron-microscopic equivalent of the granules as seen by light microscopy. These lipids, which have been changed in their molecular structure, cannot be hydrolized by lysosomal enzymes. They remain as an indigestible material, as a waste product in lysosomal residual bodies. Both lipofuscin and ceroid are lysosomal structures containing oxidized and polymerized lipids. The differences between these lipogenous pigments are due to their different formal and causal genesis. Lipofuscin develops in parenchymal and muscle cells by autophagocytosis and by subsequent oxidation and polymerization of segregated membrane lipids. Ceroid is formed in macrophages by heterophagocytosis of unsaturated lipid material which is also oxidized and polymerized.     

The life cycle of the gastrin granule

Summary The ultrastructure of gastrin cells in the rat antrum was analyzed with standardized and quantitative planimetric methods. Resting and active cells were compared. The gastrin cells were activated by removal of the acidproducing part of the stomach (fundectomy). As a result the serum gastrin concentrations were greatly elevated. Compared with gastrin cells in fasted control rats the gastrin cells in fundectomized rats were increased in number, contained fewer cytoplasmic granules, increased amount of endoplasmic reticulum, and an enlarged Golgi area.Generally, the secretory granules of the gastrin cell displayed a wide range of electron density from highly electron-dense to electron-lucent. They exhibited certain characteristic features: 1) Electron-dense granules made up a greater proportion of the total granule population in active gastrin cells than in resting cells. 2) Electron-dense granules were more frequent near the Golgi stacks than in the periphery of the cell. 3) Electron-dense granules were smaller in size than the electron-lucent granules; hence, small electron-dense granules probably represent young granules (progranules), while large, electron-lucent granules represent mature (old) granules. 4) Electron-dense granules invariably displayed a more intense immunoreactivity than electron-lucent granules.The gastrins are generated from a large precursor molecule. The posttranslational processing of this precursor is reflected in the gastrin-component pattern. The gastrin-component pattern in antral extracts of fundectomized and normal fasting rats differed in that the proportion of the gastrin-4-like component was reduced, whereas the gastrin-34-like component was increased in the fundectomized rats. The results suggest a greater proportion of small gastrin components in the mature granules than in the newly formed ones, presumably due to more extensive conversion of larger forms into smaller forms with a longer granule half-life. As a result gastrin-17-and gastrin-34-like components make up a larger proportion of total gastrin in active gastrin cells than in resting gastrin cells.The findings were presented at the Meeting of the European Gastro-Club, Erlangen, October 1980 (R. Håkanson, J.F. Rehfeld, M. Ekelund, and F. Sundler 1981)     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

A cytochemical, light and electron microscopic study of the eosinophilic granule cells in the gut of the rainbow trout, Salmo gairdneri Richardson

The distribution, morphology and cytochemistry of the eosinophilic granule cells in the gut of the rainbow trout, Salmo gairdneri , were studied using light and electron microscopes. Granule cells were associated generally with the propria of the gut wall, the majority being arranged in a definitive layer, as a stratum granulosum. The cytoplasm of the eosinophilic granule cells contained numerous spherical, membrane-bound, homogeneous electron-dense granules, ranging from 0·1–1·3 urn in diameter. A portion of the cytoplasm located towards one pole of the cell was usually devoid of organelles other than ribosomes. Whatever their location, the eosinophilic granule cells were always associated with fixed connective tissue cells, referred to here as 'ensheathing' cells. Phospholipid, acid mucopoly-saccharide and the activities of alkaline phosphatase, acid phosphatase, arylsulphatase and 5-nucleotidase were demonstrated in the granules. It is hypothesized that the 'ensheathing' cells and eosinophilic granule cells constitute a part of the body defence mechanism.     

Morphometric and densitometric study of the biogenesis of electron-dense granules in Fonsecaea pedrosoi

Fonsecaea pedrosoi is a polymorphic pathogenic fungus, etiological agent of chromoblastomycosis, that synthesizes a melanin-like pigment. Although this pigment has been described as a component of the outer layers of the cell wall, electron-dense cytoplasmic bodies have also been visualized. In this work, we have correlated the appearance of intracellular electron-dense granules with the melanization process in F. pedrosoi. For this, conidial forms were grown under conditions where melanin was not synthesized. Afterwards, cells were incubated in Hank's medium supplemented with bovine fetal serum, at 37 degrees C, to stimulate the pigment production. The genesis of cytoplasmic bodies, with different stages of electron density, was demonstrated by transmission electron microscopy. The appearance of fungal acidic compartments, visualized by confocal laser scanning microscopy in cells stained with acridine orange, was time coincident with the formation of electron-dense granules observed by transmission electron microscopy. The quantification of granule numbers as well as morphometric and densitometric studies were performed.     

Pregnant mouse corpora lutea: immunocytochemical localization of relaxin and ultrastructure

Relaxin was localized in corpora lutea of pregnant mouse ovaries by using the unlabeled antibody peroxidase-antiperoxidase technique and a highly specific rabbit antirat relaxin serum. Relaxin immunostaining was first observed in luteal cells located at the periphery of corpora lutea on Day 10 of gestation. The number of relaxin immunostained cells and the intensity of the stain gradually increased to reach a maximum between Days 16 and 18 of gestation. While a few luteal cells were specifically stained for relaxin on Day 1 postpartum, no luteal cells were stained on Day 2 postpartum. Ultrastructural studies of luteal cells from pregnant mouse ovaries revealed the presence of a distinct electron-dense, membrane-bound granule population, which was first observed on Day 12 of gestation. The granules increased in number to reach a maximum between Days 16 and 18 of gestation, and were absent by Day 2 postpartum. The appearance and disappearance of this granule population closely paralleled the relaxin immunostaining in the luteal cells. We suggest that the granules may be the subcellular sites of relaxin storage in the pregnant mouse ovary.     

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.     

Electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of the calf fundic glands

The electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of calf fundic glands was studied using specific antisera for prochymosin and pepsinogen with a protein A-gold method. Prochymosin and pepsinogen immunoreactivities were detected in the same secretory granules of the chief, mucous neck and transitional cells, simultaneously using small and large colloidal gold particles. In chief cells, both immunoreactivities were distributed uniformly over the same zymogen granules showing a round, large, homogeneous and electron-dense appearance. In mucous neck cells, both immunoreactivities were found exclusively on the same electron-dense core located eccentrically in the mucous granule showing light or moderate electron density. In transitional mucous neck/chief cells, electron-dense cores became larger in size and some granules were occupied by the electron-dense core without a halo between the core and the limiting membrane. Both immunoreactivities were found uniformly over the electron-dense core. The granules having no halo in the transitional cells could not be distinguished from the typical zymogen granules in the chief cells.     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

Different Requirements for Early and Late Phases of Azurophilic Granule–Phagosome Fusion

Phagocytosis and killing of microorganisms are complex processes that involve tightly regulated membrane traffic events. Because many signaling molecules associate with membrane rafts and because these structures can be found on azurophilic granules, we decided to investigate raft recruitment and the signaling requirements for azurophilic granule secretion during phagosome maturation. At the site of phagocytosis of immunoglobulin G-opsonized prey in human neutrophils, we found that early secretion of azurophilic granules was both raft- and calcium-dependent. Subsequently, rafts at the phagocytic site were internalized with the prey. At the fully formed phagosome, the fusion of azurophilic granules was no longer dependent on rafts or calcium. These findings were found to be true also when using Streptococcus pyogenes bacteria as prey, and depletion of calcium affected the kinetics of bacterial intracellular survival. These findings suggest that the mechanisms for delivery of azurophilic content to nascent and sealed phagosomes, respectively, differ in their dependence on calcium and membrane rafts.     

Ultrastructural changes associated with renin secretion from the juxtaglomerular apparatus of mice

Summary Thin sections and freeze-fracture replicas were used to investigate the ultrastructural changes associated with renin secretion from the juxtaglomerular part of the afferent arteriole of male mice. Adrenalectomized animals in which renin secretion was stimulated by furosemide application and bleeding were also studied. Exocytosis of mature electron-dense granules was found in all experimental groups. Before extrusion, the region of granule facing the cell membrane changed, with vesicular and/or stacked membrane-like profiles and a small local protrusion of the granule membrane appearance of. Concomitantly, punctuate sites of fusion between the cell and granule membranes were observed. Later, unaltered amorphous, and altered membrane-like granule content was released from omega-shaped cavities into the extracellular space. In stimulated animals the alteration and extrusion of several closely apposed granules was reminiscent of compound exocytosis. Coated pits were frequently seen, suggesting specific retrieval of the former granule membrane. The collapsing silhouette of a depleted granule very rarely took the form of a saccule whose narrow membrane-bounded neck was continuous with the extracellular space.Observed were two additional events by which active and inactive renin may be released. Small electron-lucent vacuoles of undetermined origin fused with the cell membrane and, in stimulated kidneys, some epithelioid cell processes disintegrated. However, the interpretation of the related ultrastructural phenomena was uncertain.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Ultrastructure of granulocytes of teleost fish (Salmoniformes, Cypriniformes, Perciformes)

Analysis of data on ultrastructure of granulocytes of freshwater and marine bony fish of orders Salmoniformes, Cypriniformes, and Perciformes showed that in all studied species there were revealed two types of granulocytes-neutrophils and eosinophils. The exception was the bluefish Pomatomus saltatrix L. whose pronephros hemopoietic tissue was found to contain one type of the granulocyte line-neutrophils. The identification parameters of granular leukocytes are specific granules filling the cytoplasm. The main form of specific granules in neutrophils of bony fish of various phylogenetic groups is an elongated granule with different distribution of fibrils or a granule that has fibrils-formed crystalloid. Eosinophil granules are mainly of large, electron-dense, homogenous form.     

Electron microscopic observations of the phagocytosis and subsequent fate of Aeromonas salmonicida by Atlantic salmon neutrophils in vitro

Neutrophils isolated from the blood of Atlantic salmon (Salmo salar L.) were studied by electron microscopy at various time intervals after being incubated with opsonised and non-opsonised Aeromonas salmonicida strains, namely, the avirulent MT004 (A-layer negative) and the virulent MT423 (A-layer positive). After 15 min incubation with all four groups of bacteria (virulent, avirulent, opsonised or non-opsonised) a large number of neutrophils showed an elongated shape with the nucleus and all the organelles being located in one pole of the cell. Small vacuoles and clumping of glycogen granules were also observed. Neutrophils devoid of granules were noted after 30 min incubation, the majority containing engulfed bacteria. Degenerate neutrophils were also found in all the groups incubated with bacteria. Phagocytosis of bacteria was observed after 15 min incubation. The number of intracellular bacteria was very low, usually one or two per cell, although some neutrophils incubated with the opsonised avirulent strain MT004 contained a larger number of engulfed bacteria. Ingestion of bacteria was usually accompanied by the formation of phagocytic vacuoles containing an amorphous material of moderate electron-density as well as granule discharge into the vacuole. Both strains (MT004 and MT423), opsonised and non-opsonised, underwent morphological alterations after 3-7 h incubation suggesting that both A. salmonicida strains were killed by the neutrophils.     

Structure of yolk granules in oocytes and eggs of Blattella germanica and their interaction with vitellophages and endosymbiotic bacteria during granule degradation

Oocytes and embryos of the cockroach Blattella germanica were examined by optical and electron microscopy to study yolk granule degradation during embryo development. During vitellogenesis, progressively larger yolk granules are formed in the ooplasm and by chorionogenesis, the mature granules are packed so tightly that their shape is highly distorted. Throughout ovarian development, endosymbiotic bacteria lie at the follicle cell/oocyte interface. Just prior to chorionogenesis the endosymbionts transit the oocyte plasma membrane and cluster at the periphery. Bacteria become more numerous over the ventral region of the egg by day 1 postovulation and begin to invade the interior of the yolk mass from the ventral periphery. At that time, lysis of the nearby yolk granules occurs while those in the central ooplasm remain intact and free of bacteria up to day 4. Vitellophages become evident by day 2 postovulation. These cells are also distributed over the egg's periphery but are most numerous in the ventral region. Vitellophages, in association with the endosymbionts, protrude toward the yolk granules and extend filo- and lamellipodia over the granule surface. Portions of the yolk granules are then engulfed and sequestered as large vacuoles in the vitellophage's cytoplasm. The vacuoles then become vesiculated. As embryo development proceeds, the vesiculated portions partition into smaller multivesicular bodies. This study describes the dynamics of yolk granule-vitellophage interaction in embryos of B. germanica and suggests that yolk utilization entails the cooperative efforts of both vitellophages and endosymbiont bacteria.