Light and electron microscopic studies on the enzyme cytochemistry of leucocytes of eels, Anguilla species

The leucocytes of three anguillid eels were studied using enzyme cytochemistry. Leucocytes were stained for peroxidase, alkaline phosphatase, acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, lysozyme, a variety of non-specific esterases, chloroacetate esterase and two proteases. All cells were negative for aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and β-galactosidase. Very few neutrophils, thought to be mature, and all eosinophils contained peroxidase-positive granules, and some monocytes showed very weak peroxidase staining. All leucocytes lacked alkaline phosphatase, but all cells except lymphocytes and thrombocytes of A. dieffenbachii contained acid phosphatase. Neutrophil acid phosphatase released into phagosomes was associated with Escherischia coli bacteriolysis. Neutrophils also secrete lysozyme and, with monocytes, produce and secrete a variety of esterases. The possible interaction of lysozyme, acid phosphatase and esterases in bacteriolysis is discussed.     

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.     

Identification of Anguilla anguilla (L.) and Anguilla rostrata (Le Sueur) and their hybrids based on a diagnostic single nucleotide polymorphism in nuclear 18S rDNA

The variability of the nuclear 18S rDNA was examined for 10 species of freshwater eels: Anguilla anguilla, A. australis, A. dieffenbachii, A. japonica, A. marmorata, A. mossambica, A. nebulosa labiata, A. obscura, A. reinhardtii and A. rostrata. We observed that a single nucleotide polymorphism (SNP) separated A. anguilla and A. japonica from the remaining taxa. While this SNP marker may be used to identify hypothetical hybrids of A. japonica and sympatric eel species, we focused on Atlantic eels to evaluate the potential for this diagnostic chromosomal marker to discriminate between individuals of A. anguilla, A. rostrata and their hybrids.     

Effects of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity measured by use of selective chemiluminigenic substrates

Chicken heterophil polymorphonuclear leukocytes (CPMNLs) have NADPH oxidase activity, but lack myeloperoxidase (MPO). Stimulation of CPMNLs by phorbol 12-myristate 13-acetate or chicken opsonified zymosan results in luminol-dependent chemiluminescence (CL) activity, which is small relative to that of human peroxidase-positive neutrophils (HPMNLs), as well as lucigenin-dependent CL, comparable to HPMNL responses. Inhibitors were used to investigate and characterize the CL activity of CPMNLs. Inhibition constants were calculated, using Dixon inhibition analysis, or were reported as the concentration producing 50% inhibition of the magnitude of CL responses. Azide and cyanide are effective inhibitors of luminol CL in HPMNLs, although these peroxidase inhibitors do not inhibit either luminol or lucigenin CL of CPMNLs. Since these agents also inhibit eosinophil peroxidase, lack of inhibition of CPMNL CL indicates that the small percentages of peroxidase-positive eosinophils in CPMNL preparations are not responsible for the luminol CL observed. Iodoacetate and fluoride, pre-oxidase and pre-peroxidase inhibitors of glycolytic metabolism, effectively inhibit lucigenin and luminol CL activities in CPMNLs. Superoxide dismutase competitively inhibits lucigenin and luminol CL in CPMNLs, but catalase is an ineffective inhibitor. Although luminol is efficiently dioxygenated by a MPO-dependent mechanism in HPMNL, use of peroxidase-deficient CPMNLs indicates that this substrate does not exclusively measure peroxidase activity.     

Induction of myeloperoxidase and nitrotyrosine formation in a human eosinophilic leukemia cell line, EoL-1

The human eosinophilic leukemia cell line, EoL-1, differentiated with butyrate as an eosinophilic cellular model was evaluated for peroxidase-dependent tyrosine nitration. Butyrate suppressed cell growth and induced eosinophilic granules in EoL-1 cells after 9 days of culture. Peroxidase activity was detected biochemically and histochemically from 3-day cultures and it increased in a time dependent manner. This peroxidase activity was inhibited by cyanide. Nitrotyrosine formation catalysed by peroxidase using hydrogen peroxide and nitrite was detected at a high level similar to that of mature eosinophils. However, no expression of eosinophil peroxidase (EPO) was detected by RT-PCR or immunocytochemistry. In contrast, the induction of myeloperoxidase (MPO) by butyrate was clearly detected by RT-PCR, Northern blot, and immunocytochemical staining. These results suggest that butyrate induces MPO rather than EPO in EoL-1 cells and that the formation of nitrotyrosine in butyrate-induced cells is dependent on MPO.     

Digestion of the fifth component of complement by eosinophil lysosomal enzymes. Production of eosinophil specific chemotactic activity

Recently our laboratory has shown that neutrophils contain enzymatic activity within their lysosomal granules which will generate chemotactic activity for neutrophils and tumor cells from the fifth component of complement (C5). We have now expanded this initial observation and have demonstrated that eosinophils can release enzymatic activity from their lysosomal granules upon stimulation with immune complexes or opsoninized zymosan, but not with C5a or synthetic chemotactic peptides. Furthermore, the enzymatic activity released from the eosinophil lysosomal granules can cleave C5 into eosinophil-specific chemotactic activity. The generation of the eosinophil chemotactic activities from C5 is blocked by prior treatment of the eosinophil preparations with a number of protease inhibitors. The eosinophil-derived C5 cleaving activity possesses a pH optimum of 7.2, thus suggesting the enzymatic activity is a neutral protease. The demonstration that enzyme activities derived from eosinophils have the ability to generate eosinophil chemotactic factor(s) from C5 may explain why eosinophils are the predominant inflammatory cell in both nasal polyps and in the nasopharynx and bronchi of patients with allergic conditions such as hay fever and asthma.     

The identification of eosinophil colonies in soft-agar cultures by differential staining for peroxidase.

There has been a need to easily quantitate the incidence of eosinophil colonies within soft agar cultures. This has been realized by layering of the agar with benzidine dihydrochloride that permits detection of peroxidase activity in cells. Eosinophil colonies can be specifically identified by the addition to the substrate of potassium cyanide, an inhibitor of enzyme activity in neutrophils and monocytes. The enumeration of eosinophil colonies can be accomplished by scanning fresh or embedded cultures with low power magnification.     

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) I. Granulocytes

The cytochemistry and staining of granula in peripheral blood granulocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl-acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. Different granula types were shown by triazid-staining (eosinophil and neutrophil granula) and methylenblue-staining for basophil granulation. Toluidinblue-staining was used for basophil granulocytes. Lipids were shown by the Sudan-black-reaction. Four granulocyte subpopulations are described: neutrophil, heterophil, basophil and eosinophil granulocytes. Neutrophils possess all tested granula types, whereas heterophil and basophil granulocytes show only basophil granula. Neutrophils and heterophils show no activity of the tested esterases with the exception of AS-D. Only neutrophils were peroxidase-positive. Alkaline phosphatase and β-glucuronidase were not detected in granulocytes. Basophils and especially eosinophils were rarely found in peripheral blood.     

The reactivity of the circulating neutrophils in human peripheral blood]

Using cytochemical, biochemical and disc-electrophoretic methods, the degree of extracellular secretion of peroxidase-containing neutrophil granules has been investigated as an index of their functional activity when in contact with antigens of extra- and intra-circulation. It was established that in in vitro contacts of neutrophils with alive and killed microbe culture St. aur., the quantity of the granules in the cells decreased as well as the enzyme activity in them. This is partially due to extracellular secretion of the granules content confirmed by the presence of peroxidase fractions in the solution. Similar results have been obtained for circulating neutrophils and serum of patients with acute pneumonia at the height of the disease. It is hold that antigen-induced extra- and intracellular neutrophil degranulation in the peripheral human blood reflects functional activity of the neutrophils.     

嗜酸粒细胞过氧化物酶快速染色的研究

目的建立嗜酸粒细胞过氧化物酶快速染色方法,完善包括嗜酸粒细胞脱颗粒或中性粒细胞粗颗粒等各种情况下嗜酸粒细胞准确并快速计数的质量控制。方法随机选取75例血液病患者的骨髓涂片标本,要求常规瑞-姬染色骨髓分类嗜酸粒细胞≥3.5%,取材3d内。每份标本中选取两张,分别划入实验组和对照组。实验组标本进行嗜酸粒细胞过氧化物酶快速染色,对照组标本进行常规瑞-姬染色。分别计数嗜酸粒细胞百分数。结果实验组嗜酸粒细胞颗粒染成黑色,对包括中性粒细胞在内的其他细胞染色效果同瑞-姬染色,显微镜下嗜酸粒细胞显示醒目,可快速准确计数。结果与对照组比较,经t检验,P0.05,无统计学差异。结论嗜酸粒细胞过氧化物酶快速染色方法比较常规瑞-姬染色具有快速染色,对嗜酸粒细胞的显示更加醒目的优点,且对包括中性粒细胞在内的其他细胞染色兼有瑞-姬染色的效果。该方法值得推广用于快速骨髓细胞染色,且适用于包括嗜酸粒细胞形态不典型及中性粒细胞颗粒粗大等各种情况下的嗜酸细胞计数的质量控制,从而准确有效地服务于临床诊治。     

Partial characterization of the protein components of eosinophil granules isolated from guinea pig exudates

Peritoneal exudates enriched in eosinophils were induced in guinea pigs by serial intraperitoneal injections of Trichinella antigen. A method is described whereby highly purified eosinophil granules were obtained in good yield from these exudates. The granules were shown by electron microscopy to be intact and comparable to those of the mature eosinophil. The proteins of the purified granules were completely extracted with cetyltrimethylammonium bromide (CETAB) and were examined by polyacrylamide gel electrophoresis. Four major and several minor proteins, all of them basic, were resolved. One of the major proteins was identified as eosinophil peroxidase. Acid phosphatase, β-glucuronidase, and arylsulfatase activities were also present as minor components.     

Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

成年草鱼外周血细胞的超微结构

用透视电镜技术研究了成年草鱼外周血细胞的超微结构，结果表明：在外周血细胞中可区分出红细胞，淋巴细胞，单核细胞，嗜中性白细胞，嗜酸性白细胞和血检细胞；嗜碱性白细胞无论在血涂片上或超薄切片上都没有发现。电镜图像显示，在红细胞中，仅能看到少数线粒体和液泡。淋巴细胞以短的伪足，较多的游离核糖体和滑面内质网为其特征。单核细胞的特征是具有较多的伪足和胞质液泡以及偏位核。在外周血液中发现了嗜中性白细胞生长、发育、功能成熟和衰退的全过程，描述了嗜中性白细胞的幼稚型、成熟型和衰退型细胞的超微结构。在成年草鱼外周血中，还发现了一种新型的颗粒细胞，它的特殊颗粒为大的六角形或棒状致密结晶颗粒，对上述特殊颗粒细胞的属性也进行了讨论。     

Studies on the morphology of blood of the European eel (Anguilla agnuilla). III. Studies on monocytic and lymphoid cells]

Investigations concerning the monocytic and lymphocytic cell series were carried out on the peripheral blood and dabbings of the kidney, the spleen and intestines of healthy eels (anguilla anguilla), and eels taken ill with vibrio anguillarum. By means of histological, cytochemical, phaseoptical, and electronmicroscopical examinations independent development lines of the monocytes and lymphocytes were established. The place of origination for the monocytes was found to be the kidney, for the lymphocytes the spleen and the intestines additionally. The evidence of the cytochemical assay of enzymes for the differentiation of these blood cells and their cellular systems is primarily dependent on the distribution of the activity of the unspecific esterase, the naphthol-AS-D-chloroacetate-esterase, the peroxidase and the alkalic phosphatase. Macrophages were - on the basis of enzymocytochemical results - seen as derivatives of the monocytes. Plasma cells and lymphoid cells are transformation forms of the lymphocytes. Our results emphasize the significance especially of cytochemistry for the hematological diagnostics of fish diseases.     

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.     

Identification of endogenous peroxidase-containing cells as eosinophils in the gastrointestinal system

 Endogenous peroxidase (EPX) activity in certain cells in the gastrointestinal system interferes with immunohistochemical methods based on the horseradish peroxidase-catalyzed substrate deposition. We studied the distribution and characteristics of these cells. We also report an effective and antigen-preserving EPX blocking method, to make possible the evaluation of immunoperoxidase stainings in cryostat sections. The EPX-containing cells (EPX cells) are present in every part of the gastrointestinal tract, predominantly in the tunica propria. We identified them as eosinophil cells in May-Grünwald-Giemsa stained sections. The complete match was confirmed by different fluorescence techniques. Firstly, the EPX cells were labeled by a red fluorochrome-conjugated substrate of peroxidase enzymes, rhodamine-tyramide, whereas the eosinophil cells were labeled by the green fluorochrome, 1-hydroxy-3,6,8-pyrenetrisulfonic acid, which is known to label exclusively eosinophilic granules at pH 10. Secondly, all the EPX cells reacted with a monoclonal antibody against the eosinophil peroxidase enzyme. Finally, a set of commercially available leukocyte markers was used to characterize the EPX cells colabeled by fluorochrome-tyramides. Neither macrophages nor mast cells showed EPX activity. Increased numbers and altered distribution were seen in stressed rats and in ulcerated human stomach. Accepted: 16 July 1996     

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.     

Human eosinophil peroxidase: purification and characterization

Human eosinophil peroxidase (EPO) was isolated from granules from granulocytes of a patient with hypereosinophilia. The granules were extracted by means of 0.2 M NaAc, pH 4.0. The purification steps included gel filtration chromatography on Sephadex G-75 superfine and ion-exchange chromatography on CM-Sephadex G-50. The purified protein showed one band on agarose-electrophoresis, a high peroxidase activity, and a 415-nm/280 nm ratio of 1.15. After reduction, EPO showed two bands on SDS-PAGE of m.w. 52,000 and 15,000, respectively. On gel filtration, the unreduced protein had a m.w. of approximately 77,000. Amino acid analyses showed a high content of arginine and aspartic acid. Monospecific antibodies to EPO were prepared in rabbits, and a specific radioimmunoassay was developed. There was an almost linear correlation between the content of EPO measured by the radioimmunoassay and the number of eosinophils in a mixed cell extract from reference material, indicating the eosinophil origin of EPO. The content of EPO was estimated to be 15.0 micrograms/10(6) eosinophils.     

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.