ACID PHOSPHATASE LOCALIZATION IN RABBIT EOSINOPHILS

Eosinophil (and heterophil) leukocytes of glycogen-induced rabbit peritoneal exudates were fixed for 1½ min in 2% glutaraldehyde and examined for acid phosphatase activity both biochemically and cytochemically. Biochemical assays showed that enzymatic activity had been inhibited by only ~10% under these conditions. The cytochemical reaction in the eosinophil was confined to the granules in which the reaction product appeared in the matrix, not in the crystalline core (or in the core region after the latter's extraction). Granules wherein the matrix was disrupted and the crystalline core degraded or extracted showed the most intense deposition of reaction product, whereas well preserved granules with morphologically intact matrix and crystals were unreactive. Yet, not all disrupted granules gave a positive reaction, indicating that disruption was a necessary but not sufficient condition for reactivity. In many eosinophil leukocytes, most if not all granules were acid phosphatase-positive, provided they had become disrupted to a certain degree. Factors possibly involved in converting the granules from an unreactive to a reactive state are discussed.     

Ultrastructure of the eosinophil granule-internum; the problem of reversed relative density

Summary An electron microscope study was made of the specific granules of eosinophil leucocytes in mouse bone marrow. A crystal lattice was apparent in the granule-internum, with a repeating pattern of about 30 Å. The presence of this lattice in stained material in which the usual densities of internum and externum were reversed is taken as supporting the hypothesis that the reversal is a negative staining effect. Very occasionally a myelin-like inclusion was also seen in eosinophil granules.     

Sulphated Glycosaminoglycans in Guinea Pig Eosinophils Studied by Means of Cationic Colloidal Gold

Using bone marrow embedded in hydrophilic resin Lowicryl K4M and cationic colloidal gold pH 1.0 labelling, we studied sites of sulphation and sulphated glycosaminoglycans ultrastructurally in various maturational stages of both eosinophil granulocytes and eosinophil granules of guinea pig. Eosinophil granules reacted positively to cationic gold, the pattern of labelling varying according to the degree of cell maturation. The formation of eosinophil granules takes place throughout the myelocyte stage. Early eosinophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late ones contain a small and less active Golgi apparatus. All the immature granules were labelled positively. However, mature granules with a central crystal bar lost their affinity towards colloidal gold. Interestingly, strong colloidal gold labelling was also observed in the trans to transmost Golgi apparatus, especially in immature eosinophil granulocytes. This indicates that sulphation of glycosaminoglycans occurs in the trans to transmost Golgi apparatus of eosinophil granulocytes. Prior absorption with poly-L-lysine prevented colloidal gold labelling of tissue sections. Methylation of sections at 37°C did not alter the gold labelling, whereas the labelling disappeared after methylation at 60°C. Prior treatment with chondroitinase ABC or heparinase I abolished the majority of colloidal gold labelling in immature eosinophil granules. Taking these results together, we conclude that immature eosinophil granules contain sulphated glycosaminoglycans including chondroitin sulphate or heparan sulphate or both.     

Localization of the guinea pig eosinophil major basic protein to the core of the granule

The localization of the guinea pig eosinophil major basic protein (MBP) within the cell was investigated by the use of immunoelectron microscopy and by isolation of the granule crystalloids. First, by immunoperoxidase electron microscopy, we found that the MBP of eosinophil granules is contained within the crystalloid core of the granule. Specific staining of cores was present when rabbit antiserum to MBP was used as the first stage antibody in a double antibody staining procedure, whereas staining was not seen when normal rabbit serum was used as the first stage antibody. Second, crystalloids were isolated from eosinophil granules by disruption in 0.1% Triton X-100 and centrifugation through a cushion of 50% sucrose. Highly purified core preparations yielded essentially a single band when analyzed by electrophoresis on polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The E1%1cm of the core protein was 26.8 +/- 1.0 (X +/- SEM); the E1%1cm for the MBP was 26.3. The core protein could not be distinguished from the MBP by radioimmunoassay (RIA) and essentially all of the protein in the core preparations could be accounted for as MBP. The results indicate that the MBP is contained in the core of the guinea pig eosinophil granule and that it is probably the only protein present in the core.     

The proform of the eosinophil major basic protein binds the cell surface through a site distinct from its C-type lectin ligand-binding region

The highly basic eosinophil major basic protein (MBP), present in the crystalloid core of eosinophil leukocyte granules, has both cytotoxic and cytostimulatory properties and is directly implicated in a number of diseases. The crystal structure of MBP resembles that of the C-type lectin (CTL) superfamily, and recent data showed that MBP binds heparan sulfate glycosaminoglycan (GAG), with the CTL ligand-binding region as the binding site. MBP is synthesized as a proform (pro-MBP) containing an acidic propiece believed to neutralize the basic MBP domain. Using flow cytometry and site-directed mutagenesis, we demonstrate here that the MBP domain of pro-MBP binds to heparan sulfate GAG on the cell surface and that this is independent of GAG covalently bound to pro-MBP. Eight basic residues located in the CTL ligand-binding region of MBP were hypothesized previously to mediate GAG binding, but we found that surface binding was not compromised by the substitution of these residues with alanine. However, the analysis of a series of mutants with surface-exposed residues substituted with alanine showed that Ser-166, Arg-168, and Arg-171 are involved in surface binding. A binding site formed by these residues is located in the MBP domain between loop 1 and beta-strand 5, outside the CTL ligand-binding region. The binding of a cell-surface heparan sulfate proteoglycan may be important in MBP action, and our findings suggest that two regions shown previously to contain the cytotoxic and cytostimulatory properties of MBP are accessible for ligand interaction in cell surface-bound MBP.     

乌龟外周血细胞的显微和超微结构

乌龟血细胞的显微和超微结构研究表明;在外周血细胞中,可分辨出红细胞、单核细胞、淋巴细胞、血栓细胞、嗜酸性粒细胞、嗜碱性粒细胞、嗜中性粒细胞等7种细胞.红细胞核圆形,胞质均匀无细胞器.单核细胞的特征是核内异染色质多聚集于周边,胞质中含有许多囊泡.淋巴细胞的核质比例大.血栓细胞以具细长的指状突起,基本无细胞器为其特征.嗜酸性粒细胞仅含一种圆形颗粒,颗粒质地均匀,电子致密,大小不等.嗜碱性粒细胞颗粒环绕在胞核周围,有三种电子密度、颗粒大小不一的类型.嗜中性粒细胞含有两种电子密度、形态不一的颗粒.       

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.     

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.     

SPECIFIC GRANULES IN ATRIAL MUSCLE CELLS

Large populations (up to 600/cell) of spherical, electron-opaque granules ~0.3 to 0.4 µ in diameter are characteristically found in muscle fibers of mammalian atria. They are absent in muscle fibers of the ventricles. The granules are concentrated in the sarcoplasmic core and occur in lesser numbers in the sarcoplasmic layers between myofibrils and under the plasma membrane. Their intimate association with a central voluminous Golgi complex and the frequent occurrence of material reminiscent of the granular content within the cisternae of the Golgi complex suggest that the latter is involved in the formation of the atrial granules. Atrial granules are larger and more numerous in smaller species (rat, mouse), and generally smaller and less numerous in larger mammals (dog, cat, human); they are absent from the atrial fibers of very young fetuses (rat) but are present in those of newborn animals. A small population of bodies containing glycogen particles and remnants of the endoplasmic reticulum and mitochondria occurs in the sarcoplasmic cores of atrial as well as ventricular muscle fibers in the rat; they contain acid phosphatase and thus appear to be residual bodies of autolytic foci. Their frequency increases with the age of the animal. Typical lipofuscin pigment granules, which are known to contain acid phosphatase and are found in the sarcoplasmic cores in old animals (cat, dog and human), are presumed to arise by progressive aggregation and fusion of small residual bodies.     

二叠纪古纺锤(Palaeo fusulina)旋壁超微构造及其分类位置

电子显微镜下研究表明 ,Palaeofusulina的旋壁由致密层及“透明层”组成 ,“透明层”的晶粒形状、大小和排列方式与旋壁四层式 (Fusulinella型 )中的透明层超微特征有较大的差异。文中应用Palaeofusulina旋壁的透明层一词 ,加以引号 ,以示两者之间的区别。晚二叠世长兴阶的Palaeofusulina属及其旋壁构造相同的若干属均可归于PalaeofusulininaeM . Maclay ,196 3亚科     

Stimulation of basophil and rat mast cell histamine release by eosinophil granule-derived cationic proteins

Major basic protein (MBP), an arginine-rich basic polypeptide that constitutes the crystalloid core of the large specific eosinophil granule, has previously been shown to stimulate noncytolytic histamine release from human basophils and rat mast cells by an IgE-independent mechanism. Two additional basic polypeptides present in eosinophil granules, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), were examined for similar activity in the present study. Acid-solubilized eosinophil granules were fractionated by chromatography on a Sephadex G-50 column. Incubation of basophil-containing human mononuclear cells with the individual column fractions demonstrated that histamine release occurred only with the fractions that contained MBP. The selectivity of the basophil response for MBP was confirmed by using equimolar concentrations of purified MBP, ECP, and EDN. In contrast, both MBP and ECP, but not EDN, stimulated histamine release from purified rat peritoneal mast cells. Reduction and alkylation of the MBP molecule diminished the response of human basophils to MBP but enhanced the potency of the molecule with rat mast cells. The distinct potency of MBP as a stimulus for histamine secretion from human basophils suggests that eosinophil release of MBP may be a specific event in the augmentation of immediate hypersensitivity reactions and other disorders characterized by eosinophilia.     

Type 4A cAMP-specific phosphodiesterase is stored in granules of human neutrophils and eosinophils

Persistent elevations of cAMP levels are generally accompanied by an inhibition of granulocyte functions. Phosphodiesterases play a critical role in regulating intracellular levels of cAMP. The expression of three isoforms of type 4 cAMP-specific phosphodiesterase (PDE4) in neutrophils suggests diversity of isoform localization and targeting in regulating cell function. The sites of cAMP regulation in granulocytes by the PDE4A isoform were investigated by immunoelectron microscopy. PDE4A was localized uniformly in all granule classes of eosinophils, but was restricted in neutrophils to a subset of myeloperoxidase (MPO)-containing granules that were round or elongated with a central crystalloid core. Granulocytes were stimulated with fMLP to investigate the sites of PDE4A targeting during cell activation. In neutrophils, fMLP induced a rapid (1 min) translocation of granules containing PDE4A to the plasmalemma, where some PDE4A and MPO were exocytosed. In these cells, PDE4A labeling within granules was focal and no longer homogeneous. While immunogold labeling of PDE4A was reduced after fMLP stimulation, staining of MPO-containing granules remained high. Extracellular release of PDE4A was also observed in eosinophils stimulated with fMLP. Morphometry revealed that Au labeling was significantly reduced within 1 min, and that there was a shift in PDE4A localization within eosinophil granules from the crystalline core to the matrix. Fluctuations of cAMP levels and ectoprotein kinase activity with PKA properties occur in blood under normal and pathological conditions. The exclusive localization of PDE4A within granules of neutrophils and eosinophils suggests that PDE4A may function to downregulate cAMP signaling at the cell membrane and/or in the extracellular space at the time of granule release.     

Tethering forces of secretory granules measured with optical tweezers

Fusion of a vesicle with its target membrane is preceded by tethering or docking. However, the physical mechanism of vesicle-tethering is unknown. To study this mechanism, we used eosinophil secretory granules, which undergo stimulated homotypic fusion events inside the cell during degranulation. Using a dual optical trap system, we observed tether formation between isolated eosinophil secretory granules. The results show that secretory granules interact stochastically with a target membrane forming physical tethers linking the vesicle and target membrane, rather than via interactions with the cytoskeleton. The necessary components are membrane-associated, and the addition of cytosolic components is not required. Tether-lifetime measurements as a function of applied mechanical force revealed at least three kinetically distinct tethered states. The tethered-state lifetimes of isolated eosinophil granules match the residence times of chromaffin granules at the plasma membrane in intact cells, suggesting that the tethering mechanisms reported here may represent the physiological mechanisms of vesicle-tethering in the cell.     

Crystal structure of the eosinophil major basic protein at 1.8 A. An atypical lectin with a paradigm shift in specificity

The eosinophil major basic protein (EMBP) is the predominant constituent of the crystalline core of the eosinophil primary granule. EMBP is directly implicated in epithelial cell damage, exfoliation, and bronchospasm in allergic diseases such as asthma. Here we report the crystal structure of EMBP at 1.8 A resolution, and show that it is similar to that of members of the C-type lectin superfamily with which it shares minimal amino acid sequence identity (approximately 15--28%). However, this protein lacks a Ca(2+)/carbohydrate-binding site. Our analysis suggests that EMBP specifically binds heparin. Based on our results, we propose a possible new function for this protein, which is likely to have implications for EMBP function.     

THE BIOSYNTHESIS, INTRACELLULAR TRANSPORT, AND PACKAGING OF MELANOCYTE-STIMULATING PEPTIDES IN THE AMPHIBIAN PARS INTERMEDIA

Experiments in which glycine-³H has been introduced into excised neurointermediate lobes of Xenopus laevis incubated in a modified Krebs-Ringer bicarbonate medium have shown that ~ 50% of the incorporated radioactivity is present in small peptides which have an electrophoretic mobility characteristic of the melanocyte-stimulating (MSH) peptides shown to be elaborated within the tissue. Based on these results and the demonstration that a discrete ~ 7 min pulse of the label can be introduced into the tissue, electron microscope radioautography has been employed to follow the subcellular events concerned with the synthesis, intracellular transport, and packaging of the labeled secretory product. Together, these studies indicate that the newly synthesized material arises in peptide form, rather than as part of a larger prohormone molecule, on the ribosomes of the rough endoplasmic reticulum within the parenchymal cells of the intermediate portion of the lobe. A proportion is then incorporated into and remains for an extended period within the intracisternal granules which are a feature of the rough endoplasmic reticulum within these cells in vitro Most (~ 60%) of the labeled secretory product, however, is transferred to the Golgi complex within 30 min and, within a further 10 min, becomes packaged into small (~ 200 mµ) electron-opaque secretory granules. It is probable that under the conditions employed these granules represent the final intracellular location of secretory product before it is released     

Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin

The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.     

Calcium ionophore A23187 calcium-dependent cytolytic degranulation in human eosinophils

The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.     

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.     

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.     

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。