Endogenous peroxidase activity in mononuclear phagocytes

The diaminobenzidine (DAB) technique has been used to visualize the subcellular localization of peroxidatic enzymes in mononuclear phagocytes. The latter cells are part of the mononuclear phagocyte system (MPS), which includes the monocytes in the bone marrow and blood, their precursors in the bone marrow, and the resident macrophages in the tissues. The DAB cytochemistry has revealed distinct subcellular distribution patterns of peroxidase in the mononuclear phagocytes. Thus the technique facilitates the identification of the various phagocyte types: Promonocytes contain peroxidase reaction in the nuclear envelope, endoplasmic reticulum, Golgi apparatus, and cytoplasmic granules. Monocytes exhibit the reaction product only in cytoplasmic granules. Most resident macrophages show the activity only in the nuclear envelope and endoplasmic reticulum. Furthermore, new phagocyte types have been detected based on the peroxidase cytochemistry. Intermediate cells between monocytes and resident macrophages contain reaction product in the nuclear envelope, endoplasmic reticulum and cytoplasmic granules. The resident macrophages can be divided into two subtypes. Most of them exhibit the pattern noted above. Some, however, are totally devoid of peroxidase reaction. Most studies on peroxidase cytochemistry of monocytes and macrophages agree that the peroxidase patterns reflect differentiation or maturation stages of one cell line. Some authors, however, still interpret the patterns as invariable characteristics of separate cell lines. As to the function of the peroxidase in phagocytes, the cytochemical findings imply that two different peroxidatic enzymes exist in the latter cells: one peroxidase is synthesized in the endoplasmic reticulum of promonocytes and transported to granules via the Golgi apparatus. The synthesis ceases when the promonocyte matures to the monocyte. Upon phagocytosis the peroxidase is discharged into the phagosomes. Biochemical and functional studies have indicated that this peroxidase (myeloperoxidase) is part of a microbicidal system operating in host defence mechanisms. The other enzyme with peroxidatic activity is confined to the nuclear envelope and endoplasmic reticulum of resident macrophages in-situ and of monocytes at early stages in culture. As suggested by the subcellular distribution, the inhibition by peroxidase blockers, and the localization during phagocytosis studies, the latter peroxidase is functionally different from the myeloperoxidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endogeneous peroxidase activity in the frog thyroid gland

Summary The fine structural localization of the endogeneous peroxidase activity in the thyroid of the young frog was studied. The reaction product for peroxidase was observed over the peripheral luminal colloid and apical region of the follicular epithelial cell. Most apical small granules and some parts of Golgi lamellae and a few Golgi vesicles were specifically stained. The cisternae of rough endoplasmic reticulum and the nuclear cisternae did not demonstrate any positive reaction for peroxidase activity with difference from that of various cells of mammalia. In this study, only mature peroxidase seems to be positive for its reaction and the enzyme in the rough endoplasmic reticulum is considered to be too immature to react for DAB method in the frog thyroid cell. The relationship between the localization of peroxidase reaction and the site of the iodination of thyroglobulin was discussed.     

Endogenous peroxidase in the lacrimal gland of the rat and its differentiation against injected catalase and horseradish-peroxidase

Summary The localization of endogenous peroxidase was studied in the glandula orbitalis (lacrimalis) externa of the rat by the method of Graham and Karnovsky (1966). Reaction product is visible in all cisternae of the rough endoplasmic reticulum including the perinuclear cisternae, in condensing vacuoles, and in all secretion granules. The Golgi cisternae seldom are peroxidase positive. Intercalated duct cells rarely contain reaction product in a few scattered cisternae of the rough endoplasmic reticulum and in secretion granules.After the injection of beef liver catalase reaction product is found in the capillary lumen. Both the injected catalase and the endogenous peroxidase are completely inhibited by 10^–2M aminotriazole, while the pseudoperoxidatic activity within the erythrocytes persists. After injection of horseradish-peroxidase reaction product is visible within the capillary lumen and also in the intercellular spaces between lacrimal gland cells. 10^–2M aminotriazole completely inhibits the endogenous peroxidase while the exogenous horseradish-peroxidase remains unaffected. The inhibitory effect of aminotriazole is not specific for catalase since lacrimal gland peroxidase is also inhibited.     

INDUCTION OF ENDOMETRIAL PEROXIDASE SYNTHESIS AND SECRETION BY ESTROGEN AND ESTROGEN ANTAGONIST

Synthesis of peroxidase was induced in the uterine epithelium of immature rats by multiple doses over a 24–96-h period of either 17 β-estradiol, the estrogen-antagonist Parke-Davis CI-628, or a combination of estradiol plus antagonist. Endogenous peroxidase activity first appeared in the cisternae of the rough endoplasmic reticulum of surface epithelial and glandular cells within 24–48 after the initial injection. Uterine peroxidase activity was also visible in the cisternae of the Golgi apparatus, in Golgi-derived secretory granules, and within the uterine and glandular lumen. Some cells of the epithelium produced little or no peroxidase, even after 96 h. Whereas the antagonist appeared to induce synthesis and secretion of peroxidase, neither the antagonist alone nor the combined treatment (estradiol plus antagonist) reproduced the estradiol-mediated growth in organ size and increased lumen diameter.     

Ultrastructural localization of peroxidase activity in normal human bone marrow cells

Summary The ultrastructural localization of peroxidase activity has been studied in the cells of normal human bone marrow using the diaminobenzidine peroxidase technique. Peroxidase activity has been localized within the primary (azurophil) granules of the neutrophilic series as well as in the cytoplasmic granules of eosinophils, basophils and monocytes. Peroxidase activity appears within the cisternal system (nuclear envelope, Golgi complex and rough endoplasmic reticulum) of these cells during the period of peroxidase-containing lysosome production. With the cessation of granulogenesis, peroxidase activity disappears from the cisternal system and does not reappear in subsequent developmental stages. In cells incubated in peroxide-free media, staining of granular components, but not of cisternae, is reduced. The inclusion of catalase in peroxide-free media eliminates all staining. This indicates that an endogenous peroxide is present within the cisternae and granules of these cell types.Supported by Grant No. AM-HE-12084-12 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson and Barbara Jordan for their technical assistance and to Dr. Arthur Sagone who performed the marrow aspirations.     

The localization of endogenous peroxidase in the lacrimal gland of the rat during postnatal development. Electron microscope cytochemical and biochemical studies

The distribution of endogenous peroxidase activity in the lacrimal gland of the rat during postnatal development was investigated by electron microscope cytochemistry Peroxidase activity is first found 6 hr after birth in only a few acinar cells At this stage, reaction product fills only localized segments of the scant rough endoplasmic reticulum and of the perinuclear cisternae. Peroxidase activity thus develops asynchronously in a given cell as well as in the secretory cell population as a whole 2 days after birth, all cisternae of the rough endoplasmic reticulum of a peroxidase-positive cell contain reaction product, but the majority of the acinar cells is still negative During the next days, the number of peroxidase-positive cells and the amount of the rough endoplasmic reticulum increase rapidly. By 15 days postparturition, all secretory cells are peroxidase-positive. Reaction product is then found in all cisternae of the rough endoplasmic reticulum including the perinuclear cisternae, in smooth surface vesicles located mainly between the rough endoplasmic reticulum and the Golgi stacks, in condensing vacuoles, and in all secretory granules The Golgi cisternae rarely contain reaction product In total homogenates and in fractions of glandular tissue of adult rats, peroxidatic and catalatic activities are demonstrable. The microsomal fractions and the postmicrosomal supernatants were used to separate peroxidase from catalase by precipitation with ammonium sulfate, and the following parameters were determined: substrate (H₂O_2-) optimum (∼ 2.0 x 10^-4 M), pH-optimum (pH 6 5), temperature-optimum (42°C), and the absorption maximum (415 nm before and 425 nm after addition of H₂O₂) The same parameters were obtained from lacrimal fluid peroxidase. Both peroxidase from lacrimal gland and that from lacrimal fluid are almost completely inhibited by 10^-3 M aminotriazole and are possibly identical enzymes. Peroxidase is secreted into lacrimal fluid, which does not contain catalase.     

Mutant vasopressin precursor in the endoplasmic reticulum of the Brattleboro rat. Ultrastructural evidence from individual “vasopressin” cells localized with the light microscope by use of a new gold/silver method for immunostain enhancement

Summary This ultrastructural study demonstrates that the vasopressin immunoreactivity found in the occasional, densely stained cells in the hypothalamus of the homozygous Brattleboro rat is localized in the rough endoplasmic reticulum. 50-m Vibratome sections were stained with anti-vasopressin serum by use of a peroxidase method with 3,3-diaminobenzidine as chromogen. The diaminobenzidine end-product has a specific capability to bind gold particles from a chloroauric acid solution and the bound gold was used to precipitate silver grains from a silver developer. The stained sections were flat embedded in resin and ultrathin sections were cut of areas containing the immuno-identified occasional cells. In these densely stained, vasopressin-immunoreactive cells of homozygous Brattleboro rats the rough endoplasmic reticulum was dilated. The lumen of the reticulum contained both end-products of diaminobenzidine and gold/silver grains, but some parts of the reticulum appeared unstained. No other cell organelles were immunostained and no secretory granules were found. In control rats, gold/silver deposits were found throughout the cytoplasm of vasopressin-immunoreactive cells. In these immunostained cells secretory granules were seen.     

Pancreatic damage induced by injecting a large dose of arginine

Male Wistar rats were injected intraperitoneally with a large dose of arginine (500 mg/100 g body weight) and were sacrificed 24, 48 and 72 h later. Pancreatic tissue was examined by electron microscopy to study the resulting process of degeneration. Degeneration started with disorganization of the rough endoplasmic reticulum into whorls with a concomitant decrease in the numbers of zymogen granules. The main changes in acinar cells after 24 h were partial distension of the endoplasmic reticulum, whorls of agranular membranes encircling zymogen granules and perinuclear vacuoles. At this time large sequestered areas in the cytoplasm contained disarranged rough endoplasmic reticulum and degraded zymogen granules. The mitochondria showed only slight changes. After 48 h, dissociation and necrosis of acinar cells were noted. Subsequently, the necrotic cells were replaced by interstitial tissue composed of leucocytes and fibroblasts. It was concluded that a large dose of arginine is toxic to the rat pancreas when injected intraperitoneally. The early morphological changes of the acinar cells may be related to metabolic alterations associated with the endoplasmic reticulum. The disorganization of the endoplasmic reticulum and the reduced number of zymogen granules may indicate disturbance of protein synthesis. The focal sequestration and degradation of the cytoplasm seemed to represent changes of the acinar cells associated with removal of damaged organelles.     

Fine structural localization of peroxidase activity in the epithelium and the gland of the rat larynx

Summary Endogenous peroxidase activity was demonstrated in ciliated cells and secretory cells of the laryngeal epithelium and gland of rats, using the diaminobenzidine method for cytochemical demonstration of peroxidase activity. The intensity of peroxidase activity was greatly varied from cell to cell, but the fine structural localization of the activity was similar in various cell types. The activity was localized in cisternae of rough-surfaced endoplasmic reticulum including nuclear envelope, some vesicles and saccules of the Golgi complex, large membrane-limited granules, multivesicular bodies and probable lysosomes. In secretory cells, the activity was also found in secretory granules.The significance of peroxidase activity is not unclear, while the activity, at least a part of it, seems to be secreted into the cavity of the larynx. The possibility that peroxidase participates bactericidal mechanism, deserves further investigation.     

An immunocytochemical study of TSH beta storage in rat thyroidectomy cells with and without D or L thyroxine treatment.

Binding sites to the beta chain of thyroid stimulating hormone (TSH) were localized in pituitaries of thyroidectomized rats. Immunocytochemical staining was observed in hypertrophied TSH cells ("thyroidectomy cells") and primarily located in dilated rough endoplasmic reticulum. Staining was also found on the few secretory granules and on some of the intracisternal granules. Some of the thyroidectomy cells stained intensely, while others exhibited very little staining. When thyroidectomized rats were treated with thyroxine 4 days before death, the TSH cells contained more secretory granules, and the intracisternal granules were larger and more numerous. L-thyroxine was 10 times as potent as D-thyroxine in promoting the build-up of granules. Both types of granules stained intensely.     

Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material

The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.     

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.     

Immunogold identification of prolactin cells of goats in anoestrus, pregnancy and milk production: ultrastructural variations.

Prolactin (PRL) cells of the goat adenohypophysis have been identified by the IgG-gold procedure with anti-sheep PRL serum. The secretion of these cells show differences in size and labelling in the three reproductive stages under study. Cells containing PRL can be grouped into low secretory activity cells (PRL-I) and high secretory activity cells (PRL-II) regarding their ultrastructure and functional significance. PRL-I were the most frequent cells in animals at the anoestrus stage, presenting numerous secretory granules and scarce development of the rough endoplasmic reticulum (RER) and Golgi complex (GC). At anoestrus and pregnancy stages there are frequent granule fusions, and the hormonal content partially disappears, perhaps by digestion. PRL-II cells were the most numerous at the lactating stage, presenting a moderate number of secretory granules and well-developed GC and RER. Some PRL-II cells of lactating animals exhibiting scarce granules and numerous exocytosis suggesting a high secretory activity. In both anoestrus and pregnancy stages most granules range in diameter from 450 to 750 nm, in contrast to the lactating stage in which most granules range in diameter from 150 to 450 nm.     

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)     

The fine structural localization of acid phosphatase in pore cells of embryonic and newly hatched Deroceras reticulatum (Pulmonata: Stylommatophora)

Summary The fine structure of the pore cells in pre- and post-hatched Deroceras reticulatum is described. The cells have been divided into three main types on morphological grounds, one type being particularly rich in glycogen. Certain pore cells contain haemocyanin granules in grooves below cytoplasmic tongues, and in characteristic double-membrane-bounded vesicles within dilated cisternae of rough endoplasmic reticulum, as well as in other identified areas. All types of pore cells show fine fibres reminiscent of collagen associated with the basal lamina and pore complexes.In addition to acid phosphatase activity in lysosomes and Golgi elements, intra- and extracisternal activity has been demonstrated in association with the rough endoplasmic reticulum. The intracisternal activity is in close proximity to the Golgi apparatus and may represent enzyme that is about to enter the GERL system. Extracisternal activity may be associated with cellular lysis and death, or may represent local areas of degradation leading to cytodifferentiation. Remnants of lysed pore cells appear to be taken up by connective tissue amoebocytes.The authors wish to acknowledge the financial support of the Agricultural Research Council (G.B.) Grant No. AG 72/21, the photographic assistance of Mr. Nigel Green, and some technical assistance from Miss Jane Morgans     

Immunohistochemical, electron microscopic and morphometric studies of estrogen-induced rat prolactinomas after bromocriptine treatment

To clarify the effects of bromocriptine on prolactinoma cells in vivo, immunohistochemical, ultrastructural and morphometrical analyses were applied to estrogen-induced rat prolactinoma cells 1 h and 6 h after injection of bromocriptine (3 mg/kg of body weight). One h after treatment, serum prolactin levels decreased markedly. Electron microscopy disclosed many secretory granules, slightly distorted rough endoplasmic reticulum, and partially dilated Golgi cisternae in the prolactinoma cells. Morphometric analysis revealed that the volume density of secretory granules increased, while the volume density of cytoplasmic microtubules decreased. These findings suggest that lowered serum prolactin levels in the early phase of bromocriptine treatment may result from an impaired secretion of prolactin due to decreasing numbers of cytoplasmic microtubules. At 6 h after injection, serum prolactin levels were still considerably lower than in controls. The prolactinoma cells at this time were well granulated, with vesiculated rough endoplasmic reticulum and markedly dilated Golgi cisternae. Electron microscopical immunohistochemistry revealed positive reaction products noted on the secretory granules, Golgi cisternae, and endoplasmic reticulum of the untreated rat prolactinoma cells. However, only secretory granules showed the positive reaction products for prolactin 6 h after bromocriptine treatment of the adenoma cells. An increase in the volume density of secretory granules and a decrease in the volume densities of rough endoplasmic reticulum and microtubules was determined by morphometric analysis, suggesting that bromocriptine inhibits protein synthesis as well as bringing about a disturbance of the prolactin secretion.     

Electron-immunocytochemical localization of calcitonin and calcitonin-gene-related peptide in human c cells of the thyroid

A preembedding immunocytochemical technique enabled us to demonstrate normal human parafollicular (C) cells at the electron-microscopic level. The normal human C cells had numerous large secretory granules with a diameter of approximately 200 nm, well-developed rough endoplasmic reticulum and Golgi complex in their cytoplasm. Calcitonin immunoreactivity and calcitonin-gene-related peptide (CGRP) immunoreactivity were present only in the C cells whose secretory granules were heavily labeled. Both calcitonin and CGRP immunoreaction deposits were seen in the cytosol but not in the cisterna of endoplasmic reticulum, Golgi apparatus or mitochondrial matrix. The two peptides produced from a single calcitonin gene were stored in the secretory granules of the C cells.     

A fine-structural analysis of mouse molar odontoblast maturation.

The first mandibular molars of the Swiss albino mice, 1 through 4 days of age, were fixed in glutaraldehyde or Karnovsky's fixative. The tissues were postfixed in OSO4, dehydrated and embedded in Epon. The prepolarizing, polarizing and secretory odontoblasts were described. The prepolarizing cells, located in the vicinity of the cervical loop, were mesenchymal-like in morphology. The cells of the polarizing stage possessed organelles indicative of protein synthesis. The nucleus was located proximally. Aperiodic fibers were evident in the wide basement membrane. The secretory odontoblasts were long, slender, polarized cells closely adjoining one another. Each odontoblast possessed six morphologically discernible regions: (1) an infranuclear region, limited in size and containing few cellular organelles; (2) a nuclear region, housing the oval nucleus and a few associated lamellae of rough endoplasmic reticulum as well as a limited number of mitochondria; (3) a supranuclear rough endoplasmic reticulum region, possessing an abundance of these organelles as well as some mitochondria and secretory vesicles; (4) a Golgi region, occupying the middle third of the cell, housing the elements of an extensive Golgi apparatus which was surrounded by peripherally located profiles of rough endoplasmic reticulum; additionally, this region contained smooth endoplasmic reticulum, mitochondria, numerous secretory granules and vesicles and occasional intracellular collagen fibers; (5) an apical rough endoplasmic reticulum region, containing a rough endoplasmic reticulum component that was less extensive than its supranuclear counterpart; in addition, this region was the one richest in mitochondria and contained a plethora of secretory vesicles and granules; (6) the odontoblastic process, a region mostly void of organelles, containing various secretory products, some of which appeared to be in the process of being released extracellularly into the surrounding dentin matrix.     

The fine structural localization of endogenous and exogenous peroxidase activity in human bone marrow mast cells under pathological conditions

We have examined the ultrastructural characteristics of peroxidase activity in human bone marrow mast cells. These studies were performed in three patients with systemic mast cell disease, and in another six patients showing bone marrow mast cell hyperplasia. Endogenous peroxidase activity was localized in the perinuclear cisternae and strands of endoplasmic reticulum, but never in the granules. We have also demonstrated the "in vivo" existence of exogenous peroxidase activity in two of the three cases of systemic mast cell disease. The peroxidase internalization involved its binding to the plasma membrane, followed by its incorporation into the cell by a general endocytic process comprising the uptake of dispersed peroxidase-positive material mainly by phagocytosis of granular structures containing peroxidase. The exogenous peroxidase appeared in non-membrane bound granules, vacuoles or aggregates, but we have never seen the enzyme linked to the mast cell granules.     

幼龄皱纹盘鲍唾液腺和消化腺的超微结构与组织化学

以透射电镜观察和组织化学方法研究了45日龄皱纹盘鲍的唾液腺和消化腺。唾液腺由粘液细胞和纤毛细胞组成,粘液细胞含发达的粗面内质网和大量的粘原颗粒,分泌中性和酸性混合粘多糖。消化腺由消化细胞和嗜碱性细胞组成,消化细胞呈现活跃的内吞和细胞内消化,并具蛋白酶和非特异性酯酶活性。嗜碱性细胞含发达的粗面内质网和大量含铁的折光小体,折光小体的电子密度较低。