Analysis of human neutrophil granule protein composition in chronic myeloid leukaemia by immuno-electron microscopy

Summary In this study immuno-electron microscopy was used to assay, semi-quantitatively, the granule contents of elastase, lactoferrin, lysozyme and myeloperoxidase in human peripheral blood neutrophils from 13 chronic myeloid leukaemia patients in the chronic phase of the disease and from normal non-smoking donors. The fixation conditions that adequately preserved the antibody binding capacities of these antigens and reasonably preserved the ultrastructure of the neutrophils were selected by light-microscopic immunoperoxidase cytochemistry on cytospin smears. Immunogold cytochemistry on LR White resin sections localised elastase and myeloperoxidase to the primary granules, lactoferrin to the secondary granules and lysozyme to both types of granule. When applicable, peroxidase cytochemistry was combined with immunogold staining making it easier to distinguish the primary from the secondary granules. A comparison of the immunolabelling density values obtained for the leukaemic and normal states revealed no significant abnormalities in the immunoreactivity patterns for any of these neutrophil granule antigens in the leukaemic patients. All 13 patients gave normal immunostaining reactivities for these neutrophil granule proteins. Consequently the distribution patterns of these proteins, as shown in this study, cannot be used as indices in distinguishing chronic myeloid leukaemic neutrophils from normal neutrophils.     

Freeze-fracture cytochemistry: a new method combining immunocytochemistry and enzyme cytochemistry on replicas.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.     

Quantitative cytochemistry of blood neutrophils in myelodysplastic syndromes and chronic granulocytic leukaemia

Quantitative cytochemistry of components of blood neutrophil azurophilic granules (myeloperoxidase, chloroacetate esterase, beta-glucuronidase, and acid phosphatase) and specific granules (lactoferrin) has been performed by scanning and integrating microdensitometry in 13 patients with a myelodysplastic syndrome and 11 patients with chronic granulocytic leukaemia. Both patient groups showed a reduction of enzyme activity in azurophilic granules, and also of lactoferrin, consistent with abnormal development of neutrophil granules. These cytochemical changes in blood neutrophils are similar to those found in acute myeloid leukaemia, are consistent with a leukaemic maturation defect, and may be of diagnostic value.     

Pyridoxal 5'-phosphate: a possible physiological substrate for alkaline phosphatase in human neutrophils

The intracellular localization of pyridoxal phosphatase activity was demonstrated in human neutrophils by electron microscope cytochemistry. Under alkaline conditions, an enzyme active against pyridoxal phosphate was localized to a cytoplasmic granule population, the phosphasome. These granules have previously been shown by electron microscope cytochemical techniques and by subcellular fractionation to be rich in alkaline phosphatase. Under acidic conditions, a phosphatase activity against pyridoxal phosphate was localized to intracellular multilamellar bodies resembling secondary lysosomes. These were quite distinct from the primary, secondary and phosphasome granules and this unique localization corresponds to that previously demonstrated (tertiary granules) by subcellular fractionation studies of these cells. The similarity in the enzyme reaction requirements of alkaline pyridoxal phosphatase and alkaline phosphatase, and their localization to the same subcellular organelle, suggests that pyridoxal phosphate may be a physiological substrate for human neutrophil alkaline phosphatase.     

FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy.

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for high-resolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.     

Immunocytochemical demonstration of quantitative differences in the distribution of lysozyme in human airway secretory granule phenotypes

The distribution of lysozyme in the different secretory granules (SG) of human tracheal and bronchial submucosal gland serous cells was studied by light and electron microscopy, using a post-embedding immunogold technique. SG were differentiated into 5 phenotypes according to their structure and staining electron density. All the SG-phenotypes were reactive to lysozyme. In the heterogeneous SG-phenotypes, quantitative immunocytochemistry showed that the density of lysozyme labeling was significantly higher in the electron-dense central core compared to the electron-lucent peripheral rim. At the tracheal level, the density of lysozyme did not vary significantly within the different SG-phenotypes, whereas at the bronchial level, the differences were significant. Moreover, the lysozyme labeling density was much higher in the bronchial than in the tracheal SG.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

Immunocytochemical demonstration of quantitative differences in the distribution of lysozyme in human airway secretory granule phenotypes

The distribution of lysozyme in the different secretory granules (SG) of human tracheal and bronchial submucosal gland serous cells was studied by light and electron microscopy, using a post-embedding immunogold technique. SG were differentiated into 5 phenotypes according to their structure and staining electron density. All the SG-phenotypes were reactive to lysozyme. In the heterogeneous SG-phenotypes, quantitative immunocytochemistry showed that the density of lysozyme labeling was significantly higher in the electron-dense central core compared to the electron-lucent peripheral rim. At the tracheal level, the density of lysozyme did not vary significantly within the different SG-phenotypes, whereas at the bronchial level, the differences were significant. Moreover, the lysozyme labeling density was much higher in the bronchial than in the tracheal SG.     

Use of immunogold electron microscopy and monoclonal antibodies in the identification of nuclear substructures

A cytochemical technique for the ultrastructural localization of unique nuclear antigens is reported. Using a post-embedding indirect immunogold labeling procedure, nuclear antigens in electron-dense regions of the nucleus are localized with a minimum of nonspecific staining. Using this technique and indirect immunofluorescence, a panel of antinuclear monoclonal antibodies is shown to recognize preferentially cell cycle-dependent nuclear substructures. The antigenic domains recognized include specific regions in condensed chromatin, interchromatin granules, euchromatin, and chromosomes. The specificity of antigen recognition is demonstrated with qualitative and quantitative immunogold electron microscopy and immunoblot analysis. These results reveal the existence of previously undefined supramolecular organization within the nucleus and demonstrate the utility of the immunogold procedure when monoclonal antibodies are used.     

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.     

Lysozyme expression in the rat parotid gland: light and electron microscopic immunogold studies

 Lysozyme (muramidase) is capable of direct bacteriolytic action by hydrolyzing glycosidic bonds in bacterial cell walls. Although it is broadly distributed in vertebrate tissues and secretions, the cellular and subcellular localizations of the enzyme are still not well known. The present study examines the distribution of lysozyme expression in the various cell types of LR gold-embedded rat parotid gland, applying a postembedding immunogold-silver staining technique for light microscopy. Simultaneously, a postembedding immunogold method for electron microscopy was used to determine the cellular compartments engaged in the biosynthesis and exocytosis of lysozyme. Silver-amplified immunogold staining for lysozyme demonstrated identical localization in both paraffin and semithin LR-gold sections: in the supranuclear parts of acinar and intercalated duct cells. Staining intensity varied even between adjacent cells. In the electron microscope, immunogold labeling was detected over the cell compartments associated with protein synthesis and exocytosis in acinar and intercalated duct cells. Lysozyme antigenic sites were visible over endoplasmic reticulum and throughout the Golgi apparatus, being intense over the trans-Golgi network, but even stronger in the condensing vacuoles and most prominent over secretory granules in both cell types. The findings provide the first immunocytochemical evidence of the synthesis and secretion of lysozyme in parotid acinar and intercalated duct cells. Accepted: 3 December 1996     

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 study of periodic lamellar granules in human neutrophils

Granules consisting of periodically arranged membranous lamellae and amorphous electron-opaque material, i.e., periodic lamellar granules, are present in human neutrophils. To date, no extensive ultrastructural studies have been carried out on these granules because of their infrequent presence in neutrophils. The bone marrow of 18 cases of chronic myeloproliferative disorders, including one case of chronic neutrophilic leukemia in which periodic lamellar granules were frequently seen in neutrophils, was investigated by electron microscopy. Periodic lamellar granules were seen in neutrophils in 12 of the 18 cases at varying frequencies. They were preferentially seen in immature neutrophils. The transverse profiles of these granules revealed concentric complete/incomplete rings or periodic parallel straight lines, i.e., various patterns of lamellar arrangement were present. Periodic lamellar granules were positive for myeloperoxidase and lysozyme at the electron-microscopic level. These results suggest that these granules represent a primary neutrophil granule subtype. However, their functional and pathologic significance remains unknown.     

Kinetics of fusion of the cytoplasmic granules with phagocytic vacuoles in human polymorphonuclear leukocytes. Biochemical and morphological studies

This study on human neutrophils was conducted to measure the kinetics of degranulation of the different cytoplasmic granules into phagocytic vacuoles, and to relate the timing of these events to the burst of respiration that accompanies phagocytosis by these cells. Purified neutrophils were incubated with latex particles opsonized with human immunoglobulin (Ig)G, and phagocytosis was stopped at timed intervals. The cells were examined by electron microscopy to document the sequence of degranulation of the cytoplasmic granules. The azurophil granules and lyosomes were identified by histochemical staining for peroxidase and acid phosphatase, respectively. Phagocytic vacuoles were separated from cell homogenates by floatation on sucrose gradients and assayed for contained lactoferrin, myeloperoxidase, and acid hydrolases. The conclusions drawn from the biochemical and morphological studies were in agreement and indicated: particle uptake and vacuole closure can be completed within 20 s; both the specific and azurophil granules fuse with the phagocytic vacuole much earlier than is generally appreciated, with half-saturation times of 39 s (99% confidence limits, 15-72); oxygen consumption has kinetics similar to those of the fusion of these granules with the phagosome; degranulation of the acid hydrolases beta- glucuronidase, N-acetyl-beta-glucosaminidase (biochemical assays), and acid phosphatase (biochemical assay and electron microscopic cytochemistry) have kinetics of degranulation that are similar to each other but totally different from and much slower than that of myeloperoxidase with half-saturation times of between 354 and 682 s (99% confidence limits, 246-883). This suggests that the acid hydrolases are not co-located with myeloperoxidase in the azurophil granule but are contained in distinct lysosomes, or "tertiary granules".     

Freeze-fracture cytochemistry: a new fracture-labeling method for topological analysis of biomembrane molecules

Freeze-fracture cytochemistry allows visualization of cellular and molecular characteristics of biomembranes in situ. In this review, we discuss freeze-fracture cytochemistry with special reference to a new cytochemical labeling of replicas, the detergent-digestion fracture-labeling technique. In this procedure, unfixed cells are rapidly-frozen, freeze-fractured, and physically stabilized by evaporated platinum/carbon. The frozen cells are then removed from the freeze-fracture apparatus to thaw and are subsequently treated with detergents. After detergent-digestion, replicas are labeled with cytochemical markers. We demonstrate that the technique is a versatile tool for direct analysis of the macromolecular architecture of biomembranes and allows identification of particular intracellular membrane organelles. In addition, we demonstrate the application of ultrasmall gold to freeze-fracture immunocytochemistry. Freeze-fracture cytochemistry is a valuable technique for investigating topology and dynamics of membrane molecules.     

Endogenous peroxidase activity in mononuclear phagocytes

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

Localization of cellular regulatory proteins using postembedding immunogold labeling

Cyclic AMP-dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP-binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RII) of differing molecular weight, primary structure, and cyclic AMP-binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK subunits in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP-binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK subunits in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory cell function.     

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.     

Myeloperoxidase in human neutrophil host defence

Human neutrophils represent the predominant leucocyte in circulation and the first responder to infection. Concurrent with ingestion of microorganisms, neutrophils activate and assemble the NADPH oxidase at the phagosome, thereby generating superoxide anion and hydrogen peroxide. Concomitantly, granules release their contents into the phagosome, where the antimicrobial proteins and enzymes synergize with oxidants to create an environment toxic to the captured microbe. The most rapid and complete antimicrobial action by human neutrophils against many organisms relies on the combined efforts of the azurophilic granule protein myeloperoxidase and hydrogen peroxide from the NADPH oxidase to oxidize chloride, thereby generating hypochlorous acid and a host of downstream reaction products. Although individual components of the neutrophil antimicrobial response exhibit specific activities in isolation, the situation in the environment of the phagosome is far more complicated, a consequence of multiple and complex interactions among oxidants, proteins and their by‐products. In most cases, the cooperative interactions among the phagosomal contents, both from the host and the microbe, culminate in loss of viability of the ingested organism.     

Immunogold detection of co-localized neuropeptides: methodological aspects.

Whatever the protocol used, electron microscopic immunogold detection still suffers from a lack of sensitivity. In rat supraoptico-posthypophyseal neurons, unlabeled secretory granules are always detectable after electron microscopic immunocytochemistry, and their real status remains questionable. To improve the sensitivity of this approach, we assessed a protocol to visualize either one or the other of co-localized neuropeptides, i.e., vasopressin or galanin, after two successive rounds of immunogold with the same primary antibody performed on both faces of the grid. The use of different-sized gold particles enabled us to visualize the respective contribution of each face of the section to the final labeling. Our results showed a moderate but significant increase in both the proportion of labeled granules and the labeling intensity. Although limited, this improvement of immunogold detection strengthens the relevance of quantitative studies at the electron microscopic level, likely to reveal fine variations of the neuron peptidergic content. However, this enhancement depended on the peptide studied. The present data confirmed a progressive decrease of vasopressin immunoreactivity, already suggested by the single-staining procedure, all along the hypothalamo-posthypophyseal tract. In contrast, labeling intensity for galanin remained steady. Finally, our double-face labeling supported a preferential routing of galanin-containing secretory granules towards dendrites.