The ultrastructure of guinea pig heterophil granulocytes and the heterogeneity of the granules

Summary The development of the heterophil granulocytes in the bone marrow of the guinea pig is described. During the maturation of these cells, three types of granule are formed, not only the azurophil and specific granules already described in other mammals but also a third type of granule referred to here as the nucleated granule. During the process of maturation of the cells, these three types of granule are formed successively. On this basis, two steps can be distinguished in the promyelocyte phase in which primary (nucleated and azurophil) granules are formed, i.e. an early and a late stage, nucleated granules being formed in early and azurophil granules in late promyelocytes. Secondary (specific) granules occur first in myelocytes. In mature heterophils of the guinea pig the granule population is composed of about 85% secondary granules, about 10% azurophil granules, and about 5% nucleated granules. The changes in the granule population during the maturation process were quantified. The observations and calculations point to the occurrence of three mitoses: one in the early and one in the late promyelocyte and the third in the myelocyte.     

A new type of primary granule in guinea pig heterophil granulocytes

Guinea pig heterophil granulocytes were found to have three types of granules which are formed sequentially during the development of the cells in the bone marrow and differ in shape and electron density: nucleated, azurophil and specific granules. Early promyelocytes proved to synthesize nucleated granules of medium electron density prior to the formation of azurophil granules which are highly electron dense, by late promyelocytes. Since the formation of nucleated granules and azurophil granules is restricted to promyelocytes, both can be considered to be primary granules. The moderately dense specific granules (secondary granules) appear later during granulopoiesis and are firstly present in the myelocyte.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively. The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyelocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative. Our observations highly suggest that small Golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Summary Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively.The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyolocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative.Our observations highly suggest that small golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.In honour of Prof. P. van Duijn     

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.     

Investigation of granulocytopoietic kinetics by microdensitometric evaluation of primary granule naphthol-AS-D-chloroacetate esterase activity

Using a scanning microscope photometer we determined quantitatively the enzymecytochemical reaction product for naphthol-AS-D-chloroacetate esterase in neutrophilic granulocytes and their precursors in man. Evaluation of neutrophilic cells from three healthy donors resulted in a logarithm-normal distribution. After subdivision of these cells in their morphologically defined maturational stages no statistically bimodal distribution was shown within the single cell groups. Myelocytes showed twice the amount of the polymorphonuclear neutrophil absorption values. The highest promyelocyte obsorptions were double the values of the myelocyte absorptions. The standard deviation of the absorbance obtained with promyelocytes (which encompass cells already producing granules up to cells reaching their maximal granule content) was significantly higher than the standard deviation of the myelocytes. As already known, primary granules are only synthesized at the promyelocyte stage and - according to the present knowledge - their chloracylesterase and peroxidase activities are not lost during further maturation. Consequently, our results indicate that only enzyme-rich, late promyelocytes undergo mitosis transforming into myelocytes. Correspondingly, their absorption value was halved. Since the absorbance from myelocytes to polymorphonuclears is again halved, myelocytes divide only once. Metamyelocyte absorptions in part correspond to that of myelocytes. This indicates that no distinction can be made between myelocytes with mitotic capacity and "true" if only the size and the nuclear shape are considered metamyelocytes which are not longer capable of undergoing mitosis.     

Defensin-rich granules of human neutrophils: characterization of secretory properties

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.     

Selective abnormalities of azurophil and specific granules of human neutrophilic leukocytes

Human neutrophilic granulocytes (PMN) contain two chemically distinct granule types, which appear at different stages of maturation. The azurophilic granule (or primary granule) is formed during the promyelocyte stage and is known to contain myeloperoxidase in addition to numerous lysosomal enzymes, neutral proteases, glycoaminoglycans, cationic bactericidal proteins, and lysozyme. The specific granule (or secondary granule) is formed during the myelocyte stage. It is defined by the absence of peroxidase and has been shown to contain lysozyme, lactoferrin, and B12-binding proteins. The mature PMN contains both types of granules: 33% azurophilic and 67% specific granules. There are now a few well-documented examples of pathological PMN granulations that can be classified as a selective abnormality of one granule type or the other.     

ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES : Two Types Derived from Opposite Faces of the Golgi Complex in Developing Granulocytes

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO₄. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (~800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (~500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.     

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.     

Sulfated glycosaminoglycans in guinea pig neutrophils studied by use of cationic colloidal gold.

Using a high electron resolution staining method, cationic colloidal gold (CCG, pH 1.0) staining, we studied the fine structural localization of sulfated glycosaminoglycans (GAGs) in various maturational stages of guinea pig neutrophils. Azurophil and specific granules of neutrophils reacted positively to CCG, with variety in labeling according to maturation. All immature azurophil and specific granules were labeled selectively. Mature granules lost their affinity with CCG. CCG-positive labeling was also observed in the trans to trans-most Golgi apparatus of promyelocytes and myelocytes. Prior absorption with poly-l-lysine prevented CCG labeling of tissue sections. Mild methylation of ultrathin sections at 37C did not alter CCG labeling, whereas CCG labeling disappeared after active methylation at 60C. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG labeling. These findings suggest the existence of sulfated GAGs not only in immature azurophil but also in immature specific granules of neutrophils. Sulfation of GAGs occurs in the trans- to trans-most Golgi apparatus of neutrophil granulocytes. A possible correlation between accumulation of sulfated GAGs and maturation of specific granules in neutrophils is also discussed.     

A light and electron microscopic study of myelopoietic cells in the perihepatic subcapsular region of the liver in the adult aquatic newt,Notophthalmus viridescens

Summary The liver of the newt, Notophthalmus viridescens, consists of several incompletely separated lobes of parenchymal tissue each of which is covered by a perihepatic subcapsular region (PSR) of myeloid tissue. This tissue contains neutrophils and eosinophils in various stages of differentiation. As neutrophils develop from myeloblasts to late neutrophilic myelocytes, two types of granules appear. The primary granules (type of granules formed first) are more electron dense and smaller than the secondary granules (type of granules formed later). The primary granules first appear at the stage designated early neutrophilic myelocyte, and the secondary granules appear at the stage of the maturing neutrophilic myelocyte. The eosinophils present are characterized by much larger granules than those observed in neutrophils. Cells in the PSR which superficially resemble small lymphocytes are primitive stem cells that give rise to neutrophils and eosinophils. The liver PSR is invested by a visceral peritoneum of simple squamous mesothelial cells some of which are ciliated.Supported by ACS IN-105.     

Ultrastructural cytochemistry of complex carbohydrates in developing feline neutrophils

The feline species provides animal models for at least six congenital lysosomal disorders. Since knowledge of normal feline neutrophils is a prerequisite for studies of their abnormalities, the present report describes the morphology and cytochemistry of normal feline neutrophils and compares the subcellular distribution of sulfate- and vicinal-glycol-containing complex carbohydrates to that of peroxidase and acid phosphatase. Immature feline primary granules, formed in promyelocytes, were stained for peroxidase, acid phosphatase, sulfate, and vicinal glycols. During maturation, primary granules retained strong staining for peroxidase, but staining for vicinal glycols decreased, and acid phosphatase and sulfate reactivity was lost. Secondary granules formed in myelocytes lacked peroxidase, acid phosphatase, and sulfate staining, but stained intensely for vicinal-glycol-containing complex carbohydrates. No analogues of tertiary granules previously described in rabbits and humans were demonstrated in feline neutrophils. However, a new sequential staining technique for peroxidase and vicinal glycols has suggested the formation in myelocytes and late neutrophils of a third granule type that contained peroxidase, acid phosphatase, and vicinal glycols but lacked sulfate staining. Thus, the staining characteristics of primary and secondary granules in cats closely resembled those in humans and rabbits. The third (late-forming) type of granule has not previously been described in other species.     

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.     

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.     

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.     

Subcellular distribution of glycosidases in human polymorphonuclear leucocytes.

The subcellular distribution of nine glycosidases were studied in fractions of homogenized human polymorphonuclear leucocytes (neutrophils) obtained by isopycnic centrifugation through linear sucrose density gradients. The substrates were 4-methylumbelliferyl glycosides. All nine glycosides were hydrolysed by enzymes in neutrophil cytosol fractions, and by enzymes in at least one granule population. alpha-Glucosidase activity sedimented in sucrose density gradients to a point (p = 1.180 g/ml) just above the specific granules, possibly the 'tertiary' granule population. The peak corresponding to alpha-glucosidase did not co-sediment with, but considerably overlapped, the peak corresponding to lactoferrin, a marker for specific granules (p = 1.187 g/ml). alpha-Galactosidase activity was found primarily in heavy azurophil granules (p = 1.222 g/ml). alpha-Mannosidase activity was found primarily in light azurophil granules (p = 1.206 g/ml), following the distribution of myeloperoxidase, the commonly used azurophil granule marker. beta-Glucosidase activity was concentrated in mitochondrial fractions (p = 1.160 g/ml). All other glycosidases presented complex distributions, with activities not restricted to one granule class. Granule-associated glycosidase activities were increased 2--38 times when measured in the presence of 0.05% Triton X-100, indicating latency of the enzymes within granules.     

Characterization of two azurphil granule proteases with active-site homology to neutrophil elastase

Much of the tissue damage associated with emphysema and other inflammatory diseases has been attributed to the proteolytic activity of neutrophil elastase, a major component of the azurophil granule. Recently, two additional azurophil granule proteins with NH2-terminal sequence homology to elastase were isolated (Gabay, J. E., Scott, R. W., Campanelli, D., Griffith, J., Wilde, C., Marra, M. N., Seeger, M., and Nathan, C. F. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5610-5614) and designated azurophil granule protein 7 (AGP7) and azurocidin. Azurocidin and AGP7 represent significant protein components of the azurophil granule, together comprising approximately 15% of the acid-extractable protein as judged by reverse-phase high performance liquid chromatography analysis. AGP7 migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as four distinct glycoforms of molecular mass 28-34 kDa, whereas azurocidin exhibits three predominant bands with molecular mass of 28-30 kDa. Treatment of intact azurophil granules with [3H]diisopropyl fluorophosphate resulted in labeling of elastase, cathepsin G, and AGP7, whereas azurocidin was not labeled. Tryptic mapping of 3H-labeled AGP7 allowed us to identify and sequence the active-site polypeptide that has 70% identity to elastase over 20 residues. The active site peptide of azurocidin was also identified by sequence analysis of tryptic fragments and showed 65% identity to the active site of elastase. Surprisingly, the catalytic serine of azurocidin is replaced by glycine, explaining its inability to label with [3H]diisopropyl fluorophosphate. Thus, we have identified two azurophil proteins closely related to neutrophil elastase, one of which has apparently lost its proteolytic activity due to mutation of the catalytic serine.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : I. Histochemical Staining of Bone Marrow Smears

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, β-galactosidase, β-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.     

Differentiation of eosinophilic granulocytes of carp (Cyprinus carpio L.).

Electron-microscopic studies were conducted to observe ultrastructural changes during differentiation of eosinophilic granulocytes in carp (Cyprinus carpio L.). Differentiation at the myelocyte stage was found to relate to specific granules made of dense and light fields. By maturation they assume a mosaic-like texture and in each granule of mature granulocytes, a light, central "internum" and a peripheral dense wrapper can be distinguished. The activity peroxidase and acid phosphatase is located in the internum and of peroxidase in the wrapper of the granules.