THE STRUCTURE OF EOSINOPHIL LEUKOCYTE GRANULES IN RODENTS AND IN MAN

The structure of the specific granules of eosinophil leukocytes has been studied by electron microscopy in sections of tissues, buffy coats, and sediments of peritoneal washings of rats, mice, guinea pigs, and men. The core of eosinophil granules is a crystal which has a cubic lattice with a repeat of ~30 A in rodents and ~40 A in man. The chemical composition of the core is discussed in connection with recent cell fractionation studies, and the hypothesis that the core is a crystal of peroxidase is considered.     

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.     

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.     

The distribution and localisation of acid trimetaphosphatase in developing heterophils and eosinophils in the bone marrow of the fowl and the duck

Summary The distribution and localisation of acid trimetaphosphatase was investigated in developing heterophils and eosinophils from fowl and duck. In the heterophils of both species, trimetaphosphatase activity progressively increased in concentration from a thin peripheral band in the round immature primary granules to a fairly dense uniform reaction product in most of the mature specific spindle-shaped granules. Fowl and duck primary eosinophil granules had a similar distribution of reaction product as heterophils. In duck specific eosinophil granules the crystalline interna or externa, or both regions, contained strong activity whereas in the fowl, the activity of the specific granules was strongly-uniform in appearance.     

Electron cytochemistry of developing and mature eosinophils in the bone marrow of the fowl and the duck

Synopsis Enzyme cytochemical studies have been carried out on eosinophils in the fowl and the duck. Peroxidase was found in all regions of the Golgi apparatus, the rough endoplasmic reticulum and the perinuclear cisternae of the early cells. In fowl eosinophil granules irregular deposits of peroxidase and arylsulphatase final reaction product were found, but the acid phosphatase deposits were even. In the duck in contrast, peroxidase was demonstrated in the external part of the granule only. Acid phosphatase and arylsulphatase were found in both the interna and the externa of the duck eosinophil granules. An ammoniacal silver nitrate reaction for the presence of the histone arginine was also studied. Silver deposits were found occupying all regions of the granules of eosinophils from both species of bird.The presence of the hydrolytic enzymes acid phosphatase and arylsulphatase in avian eosinophil granules supports the theory that these structures are lysosomal in nature and that they correspond with mammalian eosinophils in this respect.     

Localization of eosinophil cationic protein, major basic protein, and eosinophil peroxidase in human eosinophils by immunoelectron microscopic technique

An immunoelectron microscopic technique using protein A-gold as a specific marker was used for precise intracellular localization of eosinophil granule proteins. Eosinophils from healthy individuals were isolated in metrizamide gradients. Eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were clearly located in the matrix of the large crystalloid-containing granules. In addition, ECP was probably present in the small granules of eosinophils. Major basic protein (MBP) was present in the crystalloid structure of specific granules. This method can be applied in studies of eosinophil degranulation to trace the release of biological effector molecules.     

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.     

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.     

Increased acidophilia of eosinophil granules after EDTA treatment

Summary The acidophilic reaction of eosinophil leucocyte granules from human, pig and horse blood smears was investigated by using May—Grünwald—Giemsa staining after previous treatment with EDTA and sodium citrate solutions. The same peak at 530 nm, but absorption values considerably higher than those of controls, were found in eosinophil granules after application of chelating agents, indicating that removal of metal cations could unmask basic groups in these structures.     

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.     

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.     

Granule formation in rat myeloid cells

Summary Rat bone marrow was fixed in glutaraldehyde, postfixed in osmium tetroxide, and processed for electron microscopy. The myeloid cells were arranged in order of maturation according to their successive compartments.On the basis of their differences in form, substructure, volume, and density five morphologically distinct types of developing granules are to be observed in neutrophil, two in eosinophil, and four in basophil, cells. Primordial granules appear in the interphase of the myeloblast, respectively in the early promyelocytes. The first granules in the neutrophils are pale, of homogeneous structure. These granules grow gradually denser with increasing condensation. In the myelocyte stage polymorphism is more pronounced. In the granulocytes, vacuoles and dense-cored vacuoles indicate the sites of granules. In the eosinophil line, the basophilic bodies decrease in number during differentiation. The eosinophil granules show fewer variations in the course of maturation than the neutrophils. The immature forms of the basophil granules are relatively large, pale, and of globular structure; they undergo condensation and show gradually higher density.Sites of granulogenesis in the rat are first of all the Golgi apparatus and, possibly, the cisternae in the endoplasmic reticulum. On occasion, bodies in a transitional stage between a mitochondrium and a granule can be observed, but whether they may have a bearing on the problem of granulogenesis is an open question.     

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.     

Fine structure of the bone marrow of the chicken and pigeon

The structure of bone marrow from chickens and pigeons was studied with light and electron microscopy. Erythropoiesis occurs in the lumen of the medullary sinuses. Immature erythroid cells appear to adhere to the sinus wall and may thus be prevented from entering the peripheral circulation. The wall of the medullary sinuses is formed by elongated lining cells, lacking a basement membrane, which are continous except at sites where blood cells are passing through them. When viewed with the electron microscope, developing heterophil myelocytes, which occur only in the extravascular spaces, possess two populations of granules; one type is globular in content, the other is fibrillar in content. The globular type predominates during all stages of development and appears to be the specific granule. Specific granules originate from material which is formed in the Golgi complex, pinches off, and accumulates in expanded vesicles. The origin of the material in the fibrillar granules was not determined. Like the globular granules of heterophil leucocytes, granules of eosinophil leucocytes arise from material which is formed in the Golgi complex.     

Lectins for electron microscopic distinction of eosinophils from other blood cells

Colloidal gold-labeled soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA), and Griffonia simplicifolia lectin (GS-1) were used for electron microscopic observation of blood cells. Colloidal gold-labeled SBA, HPA, and DBA showed marked deposition on eosinophil granules at all stages of maturation. Gold particles were not deposited on basophils, neutrophils, monocytes, lymphocytes, or other blood cells. Only a few colloidal gold-labeled GS-1 were deposited on eosinophil granules. Eosinophil granules are rich in N-acetyl-D-galactosamine compounds, and the colloidal gold-labeled SBA, HPA, and DBA are useful for electron microscopic detection of eosinophil granules.     

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

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

The biosynthesis of neutrophil and eosinophil granule proteins

Composition of azurophil and specific granules from human polymorphonuclear neutrophils and granules from eosinophils is presented. Biosynthesis of the granule proteins is discussed in detail with particular emphasis on neutrophil myeloperoxidase (MPO) and eosinophil cationic protein (ECP).     

Studies of the responses of basophil and eosinophil leucocytes and mast cells to the nematode Trichostrongylus colubriformis. Ultrastructural changes in basophils and eosinophils at the site of infection.

Basophil and eosinophil leucocytes infiltrate the small intestinal lamina propria of guinea-pigs infected with the nematode Trichostrongylus colubriformis. By comparing the morphology of both cell types at the site of infection with bone marrow and buffy coat cells, it was found that, after entering the lamina propria, basophils developed an electron lucent halo beneath the granule-limiting membrane while the characteristic orderly periodicity of the granules changed to a fibrillar or amorphous appearance. The granules also tended to coalesce but remained within the cell. Approximately half the eosinophils at the site of infection developed deficiencies in the amorphous outer matrix of their granules but showed no obvious change in the central electron-dense cores.     

Fluorescence of eosinophil leucocyte granules induced by 1-hydroxy-3,6,8-pyrenetrisulfonate. Visualization of differences in protein isoelectric points

After treatment of horse, rat and human blood smears with alkaline solutions of 1-hydroxy-3,6,8-pyrenetrisulfonate (HPTS), eosinophil leucocyte granules were the unique cell components which showed a bright green fluorescence. When stained with HPTS at pH 10, the whole granule of horse eosinophils showed high emission which strongly diminished after washing or staining in salt solutions or by using blocking methods for amino groups. Using HPTS at pH 12, the fluorescence reaction of house granules was specifically located in the peripheral region, appearing as fluorescent rings. These microscopic observations, which indicate differences in the isoelectric point of proteins within the eosinophil granule, were also confirmed by HPTS staining of protein blots as model substrates. Spectral analysis of HPTS at pH 10 and 12 showed practically identical absorption and emission spectra with peaks at 450 nm and 510 nm, respectively. Our results indicate that mainly ionic binding occurs between cationic proteins and HPTS in alkaline solution, and that the most cationic proteins (with isoelectric points at pH higher than 12) are located in the peripheral annular region of horse eosinophil granules.     

Eosinophil differentiating activity in sera of patients with AIDS under recombinant IL-2 substitution

An increase in circulating eosinophils was observed in patients with AIDS or ARC who were substituted for a period of 14 days with exogenous recombinant IL-2 in the context of a Phase I/II study. IL-2 exerts a broad range of biological properties and enhances the production of a variety of other cytokines, i.e., factors for haemopoietic cell growth and differentiation. After having excluded a direct effect of r IL-2 on haemopoietic precursor cells in semi-solid agar cultures, we developed a liquid culture system and studied the effect of patients' sera collected at different time intervals before, during and after r IL-2 substitution on cell differentiation of normal human bone-marrow cells in vitro. Patients' eosinophilia was preceded by a detectable activity in the sera which induced light-density, non-adherent bone-marrow cells to differentiate into the eosinophil lineage and was assessed by the presence of eosinophil primary granules or Luxol-fast blue positive granules. Thus, these in vitro data suggest the presence of circulating mediator(s) enhancing eosinophil production and differentiation in response to in vivo substitution of r IL-2.