Localization of the low-Mr subunit of cytochrome b558 in human blood phagocytes by immunoelectron microscopy

Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr subunit of 75 to 90 Kd. The present study on the subcellular localization of the low Mr subunit of cytochrome b558 (p22-phox) in resting human peripheral blood phagocytes was based on immunogold labeling with monoclonal antibody (MoAb) 449, recently characterized. In post-embedding labeled neutrophils, this subunit was found mainly in the membrane of the specific granules. This conclusion was supported by a quantitative analysis of the results obtained in immunogold double-labeled sections with a polyclonal antiserum against lactoferrin (LF) as a marker for specific granules and a polyclonal antiserum against myeloperoxidase (MPO) used to identify azurophil granules. No labeling of the plasma membrane was observed, because of limited penetration of the antibody into the cryosections, preventing the detection of low antigen concentrations. Pre-embedding labeling of digitonin-permeabilized neutrophils, which has the advantage of a better penetration of the antibody into the cells, showed intense immunoreactivity on the cytoplasmic side of intact granules and low labeling on the inner surface of the plasma membrane. These complementary findings indicate that in resting neutrophils the epitope of p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the membrane of specific granules and the plasma membrane. Similar observations were made in eosinophils, where MoAb 449 reacted strongly with the cytoplasmic side of numerous small granules, and a low level of labeling was observed on the inner surface of the plasma membrane. In monocytes, MoAb 449 labeling also occurred on the inner surface of plasma membrane, of endocytotic compartments, and the outer surface of relatively small granules differing from peroxidase-containing lysosomes, as shown by immunogold double-labeling with MPO.     

Biosynthesis and sorting of myeloperoxidase in hematopoietic cells

The neutrophil granulocytes have a critical role in innate immunity through killing of phagocytized microorganisms, in which myeloperoxidase (MPO) participates. MPO is stored in cytoplasmic azurophil lysosome-like granules together with other antibiotic proteins and digestive enzymes. During passage in the secretory pathway pro-MPO is folded, subjected to oligosaccharide modification, and retrieval from constitutive secretion to become targeted to azurophil granules for final processing and storage. Propeptide-deleted MPO precursor was found not to be processed to mature MPO and not to be targeted for storage but instead degraded or secreted. This indicated that the propeptide of the MPO precursor was a prerequisite for the final processing and granule targeting of proMPO. When the MPO propeptide was expressed as a chimera with a normally secretory protein, the ER retention of the chimera was prolonged compared with that of the native protein. Thus, the propeptide of MPO precursor may also mediate the normally long ER-residence of proMPO. Both mature MPO and secreted proMPO contained complex oligosaccharide side chains indicating that proMPO and, thus, mature MPO has passed the medial Golgi stack where complex oligosaccharides are formed, and exited at TGN like other proteins targeted for azurophil granules.     

High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions

Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity.     

Serum and Plasma Myeloperoxidase,Elastase and Lactoferrin Content in Acute Myeloid Leukaemia

Myeloperoxidase (MPO) and elastase, restricted to azurophil granules of neutrophils, as well as lactoferrin, restricted to specific granules of neutrophils, were determined in plasma and serum from patients with acute myeloid leukaemia (AML). Highly sensitive radio immuno assays were developed for detection of these proteins. Serum MPO was increased in 12/35 and decreased in 2/35 patients without correlation to WBC or neutrophil counts; these levels may reflect an abnormal production by leukaemic blasts or ineffective granulopoiesis in the bone marrow. Serum elastase was increased in 6/22 patients. Serum lactoferrin was decreased in 12/25 patients without correlation to neutrophil counts probably reflecting abnormal production. Serum elastase and MPO showed a covariation in chronic myeloid leukaemia but not in AML; the latter finding may indicate that the synthesis of these two proteins is not synchronized in AML-cells. Sequential studies of patients with AML demonstrated fluctuations of serum MPO and lactoferrin during remission most likely because of chemotherapeutic pertubation. Although a limited number of patients has been studied it is suggested that serum lactoferrin may be of help for prediction of relapse in AML.     

Serum and plasma myeloperoxidase, elastase and lactoferrin content in acute myeloid leukaemia.

Myeloperoxidase (MPO) and elastase, restricted to azurophil granules of neutrophils, as well as lactoferrin, restricted to specific granules of neutrophils, were determined in plasma and serum from patients with acute myeloid leukaemia (AML). Highly sensitive radio immuno assays were developed for detection of these proteins. Serum MPO was increased in 12/35 and decreased in 2/35 patients without correlation to WBC or neutrophil counts; these levels may reflect an abnormal production by leukaemic blasts or ineffective granulopoiesis in the bone marrow. Serum elastase was increased in 6/22 patients. Serum lactoferrin was decreased in 12/25 patients without correlation to neutrophil counts probably reflecting abnormal production. Serum elastase and MPO showed a covariation in chronic myeloid leukaemia but not in AML; the latter finding may indicate that the synthesis of these two proteins is not synchronized in AML-cells. Sequential studies of patients with AML demonstrated fluctuations of serum MPO and lactoferrin during remission most likely because of chemotherapeutic pertubation. Although a limited number of patients has been studied it is suggested that serum lactoferrin may be of help for prediction of relapse in AML.     

The heterogeneity of azurophil granules in neutrophil promyelocytes: immunogold localization of myeloperoxidase, cathepsin G, elastase, proteinase 3, and bactericidal/permeability increasing protein

Azurophil granules of myeloid cells form in promyelocytes. They store cytotoxic and digestive agents which when released are involved in the defense against infection. In order to characterize the intragranular distribution of these agents, ultrastructural methods using immunogold were used on promyelocytes. Azurophil granules were divided into nucleated, large spherical (large azurophil) and small electron-dense (small azurophil) granules. Myeloperoxidase showed a peripheral distribution of large azurophils and a uniform distribution of small and nucleated azurophils, consistent with previous findings. Likewise, the major neutral proteases of azurophils, cathepsin G, granulocyte elastase, and proteinase 3, displayed a similar distribution, with a peripheral localization in large azurophils and a uniform distribution in small and nucleated azurophils, except for proteinase 3, which was associated with the crystalloid structure in nucleated azurophils. In contrast, the bactericidal/permeability increasing protein, which is bacteristatic and bactericidal for Gram-negative bacteria, was localized to the membrane area in all types of azurophil granules, consistent with a suggested association of this protein with the granule membrane. The observed differences in intragranular distribution of the proteins investigated may reflect variations in binding to matrix structures and granule membranes.     

Partial Myeloperoxidase Deficiency in a Case of Preleukaemia

The ultrastructural localization of peroxidase activity has been studied in the circulating neutrophils and in a neutrophilic series of bone marrow cells from a patient with preleukaemia. Light microscopic examination showed 36% of the polymorphonuclear leucocytes to be totally devoid of myeloperoxidase, while 50% were normally stained and 14% were slightly positive for this enzyme. Electron microscopic studies revealed considerable heterogeneity in the promyelocyte population, since the number of peroxidase-deficient azurophil granules was seen to vary from 0 to 100% in these cells. Thus, several types of promyelocytes were identified. One cell type, which resembled that seen in normal subjects, contained myeloperoxidase within its azurophil granules and also within the cisternae of the rough endoplasmic reticulum and Golgi complex. A second type of promyelocyte, which was at an early stage of development, lacked myeloperoxidase in its secretory apparatus. These cells contained two species of azurophil granules, the first of which was devoid of peroxidase activity whereas the other reacted positively. These observations suggest that the premature arrest of myeloperoxidase synthesis in the promyelocytes from a preleukaemic patient may give rise to several populations of circulating neutrophils which can exhibit varying contents of myeloperoxidase.     

Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation

Differentiation and maturation of myeloid cells is characterized by the sequential acquisition of two distinct cytoplasmic granule subsets, azurophil granules and specific granules. We recently showed the existence of a third granule subset, gelatinase granules. To investigate whether appearance of gelatinase granules marks a further step in maturation of myeloid cells beyond the appearance of specific granules, we sorted normal human bone marrow cells into one of three groups according to maturity by centrifugation on Percoll density gradients. The biosynthesis of myeloperoxidase (MPO) (an azurophil granule marker), lactoferrin and neutrophil gelatinase-associated lipocalin NGAL (specific granules markers) and gelatinase was then studied in each of these groups. We found that gelatinase was synthesized mainly in the group containing band cells and segmented cells. This contrasted with lactoferrin and NGAL, which were synthesized almost exclusively in the group containing myelocytes and metamyelocytes, and with MPO, which was mainly synthesized in the group containing myeloblasts and promyelocytes. Immunocytochemistry was in full agreement with the biosynthesis data, and showed that gelatinase appears in band cells, whereas NGAL and lactoferrin both appear in myelocytes. Thus, acquisition of gelatinase granules marks a step in neutrophil differentiation beyond the appearance of specific granules.     

Biosynthesis and processing of lactoferrin in bone marrow cells, a comparison with processing of myeloperoxidase

The processing and intracellular transport of lactoferrin of the neutrophil specific granules was investigated by biosynthetic labeling with (14C)leucine of bone marrow cells from healthy individuals and patients with chronic myeloid leukemia. Lactoferrin was precipitated with antilactoferrin serum and the immunoprecipitates were analyzed by sodium dodecyl sulfate (SDS), polyacrylamide gel electrophoresis (PAGE) followed by fluorography. In contrast to myeloperoxidase of azurophil granules, lactoferrin was not synthesized as a larger precursor, and it was not found to be phosphorylated. The transfer to granules of newly synthesized lactoferrin was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenate in a Percoll gradient. Monensin, which exchanges protons for Na+ and NH4+ cation, blocked the transfer completely, indicating a need for acidification mechanisms. Unlike myeloperoxidase, newly synthesized lactoferrin rapidly became resistant to endoglycosidase H, indicating a transport through the medial and transcisternae of the Golgi apparatus with conversion of "high mannose" to "complex" oligosaccharide side chains. Intracellular transfer of some major neutrophil azurophil and specific granule constituents is obviously regulated differently. Lactoferrin seems to be processed like proteins destined for secretion, while myeloperoxidase is processed more or less like lysosomal enzymes.     

Azurophil and specific granules of blood neutrophils in chronic myelogenous leukemia: an ultrastructural and cytochemical analysis

That most patients with chronic myelogenous leukemia (CML) have either very low levels or no leukocyte alkaline phosphatase activity (LAP) is an established fact. In view of our new findings7 that normal mature human polymorphonuclear leukocytes (PMN) contain two types of granules, azurophils (1/3) and specifics (2/3), and that alkaline phosphatase is present only in specific granules, we undertook the present studies to determine whether these neoplastic PMN lack a specific granule population or simply lack the enzyme. The cellular buffy coats of five patients with CML (Ph1 plus, LAP minus) were fixed in glutaraldehyde, incubated for peroxidase to identify the azurophil population, and examined by electron microscopy. It was found that the specific granule population was present in all mature PMN. Counts of both azurophil and specific granules per cell were slightly lower than normal but were within an 80%-90% overlap of the normal range. We therefore conclude that the low level of LAP in patients with CML reflects a deficiency of the enzyme rather than a missing granule population. Although the mature PMN appeared relatively normal (with few exceptions), circulating myeloblasts and promyelocytes revealed several abnormalities, the most notable being the presence of large bundles of cytoplasmic microfilaments. The blood of two patients in the terminal phase of disease was reexamined. Most of their cells were immature, with aberrations similar to those in myeloblasts and promyelocytes in the chronic phase of the disorder. In addition, however, we discovered three adnormal populations of mature PMN: (1) PMN containing both populations of granules but lacking peroxidase, (2) PMN lacking specific granules, and (3) PMN lacking azurophil granules. Our findings emphasize the value of electron microscopy and cytochemistry in detecting abnormalities of maturation in the cytoplasm of leukemic PMN.     

In situ localization by double-labeling immunoelectron microscopy of anti-neutrophil cytoplasmic autoantibodies in neutrophils and monocytes

Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA-antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA-positive patients and a mouse monoclonal antibody against the ANCA-antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti-myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B-treated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocytes.     

Immunoferritin Localization of Intracellular Antigens: The Use of Ultracryotomy to Obtain Ultrathin Sections Suitable for Direct Immunoferritin Staining

A general method for the ultrastructural localization of intracellular proteins and antigens by immunoferritin techniques has been developed. The method involves direct staining of ultrathin sections of mildly glutaraldehyde-fixed and frozen tissues cut by means of a cryo-ultramicrotome. Bovine pancreatic sections were cut, mounted on grids, and stained with ferritin-rabbit antibovine RNase conjugates. After negative staining with 0.2% phosphotungstic acid, electron micrographs revealed specific labeling of all of the zymogen granules and the cisternae of the rough endoplasmic reticulum. No significant labeling was seen in the nucleus, mitochondria, or cell sap regions. The observation that no significant labeling was found in any region of rat pancreatic sections was consistent with the fact that rat RNase is immunologically non-crossreactive with bovine RNase. In addition, the labeling seen in bovine pancreas was completely absent if the sections were first incubated with free antibody. The method used here avoids prolonged fixation, dehydration, and other harsh chemical or physical treatments, and should extend the usefulness of immunoferritin techniques to the intracellular localization of many protein antigens beyond previously available methods.     

Targeting of proteins to granule subsets is determined by timing and not by sorting: The specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells.

The mechanism of protein targeting to individual granules in cells that contain different subsets of storage granules is poorly understood. The neutrophil contains two highly distinct major types of granules, the peroxidase positive (azurophil) granules and the peroxidase negative (specific and gelatinase) granules. We hypothesized that targeting of proteins to individual granule subsets may be determined by the stage of maturation of the cell, at which the granule proteins are synthesized, rather than by individual sorting information present in the proteins. This was tested by transfecting the cDNA of the specific granule protein, NGAL, which is normally synthesized in metamyelocytes, into the promyelocytic cell line HL-60, which is developmentally arrested at the stage of formation of azurophil granules, and thus does not contain specific and gelatinase granules. Controlled by a cytomegalovirus promoter, NGAL was constitutively expressed in transfected HL-60 cells. This resulted in the targeting of NGAL to azurophil granules as demonstrated by colocalization of NGAL with myeloperoxidase, visualized by immunoelectron microscopy. This shows that targeting of proteins into distinct granule subsets may be determined solely by the time of their biosynthesis and does not depend on individual sorting information present in the proteins.     

The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.     

Defensin-rich dense granules of human neutrophils

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.     

Effects of serum and cations on the selective release of granular proteins from human netrophils during phagocytosis.

The extracellular release from human neutrophils of the primary (azurophil) granule constituents, myeloperoxidase (MPO), chymotrypsin-like cationic protein (CCP), collagenase and lysozyme, and the secondary (specific) granule constituents, lactoferrin and lysozyme, was measured during ingestion of staphylococcus protein-A-IgG complexes. In buffer, lactoferrin release was consistently higher than that of the other protein. In serum, lactoferrin release increased concomitantly with ingestion, whereas the rate of lysozyme and especially of MPO release were stimulated to a higher degree than ingestion. Magnesium (0.5--2 mM) was more potent than calcium (0.5--2 mM) in promoting release but these cations worked synergistically. Zinc (0.5--4 mM) was found to be a potent and selective inhibitor of collagenase release. Manganese (0.25--4 mM), which inhibited the ingestion of SpA-IgG complexes, also inhibited release of CCP, collagenase, lysozyme and MPO, but actually stimulated lactoferrin release. The data suggests that lactoferrin and lysozyme may be confined to distinct granule populations or else released in a different fashion from the granules. When the effects on release of primary granule proteins are concerned it is suggested that the dissociation of binding of various agents to an anionic granule matrix may be affected differently by various cations.     

Phagocytosis of brushite crystals by pig neutrophils.

Neutrophils from pig blood were disrupted by homogenisation or sonication and placed on an analytical sucrose gradient. Pig neutrophil azurophil granules were less dense than the specific granules, unlike those from neutrophils of most other mammalian species. Brushite crystals, which stimulate the respiratory burst in pig neutrophils, were found by electron microscopy to be phagocytosed. Membrane-limited vesicles containing crystals were obtained from a dense region of the sucrose gradient loaded with a homogenate of crystal treated cells. Intake of crystals involved preferential mobilisation of cytochrome b from the plasma membrane and the fusion of both specific and azurophil granules with the primary phagosome. Plasma membrane and granule markers appear in a crystal-containing region of the sucrose gradient when cells are treated with crystals. They are present in much lower concentration at this location in gradients from cells untreated with a crystal stimulus.     

Effects of Serum and Cations on the Selective Release of Granular Proteins from Human Neutrophils during Phagocytosis

The extracellular release from human neutrophils of the primary (azurophil) granule constituents, myeloperoxidase (MPO), chymotrypsin-like cationic protein (CCP), col-lagenase and lysozyme, and the secondary (specific) granule constituents, lactoferrin and lysozyme, was measured during ingestion of staphylococcus protein-A-IgG complexes. In buffer, lactoferrin release was consistently higher than that of the other protein. In serum, lactoferrin release increased concomitantly with ingestion, whereas the rate of lysozyme and especially of MPO release were stimulated to a higher degree than ingestion. Magnesium (0.5-2 mM) was more potent than calcium (0.5-2 mM) in promoting release but these cations worked synergistically. Zinc (0.5-4 mM) was found to be a potent and selective inhibitor of collagenase release. Manganese (0.25-4 mM), which inhibited the ingestion of SpA-IgG complexes, also inhibited release of CCP, collagenase, lysozyme and MPO, but actually stimulated lactoferrin release. The data suggests that lactoferrin and lysozyme may be confined to distinct granule populations or else released in a different fashion from the granules. When the effects on release of primary granule proteins are concerned it is suggested that the dissociation of binding of various agents to an anionic granule matrix may be affected differently by various cations.     

Glycosaminoglycans in human neutrophils and leukemic myeloblasts: ultrastructural, cytochemical, immunologic, and biochemical characterization

Chondroitin sulfate is known to be present in normal and leukemic myeloid cells; however, its definitive subcellular location and association with other glycosaminoglycans (GAGs) has not been demonstrated. We have studied the type and distribution of GAGs in neutrophil granule subpopulations of normal and leukemic myeloid cells using ultrastructural, cytochemical, immunologic, and biochemical methods. At the ultrastructural level, high-iron diamine- thiocarbohydrazide-silver proteinate (HID-TCH-SP) stained sulfated glycoconjugates selectively in immature primary granules of normal promyelocytes and Auer rods and immature granules of leukemic myeloblasts. Staining was weak or absent in mature primary granules, whereas tertiary granules stained moderately. Primary granule staining with HID-TCH-SP was greatly diminished by prior treatment of the specimens with chondroitinase ABC and/or nitrous acid, indicating the presence of chondroitin sulfate and N-sulfated glycosaminoglycan. Immunostaining of myeloid cells with a rabbit antichondroitin 4-sulfate and ferritin-conjugated goat anti-rabbit IgG sequence resulted in staining of most primary granules. Biochemical analysis of GAGs from leukemic myeloblasts containing primary granules and Auer rods, but lacking secondary and tertiary granules, revealed 8 x 10(-17) mole of uronic acid/cell and electrophoretic and sulfaminohexose analysis showed 60%-70% chondroitin sulfate AC of heterogeneous molecular weight, 20%-30% of a GAG that most closely resembled heparan sulfate, and 10% dermatan sulfate. The lack of significant HID-TCH-SP staining of sulfate iin sites other than Auer rods and primary granules in leukemic myeloblasts indicates that these granules contain the chondroitin, dermatan, and heparan sulfate isolated from the same specimen. Similar GAGs are present in primary granules of normal cells as evidenced by their cytochemical and immunostaining properties. Thus, these studies demonstrate a heterogeneous population of GAGs not previously identified and localize these substances to the primary granule of leukemic and normal cells.     

Azurophil and Specific Granules of Blood Neutrophils in Chronic Myelogenous Leukemia: An Ultrastructural and Cytochemical Analysis

That most patients with chronic myelogenous leukemia (CML) have either verylow levels or no leukocyte alkaline phosphatase activity (LAP) is an establishedfact. In view of our new findings⁷ thatnormal mature human polymorphonuclearleukocytes (PMN) contain two types ofgranules, azurophils (1/3) and specifics(2/3), and that alkaline phosphatase ispresent only in specific granules, weundertook the present studies to determinewhether these neoplastic PMN lack a specific granule population or simply lack theenzyme. The cellular buffy coats of five patients with CML (Ph¹+, LAP-) were fixedin glutaraldehyde, incubated for peroxidase to identify the azurophil population,and examined by electron microscopy. Itwas found that the specific granule population was present in all mature PMN.Counts of both azurophil and specificgranules per cell were slightly lower thannormal but were within an 80%-90%overlap of the normal range. We thereforeconclude that the low level of LAP in patients with CML reflects a deficiency of theenzyme rather than a missing granulepopulation. Although the mature PMNappeared relatively normal (with few exceptions), circulating myeloblasts and promyelocytes revealed several abnormalities, the most notable being the presenceof large bundles of cytoplasmic microfilaments. The blood of two patients in theterminal phase of disease was reexamined. Most of their cells were immature,with aberrations similar to those in myeloblasts and promyelocytes in the chronicphase of the disorder. In addition, however, we discovered three adnormal populations of mature PMN: (1) PMN containing both populations of granules butlacking peroxidase, (2) PMN lacking specific granules, and (3) PMN lacking azurophil granules. Our findings emphasizethe value of electron microscopy and cytochemistry in detecting abnormalities ofmaturation in the cytoplasm of leukemicPMN.

Submitted on November 19, 1973 Accepted on March 30, 1974