Development ot the granule population in heterophil granulocytes from rat bone marrow

Summary The development of the heterophil granulocyte in the bone marrow of the rat is described, and an electron-microscopical analysis of the changes in the cytoplasm as well as in the granule population in several stages of maturation is reported. Three types of granule originate in consecutive stages of heterophil maturation. Granules with an internal fine structure (nucleated granules) are the first to be formed, i.e., in early promyelocytes; azurophil granules are formed in late promyelocytes; and specific granules appear in myelocytes. Quantitative analysis showed that the granule population in mature cells, i.e., about 160 granules per electron micrograph, is composed of roughly 14% nucleated granules, 10% azurophil granules, and 76% specific granules. Three cell stages were observed in mitosis: the early promyelocyte, the late promyelocyte, and the myelocyte. Granule counts in non-dividing cells confirmed the occurrence of mitosis in the late promyelocyte and myelocyte.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively. The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyelocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative. Our observations highly suggest that small Golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Summary Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively.The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyolocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative.Our observations highly suggest that small golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.In honour of Prof. P. van Duijn     

Selective abnormalities of azurophil and specific granules of human neutrophilic leukocytes

Human neutrophilic granulocytes (PMN) contain two chemically distinct granule types, which appear at different stages of maturation. The azurophilic granule (or primary granule) is formed during the promyelocyte stage and is known to contain myeloperoxidase in addition to numerous lysosomal enzymes, neutral proteases, glycoaminoglycans, cationic bactericidal proteins, and lysozyme. The specific granule (or secondary granule) is formed during the myelocyte stage. It is defined by the absence of peroxidase and has been shown to contain lysozyme, lactoferrin, and B12-binding proteins. The mature PMN contains both types of granules: 33% azurophilic and 67% specific granules. There are now a few well-documented examples of pathological PMN granulations that can be classified as a selective abnormality of one granule type or the other.     

Investigation of granulocytopoietic kinetics by microdensitometric evaluation of primary granule naphthol-AS-D-chloroacetate esterase activity

Using a scanning microscope photometer we determined quantitatively the enzymecytochemical reaction product for naphthol-AS-D-chloroacetate esterase in neutrophilic granulocytes and their precursors in man. Evaluation of neutrophilic cells from three healthy donors resulted in a logarithm-normal distribution. After subdivision of these cells in their morphologically defined maturational stages no statistically bimodal distribution was shown within the single cell groups. Myelocytes showed twice the amount of the polymorphonuclear neutrophil absorption values. The highest promyelocyte obsorptions were double the values of the myelocyte absorptions. The standard deviation of the absorbance obtained with promyelocytes (which encompass cells already producing granules up to cells reaching their maximal granule content) was significantly higher than the standard deviation of the myelocytes. As already known, primary granules are only synthesized at the promyelocyte stage and - according to the present knowledge - their chloracylesterase and peroxidase activities are not lost during further maturation. Consequently, our results indicate that only enzyme-rich, late promyelocytes undergo mitosis transforming into myelocytes. Correspondingly, their absorption value was halved. Since the absorbance from myelocytes to polymorphonuclears is again halved, myelocytes divide only once. Metamyelocyte absorptions in part correspond to that of myelocytes. This indicates that no distinction can be made between myelocytes with mitotic capacity and "true" if only the size and the nuclear shape are considered metamyelocytes which are not longer capable of undergoing mitosis.     

The ultrastructure of guinea pig heterophil granulocytes and the heterogeneity of the granules

Summary The development of the heterophil granulocytes in the bone marrow of the guinea pig is described. During the maturation of these cells, three types of granule are formed, not only the azurophil and specific granules already described in other mammals but also a third type of granule referred to here as the nucleated granule. During the process of maturation of the cells, these three types of granule are formed successively. On this basis, two steps can be distinguished in the promyelocyte phase in which primary (nucleated and azurophil) granules are formed, i.e. an early and a late stage, nucleated granules being formed in early and azurophil granules in late promyelocytes. Secondary (specific) granules occur first in myelocytes. In mature heterophils of the guinea pig the granule population is composed of about 85% secondary granules, about 10% azurophil granules, and about 5% nucleated granules. The changes in the granule population during the maturation process were quantified. The observations and calculations point to the occurrence of three mitoses: one in the early and one in the late promyelocyte and the third in the myelocyte.     

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.     

Morphometry of feline neutrophil granule genesis

During neutrophil granule genesis, the formation of primary granules is generally thought to be limited to the promyelocyte stage; whereas synthesis of secondary granules is thought to occur only at the myelocyte stage. This hypothesis was tested morphometrically in feline neutrophils that are known to contain both granule types. Marrow specimens obtained from six cats were stained with peroxidase for identification of neutrophil primary granules and counterstained with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) for identification of secondary granules. By regression analysis using arithmetic models, numbers of cytoplasmic granules in 311 cells were correlated with the degree of nuclear chromatin condensation, which was shown to be an adequate parameter for cell maturation. Promyelocytes and myelocytes had similar mean numbers of peroxidase-positive granules per unit area. A significant increase (p less than or equal to 0.0001) in the numbers of peroxidase-positive granules was noted between the metamyelocyte and the mature neutrophil stage, despite the lack of peroxidase activity in endoplasmic reticulum and Golgi lamellae. By contrast, a significant increase of peroxidase-negative granules between the metamyelocyte and the mature neutrophil stage was not clearly established with these methods. The increase in peroxidase-positive granules may indicate continued production of peroxidase-containing granules and/or redistribution of peroxidase among lysosomal organelles in late feline neutrophils.     

Hemopoietic sites and development of eosinophil granulocytes in the loach,Misgurnus anguillicaudatus

Summary Unique eosinophils, each of which contained only one eosinophilic granule, have been found in the peripheral blood of the loach (itMisgurnus anguillicaudatus). Several loach organs have been studied by light and electron microscopy to determine the hemopoietic site of this cell type. Eosinophils are produced mainly in the spleen and to a small extent in the kidney, but not in other organs.Presumed myeloblasts are identified as large lymphoid cells containing a number of small-dense granules (diameter, 0.12–0.16 m) in the cytoplasm. These granules have been observed throughout eosinophilopoiesis but they are most abundant in the promyelocyte stage. The largest cells have been identified as myelocytes which contain a number of large granules (diameter, 0.7–1.4 m) with electron-dense crystalline cores. These large granules are present from the myelocyte to metamyelocyte stage. Metamyelocytes differ from myelocytes in having more large granules. Mature eosinophils are morphologically similar to metamyelocytes but are characterized by the presence of only one very large electron-dense granule (diameter, 2.5–2.8 m) with a crystalline core.The nature of these granules has been studied by enzyme digestion using pepsin and trypsin. The results indicate that the crystalline cores are almost pure protein.     

Schistosoma mansoni: cytochemistry and morphology of the gastrodermal Golgi Apparatus

The gastrodermal Golgi apparatus of adult Schistosoma mansoni displays two distinct morphologies. In one type, there is an identifiable cis (forming) face where vesicles from the endoplasmic reticulum fuse to form the cisternae. A morphological change occurs in the cisternae as the trans (emitting) face is approached with the cisternae becoming progressively flattened. The cisternae at the emitting face produce a membrane-bound secretory granule with moderately electron-dense contents and a vacuolar structure that may be analogous to a condensing vacuole as reported in several vertebrate secretory cells. In a second type, vesicles possessing a thicker membrane than those of the transfer vesicles are observed at the emitting face. They are not observed when the secretory granules are present. Several cytochemical markers were used to aid in studying the polarity of the Golgi apparatus. Enzymes studied were thiamine pyrophosphatase (TPPase) (EC 3.6.1.1), nucleoside diphosphatase (NDPase) (EC 3.6.1.6) using uridine diphosphate as a substrate, and nicotinamide adenine dinucleotide phosphatase (NADPase) (EC 3.1.3.2). Reaction products from all enzyme markers were observed in the cisternae and, to some extent, in the transfer vesicles. At times, NADPase and TPPase reaction products were observed in all cisternae and in the transfer vesicles of the Golgi. When this distribution was evident, the latter vesicles were observed in clusters occasionally fusing with lipid-like globules dispersed throughout the gastrodermis. Heterogeneity in cisternae was observed when NDPase, TPPase, and osmium reduction techniques were used. NDPase activity was limited to the middle cisternae while reduced osmium was observed in the outer two cisternae and in some transfer vesicles. TPPase reaction product was also observed in the secretory granules and in the condensing vacuoles. It is hypothesized that a functional bipolarity may be demonstrated by the Golgi. Under certain stress conditions, the forming face of the Golgi may package lysosomal enzymes while the emitting region of the Golgi appears to be responsible for the packaging of the secretory granules. The fusion of transfer vesicles and, at times, secretory granules with lipid-like globules is postulated to represent a mechanism by which enzymes may be transported to the lumen of the cecum.     

ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES : Two Types Derived from Opposite Faces of the Golgi Complex in Developing Granulocytes

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO₄. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (~800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (~500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.     

A new type of primary granule in guinea pig heterophil granulocytes

Guinea pig heterophil granulocytes were found to have three types of granules which are formed sequentially during the development of the cells in the bone marrow and differ in shape and electron density: nucleated, azurophil and specific granules. Early promyelocytes proved to synthesize nucleated granules of medium electron density prior to the formation of azurophil granules which are highly electron dense, by late promyelocytes. Since the formation of nucleated granules and azurophil granules is restricted to promyelocytes, both can be considered to be primary granules. The moderately dense specific granules (secondary granules) appear later during granulopoiesis and are firstly present in the myelocyte.     

SEGREGATION AND PACKAGING OF GRANULE ENZYMES IN EOSINOPHILIC LEUKOCYTES

During their differentiation in the bone marrow, eosinophilic leukocytes synthesize a number of enzymes and package them into secretory granules. The pathway by which three enzymes (peroxidase, acid phosphatase, and arylsulfatase) are segregated and packaged into specific granules of eosinophils was investigated by cytochemistry and electron microscopy. During the myelocyte stage, peroxidase is present within (a) all rough ER cisternae, including transitional elements and the perinuclear cisterna; (b) clusters of smooth vesicles at the periphery of the Golgi complex; (c) all Golgi cisternae; and (d) all immature and mature specific granules. At later stages, after granule formation has ceased, peroxidase is not seen in ER or Golgi elements and is demonstrable only in granules. The distribution of acid phosphatase and arylsulfatase was similar, except that the reaction was more variable and fully condensed (mature) granules were not reactive. These results are in accord with the general pathway for intracellular transport of secretory proteins demonstrated in the pancreas exocrine cell by Palade and coworkers. The findings also demonstrate (a) that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and (b) that the entire rough ER and all the Golgi cisternae are involved in the simultaneous segregation and packaging of several proteins.     

Ultrastructural features of the developing eosinophils in bone marrow and spleen of the musk shrew, Suncus murinus

Eosinophilopoiesis in the musk shrew, Suncus murinus, a representative of the order Insectivora, was studied by light and electron microscopy. To examine biochemical features of cytoplasmic granules, extraction with proteolytic enzymes was carried out on ultrathin sections of bone marrow. In this species, eosinophils are produced in the same manner in both spleen and bone marrow. Developing eosinophils were distinguished as belonging to four stages, recognized by ultrastructural changes in cytoplasmic organelles as well as the eosinophilic granules during maturation. Granulogenesis began by budding of vacuoles containing flocculent material from the concave face of the Golgi apparatus, in the promyelocyte to myelocyte stage. The matrix of developing granules transformed into a finely granular structure, and the large spherical granules of mature eosinophils were homogeneous without crystalline cores. It was shown by proteolytic enzyme extraction that the proteinaceous cores of mature granules were uniformly removed; there was no evidence that they contained crystalloid inclusions. These results indicate that shrew eosinophils can be regarded as cells that retain a prototype of eosinophil granules, probably like those of ancestral mammals rather than those of higher living Mammalia.     

Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted proteins to the Golgi complex

Toxoplasma gondii relies on protein secretion from specialized organelles for invasion of host cells and establishment of a parasitophorous vacuole. We identify T. gondii Rab6 as a regulator of protein transport between post-Golgi dense granule organelles and the Golgi. Toxoplasma Rab6 was localized to cisternal rims of the late Golgi and trans-Golgi network, associated transport vesicles, and microdomains of dense granule and endosomal membranes. Overexpression of wild-type Rab6 or GTP-activated Rab6(Q70L) rerouted soluble dense granule secretory proteins to the Golgi and endoplasmic reticulum and augmented the effect of brefeldin A on Golgi resorption to the endoplasmic reticulum. Parasites expressing a nucleotide-free (Rab6(N124I)) or a GDP-bound (Rab6(T25N)) mutant accumulated dense granule proteins in the Golgi and associated transport vesicles and displayed reduced secretion of GRA4 and a delay in glycosylation of GRA2. Activated Rab6 on Golgi membranes colocalized with centrin during mitosis, and parasite clones expressing Rab6 mutants displayed a partial shift in cytokinesis from endodyogeny (formation of two daughter cells) to endopolygeny (multiple daughter cells). We propose that Toxoplasma Rab6 regulates retrograde transport from post-Golgi secretory granules to the parasite Golgi.     

Regulation of secretory granule formation in chronically hypersecretory melanotrophs in the rat pituitary

The formation of secretory granules in chronically hypersecretory melanotrophs in the rat pituitary was studied. Hypersecretion was induced by treatment with the dopamine antagonist haloperidol (1.5 mg/kg daily for 7 days), which releases the normal neural dopaminergic inhibition of secretion from the melanotroph. Morphometric analysis showed a 100% increase in the volume fraction of granular endoplasmic reticulum after haloperidol treatment, while the volume fractions of electron-dense granules, electron-lucent granules and the Golgi apparatus were unaltered. The mean diameter of the mature secretory granules was increased by 10%, indicating a 30% increase in mean granule volume. A similar increase in diameter was observed in condensing granules within the Golgi area. With earlier results on the effect of chronic inhibition the study shows that a main adaptive response of the melanotroph to altered secretory conditions is a change in the volume of the secretory granules, regulated by a mechanism that operates at an early stage of granule formation.     

The cytomorphology of goblet cells of the fetal intestine. Studies of the large intestine of cattle (Bos primigenius taurus)

In the region of the base of the intestinal crypts undifferentiated goblet cells display a configuration and constellation of organelles and membrane structures that are indicative of their importance for function. These images at this stage of development deliver a scenario of the mechanism of secretory granule production: aggregates of protein vesicles from the "transitional elements" (PALADE) of the granular endoplasmic reticulum are, so to speak, rolled up on the trans side of the Golgi apparatus by inversion of peripheral membrane segments of the innermost Golgi lamellae, thereby forming corpuscles. The origin of the capsulated vacuoles, which contain vesicles as single elements or as conglomerates, is well established. Their capsule consists of a trilaminar external and external and internal membrane; between them lies condensed material of the Golgi apparatus. In the opinion of the present author, the development of the ensheathed vacuoles represents a basic, more general mechanism. In contrast, the further steps of synthesis, for the formation of secretory granules, are more heterogeneous. Condensation of the vesicles and the inner capsular membrane results in the formation of a prosecretory granule, which in the basic element in the process of secretory granule production. The prosecretory granules develop singly or by fusion with other granules to give primary secretory granules. The complexity of this mechanism of secretory granule formation, however, becomes evident when considering the apposition of capsulated vacuoles and prosecretory--primary--secondary secretory granules, of prosecretory and primary secretory granules as well as prosecretory granules and secondary secretory granules. Generally, primary granules show a tendency to become secondary secretory granules or to fuse with them. During maturation of the goblet cells the secretory granules fuse to form larger mucous bodies in the theca by fusion of the laminae of the membranes; a final product, there is a homogeneous mucous mass devoid of membranes.     

Defensin-rich granules of human neutrophils: characterization of secretory properties

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.     

Ultrastructure of esophageal glands and their secretory granules in the root-knot nematodeMeloidogyne incognita

Summary The dorsal and subventral esophageal glands and their secretory granules in the root-knot nematodeMeloidogyne incognita changed during parasitism of plants. The subventral esophageal glands shrank and the dorsal gland enlarged with the onset of parasitism. While secretory granules formed by both types of glands were spherical, membrane-bound, and Golgi derived, the granules differed in morphology and size between the two types of glands. Subventral gland extensions in preparasitic second-stage juveniles were packed with secretory granules which varied in diameter from 700–1,100 nm and had a finely granular matrix. Within the matrix of each subventral gland granule was an electron-transparent core that contained minute spherical vesicles. The size and position of the core varied within different granules. Few granules were present in the dorsal gland extension in preparasitic juveniles. The matrix of dorsal gland secretory granules formed during parasitism was homogeneous and more electron-dense than the matrix of subventral gland granules. Subventral gland secretory granules of parasitic juveniles and adult females appeared degenerate.