Cytochemical characterization of leucocytes from the seawater teleost,gilthead seabream (Sparus aurata L.)

The cytochemical characterization of head-kidney and peripheral blood leucocytes of gilthead seabream (Sparus aurata L.) was studied by light and electron microscopy. Neutrophilic granulocytes show some cytoplasmic granules, which are positive for alkaline phosphatase and peroxidase but acid phosphatase negative. The scarce granules found in the cytoplasm of the circulating neutrophils and their cytochemical features seem to be indicative of an immature stage. Acidophils are also alkaline phosphatase and peroxidase positive at pH 11.0. They are strongly positive for acid phosphatase and acid phosphatase activity may thus be considered a cytochemical marker to characterize and differentiate neutrophilic from acidophilic granulocytes in this fish species. Three granule populations are characterized in the cytoplasm of the gilthead seabream acidophils: the first is positive only for peroxidase and the second contains a dense core with acid and alkaline phosphatase activities, surrounded by a thin peroxidase positive electron-dense halo. The third granule type contains an eccentric core, which is strongly positive for acid and alkaline phosphatase and peroxidase. As regards their cytochemical features, the first and second granule types seem to correspond respectively to the azurophilic and specific granules found in acidophils of mammals and could be involved in phagocytic processes, thus playing an important microbicidal role in this species. The monocytes, monocyte-macrophages and macrophages show different cytochemical features. The first have scarce acid phosphatase-positive lysosomes, while blood monocyte-macrophages and macrophages are positive for acid and alkaline phosphatases and for peroxidase; the monocyte-macrophages show scarce lysosomes.     

Cytochemical and ultracytochemical studies of peroxidase activity in rabbit blood granulocytes under hydrocortisone effect

Cytochemically peroxidase activity has been examined on the light optical and ultrastructural levels in blood granulocytes of the rabbits after a single (5 mg/kg) and multiple (1 and 5 mg/kg every 24 hrs during 4 weeks) administrations of hydrocortisone. Under electron microscope peroxidase activity was detected in the blood of intact rabbits into typical primary granules (TPG) and small polymorphic granules (SPG) of neutrophils as well as into specific granules of basophils sometimes in perinuclear space and GER channels. 6 h after hydrocortisone injection peroxidase activity in neutrophils increased, the reaction product in both kinds of cytoplasmic granules was electron denser than in the controls. After multiple hydrocortisone (1 mg/kg) administrations peroxidase general activity in granulocytes has not considerably changed, but the number of TPGs and SPGs was decreased in neutrophils. Multiple administrations of a higher dose of hydrocortisone (5 mg/kg) have induced peroxidase activity decreasing in neutrophils and a decrease in the number and electron density of TPGs and SPGs in them. In basophils there was a significant accumulation of the reaction product of high electron density in perinuclear space, in specific granules and GER channels. The conclusions has been drawn that a short-term raising of hydrocortisone level stimulates and prolonged hypercorticism inhibits peroxidase activity in neutrophils and, consequently, their function.     

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.     

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     

Fine structure of the granulocytes occurring in the hypothalamic-hypophyseal ventricle and neurohypophysis of the hagfish,Paramyxine atami

Strange granulated cells in the hypothalamic-hypophyseal ventricle and neurohypophysis of the hagfish,Paramyxine atami, were examined under light and electron microscope. Based on pleomorphic electron dense granules scattered in the entire cytoplasm and eccentrically shifted nucleus, the cells in question were diagnosed as the extravascular granulocytes.     

Electron microscope study of haemolymph cells of Metrioptera roeselii (Orthoptera,Tettigoniidae)

On the basis of patterns of haemocyte ultrastructure and functions at preimago and imago stages of Metrioptera roeselii, secretory cells of granulocyte type were recognized in the haemolymph. The development of granulocytes was traced starting from their formation up to cell death and destruction. The haemocytes develop as "dark" and "light" cells, differing in their functional activity, although their ultrastructure is similar. In the cytoplasm of granulocytes, granules of both mitochondrial and nuclear origin were detected. Simultaneously two processes occur in the cells--the accumulation and discharge of granules.     

A cytochemical, light and electron microscopical study of the leucocytes of the adult river lamprey, Lampetra fluviatilis (L. Gray)

The morphological features of the leucocytes from the blood of the river lamprey, Lampetra fluvimilis , were studied using light and electron microscopy. Four cell types were identified, namely granulocytes, lymphocytes, monocytes and thrombocytes. Enzyme cytochemical tests were also performed for the detection of acid phosphatase, β-glucuronidase and peroxidase. All the leucocyte types were positive for acid phosphatase and β-glucuronidase, to a variable extent, with the greatest activity seen in the granulocytes. None of the leucocyte types however, contained any peroxidase activity.
Only one type of granulocyte was found and this appears to be analogous to the mammalian neutrophil/heterophil. Characteristically, it has a cytoplasm containing a large number of morphologically heterogeneous granules (0.07–0.40 um in diameter). It is suggested that these granules, rather than belonging to several subpopulations, are in fact part of a single maturation series.
The results of this study show that precise identification of lamprey leucocytes can only be achieved using a combination of ultrastructural and cytochemical techniques.     

Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.     

Composition and ultrastructure of elasmobranch granulocytes. II. Rays (Rajiformes)

The blood granulocyte composition of seven species of ray is given together with ultrastructural observations made on the epigonal organ and blood of Pavoraja spinifera and the spleen of a deepwater rajid skate. Under the light microscope three granulocyte types, eosinophils, eosinophilic granulocytes and neutrophilic granulocytes could be distinguished. At the EM level two granulocyte types were apparent, one with elongated granules containing longitudinal fibrils that consolidated to form an axial rod-like inclusion, and the other with large, spherical, uniformly electron-dense granules. Correlation of light and electron microscope observations indicated that the neutrophilic granulocytes with weakly basophilic, elongated granules become weakly eosinophilic, as eosinophilic granulocytes, and these in turn develop to eosinophils with granules containing axial rods. The other granulocyte type forms another population of eosinophils with spherical granules.
The inter-relationship of these granulocytes, the identification of eosinophilic granulocytes, or heterophils, as immature eosinophils, and the co-existence of two morphologically distinct eosinophil forms are discussed.     

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.     

Induced inflammatory process in Peripatus acacioi Marcus et Marcus (Onychophora)

The inflammatory response induced by the implant of a suture thread in Peripatus acacioi muscle was characterized under light and transmission electron microscopy (TEM). After 24 and 48 h granulocytes were observed migrating through the connective tissue toward the suture thread. These cells contain cytoplasmic eosinophilic granules as well as free granules near to the thread. There were few spherule cells with eccentric smooth kidney-shaped acidophilic nuclei and basophilic granules. Cells with intermediary characteristics as well as cells with a central basophilic nucleus with scarce acidophilic cytoplasm devoid of granules were also found. Under TEM, the granulocytic coelomocytes show small and homogeneous electron dense granules, while the spherule cells possess spherules that can be heterogeneous, granular, or with myelin figures. An acute induced inflammatory process is described for the first time in Onychophora and contributes to the scarce available literature on the function of the coelomocytes within this group.     

Ultrastructural,cytochemical and functional studies on the eosinophilic granulocytes of larval lampreys

Summary Blood from larval lampreys (ammocoetes) contains a small number of eosinophilic granulocytes which are formed in the protospleen and kidney. Both immature and mature forms of this cell type are present in the blood and these are easily identified from other cell types due to the prominent eosinophilic granules that fill the cytoplasm. Ultrastructurally, these granules are electron-dense, largely unstructured, Golgi-derived and contain acid phosphatase but not peroxidase. Eosinophilic granulocytes ingest bacteria but fail to internalise colloidal carbon. The functional and phylogenetic significance of these cells is discussed.     

Gut-associated lymphoid tissue (GALT) of the goldfish,Carassius auratus

The head kidney and spleen are major sites of haemopoiesis in fish; a secondary center is found in loose connective tissue of the intestine. In this study we determined the nature of gut-associated haemopoietic tissue in the goldfish, Carassius auratus, using light and electron microscopy. This tissue is a loose stroma of reticular cells and fibers vascularized by capillaries, venules, and arterioles. The cellular population includes lymphoblasts, small and medium-sized lymphocytes, plasmocytes, macrophages, and various granulocytes. The most abundant granulocyte is the mast cell, whose large granules stain with Alcian blue and toluidine blue. Heterophils are found in the intestinal connective tissue as well as two other granulocytes: one with ovoid granules having dense parallel lamellae and another with granules containing crystalline inclusions. Immature forms of both granulocytes were also noted. Macrophages containing phagocytosed debris were often located close to the epithelium; they were observed forming clusters with lymphocytes. The epithelium contained a number of migrating leucocytes including lymphocytes and lymphoblasts, macrophages, and heterophils. Although many granulocytes were found in the connective tissue, granulopoiesis does not seem to be a major function. Gut-associated haemopoietic tissue in goldfish resembles diffuse lymphoid tissue and may be involved in intestinal immune responses.     

Composition and ultrastructure of elasmobranch granulocytes. I. Dogfishes (Squaliformes)

The blood granulocyte composition of 10 species of dogfish is given, together with ultrastructural observations made on Etmopterus baxteri Leydig organ and blood, and on spleens of Oxynotus bruniensis, Deania calcea, Scymnodon plunketi and blood of Centroscymnus crepidator . Neutrophilic granulocytes, which were common, had spherical granules that developed a dense core, which then lost contents to become lucent. Eosinophilic granulocytes had ovoid or elongated granules with a fibrillar content that became aligned longitudinally, and rarely formed an axial rod. Eosinophils had large spherical granules that were electron-dense but in early stages had a disorganised fibrillar content. These cells correspond to the neutrophils, heterophils and eosinophils, respectively, of other elasmobranchs.
Dogfish granulocytes are compared with those of other elasmobranchs, and their lack of similarity to those of higher vertebrates is noted.     

Phagocytosis and killing of Staphylococcus aureus by mouse granulocytes after interleukin-3 (IL-3) injection

Interleukin-3 is a multipotential hematopoietic growth factor, which like other colony stimulating factors (CSFs) is effective "in vitro" stimulation of the mature cells function. It was found that IL-3 synergistically with GM-CSF and G-CSF stimulated the proliferation of the granulocytes. Therefore the purpose of this investigation was the evaluation "in vivo" of the influence of IL-3 on the phagocytosis, bactericidal activity, and enzyme activities of granulocytes. IL-3 was injected into mice subcutaneously during 5 days in dose 1 microgram/kg/d. The examination of the percent of cells phagocytizing bacteria (Staphylococcus aureus), NBT test and bactericidal activity, were performed every day and evident increase of the tested parameters was found. Additionally the enzyme activities in primary granules were measured and showed on increase of acid phosphatase and peroxidase activity.     

Cytochemical identification of the leukocytes of torpedoes (Torpedo marmorata Risso and Torpedo ocellata Rafinisque)

The authors subjected peripheral blood smears of Torpedoes to cytochemical analysis of lipids, protein, neutral and acid polysaccahrides and of some enzymatic activities, i.e. adenosine triphosphatase (ATP-ase), acid and alkaline phosphatase, aliesterase and peroxidase. It was found that neutrophilic granulocytes are intensely PAS and aliesterase positive and weakly ATP-ase positive. Eosinophilic granulocytes show the presence of neutral polysaccharides in the matrix (which is PAS positive) and strong ATP-ase and acid phosphatase activities in the granules. Lymphocytes sometimes contain weakly PAS and aliesterase positive granules. Monocytes show some small PAS positive granules and weak acid phosphatase and aliesterase activities. Thrombocytes contain some peripheral granules which are PAS positive and slightly ATP-ase positive. There are no transitional forms between the various cellular types. The results confirm the classification of leukocytes of Torpedoes into neutrophilic granulocytes, eosinophilic granulocytes, lymphocytes, monocytes and thrombocytes and contribute some informations about the histoenzymatic content of Elasmobranch leukocytes.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

The haematopoietic supraneural organ of adult, sexually immature river lampreys (Lampetra fluviatilis [L.] Gray) with particular reference to azurophil leucocytes.

The haematopoietic tissue in the supraneural organ of the freshwater river lamprey (Lampetra fluviatilis L. Gray) was studied in sexually immature animals. Besides erythro- and granulopoietic elements, macrophages, reticular cells, fibroblasts and glycogen-rich fat cells were seen. Developing granulocytes of the lamprey contain one type of azurophil granules originating from small cytoplasmic (Golgi) vesicles. The lamprey's azurophil granulocytes seem to be homologous with those of fishes. However, the granulocytes of fishes, studied thus far, show granules with only one type of inclusion, whereas in lamprey the granulocyte inclusions are variable in size and shape. Thus, lamprey granulocytes are, in this respect, reminiscent of similar cells of higher vertebrates. The PAS and alkaline phosphatase reactions, common markers of vertebrate neutrophil leucocytes, are very weak in the haematopoietic tissue granulocytes of the lamprey, and intense in the blood cells of the same animal. Lamprey granulocytes, similarly to the granulocytes of Chondrostei and Elasmobranchiata, do not stain with peroxidase, naphthol-AS-D-chloroacetate esterase and sudan black B. The haematopoietic tissue contains a relatively high number of degenerated granulocytes.     

Cytochemical observations of coelomocytes from the earthworm, Lumbricus terrestris.

Synopsis Coelomocytes of the earthworm,Lumbricus terrestris, were stained by cytochemical techniques to determine the biochemical composition of the seven different cell types and subtypes. The enzymes acid phosphatase and -glucuronidase are present in all types of coelomocytes, but are especially abundant in basophils and neutrophils; the differences in enzyme amounts correlate well with the differences in phagocytic activity of the various cell types. No peroxidase is present. The cytoplasmic basophilia of basophils is due primarily to ribonucleic acid. Basophils also contain large deposits of glycogen, with neutrophils and chloragogen cells containing somewhat lesser amounts. The predominant granules of the two types of acidophils and of granulocytes are composed of a basic protein and a neutral mucopolysaccharide or glycoprotein. A second granule population, present in low numbers in acidophils and granulocytes, but in larger numbers in basophils and neutrophils, is small in size and lipid-positive and may, in part, represent lysosomes.Lipid is especially abundant in the vesicles and granules of the two types of chloragogen cells. Some granules of chloragogen cells also contain ferrous and ferric iron and a substance with pseudoperoxidase activity. The cytoplasm contains protein, glycogen, and a neutral mucopolysaccharide. In addition, acid mucopolysaccharides are variably present in the cytoplasm of chloragogen cells, the only coelomocytes to contain this class of substances.