Compound exocytosis of granules in human neutrophils

Human neutrophils are of prime importance for the immune defense. Recent data from eosinophils and pancreatic beta cells have indicated that granules, upon exocytosis, occasionally fuse with each other in the cytosol prior to their subsequent fusion with the plasma membrane. This is termed compound exocytosis. We therefore studied exocytosis of single granules from human neutrophils by the high-resolution cell-attached patch-clamp capacitance technique. We found that 1.5% of the capacitance steps was greater than 5 fF, i.e., significantly larger than steps expected for exocytosis of single granules. The mean step size of these events was 20.5 fF, corresponding to compounds formed by at least five granules. The capacitance input from compound steps contributed more than 20% of the total capacitance increase. Electron microscopy captured morphological manifestations of transient exocytic events, confirming the functional results obtained by capacitance measurements. Compound exocytosis may be a mechanism for efficient targeting of release during exocytosis.     

Fc-mediated endocytosis by human neutrophils. Ultrastructural studies

Fc Receptors (FcR) mediate the binding and uptake by polymorphonuclear leukocytes (PMN) of antibody-coated particles and soluble immune complexes. We have studied Fc-mediated endocytosis by PMN ultrastructurally using a gold-conjugated monoclonal antibody (3G8) to block or to mark the location of FcR. Phagocytosis of antibody-coated erythrocytes (EIgG) was initiated rapidly after binding to discrete foci on the PMN plasma membrane. After the phagocytosis of EIgG, we examined the distribution of FcR remaining on the PMN plasma membrane. 3G8-Colloidal gold continued to bind to PMN after ingestion of up to three EIgG, demonstrating that all PMN FcR are not utilized during a brief phagocytic event. The endocytosis of soluble immune complexes was examined by labeling plasma membrane-bound rabbit immune complexes with goat anti-rabbit IgG conjugated to colloidal gold. Gold was found in clusters randomly distributed over the plasma membrane at 4 degrees C. When cells were warmed to 37 degrees C, numerous endocytic vesicles were observed as early as 2.5 minutes after warming. After 30 minutes at 37 degrees C, large vesicles, 1 micron in diameter, were found to contain 20 to 30 gold particles. The endocytosis of 3G8 was also examined using colloidal gold. After binding of 3G8-gold at 4 degrees C, clusters of large vesicles, up to 2 micron in diameter, were rapidly formed at 37 degrees C.     

Isolation and partial characterization of human eosinophil granules. Comparison to neutrophils.

Human blood eosinophils obtained from untreated patients with large numbers of circulating eosinophils were purified and lysed. An eosinophil contains 2.65 times as much peroxidase, 2.44 times as much beta-glucuronidase, approximately two times as much acid beta-glycerophosphatase, and 1.2 times as much protein as a neutrophil. Lysate filtration allowed isolation of eosinophil granules by isopycnic ultracentrifugation in sucrose. The granules had a mean density of rho 1.24 g/ml, and contained peroxidase, beta-glucuronidase, and acid beta-glycerophosphatase. They totally lacked muramidase and alkaline phosphatase. Electron micrography confirmed the isolation.     

Degranulation of primary and secondary granules in adherent human neutrophils

The aim of this study was to investigate whether neutrophil adhesion to extracellular matrix proteins like fibronectin, fibrinogen, and albumin influence the release proteins from primary and secondary granules of neutrophils stimulated by phorbol-myristate-acetate (PMA) and formyl-methionyl-leucyl-phenylalanine (f-MLP). Isolated granulocytes plated on wells coated with fibronectin, fibrinogen, and albumin were stimulated with f-MLP (10-7 mol/l), PMA (10-9 mol/l), Mn2+ (5 mmol/l), or combinations of these stimuli, and the degree of adhesion to protein-coated surfaces and the amount of granule proteins released was quantified during 90 min of incubation. PMA, in combination with Mn2+, induced a maximum release of approximately 80% of the intracellular content of lactoferrin and human neutrophil lipocalin (HNL) and 15-20% of the myeloperoxidase (MPO) content regardless of the protein used. PMA or f-MLP alone induced 30-40% release of lactoferrin and HNL depending on the protein that the cells were plated on. Adhesion and release of lactoferrin and HNL were quantitatively related when induced by PMA and PMA plus Mn2+, but not by f-MLP. The mean release of lactoferrin and HNL showed a significant negative relationship to the viability of the cells. In conclusion, adhesion modulates neutrophil degranulation, but it is not always quantitatively related or related in time.     

Annexin I-induced aggregation of gelatinase granules in human neutrophils

Ability of neutrophil cytosol to induce aggregation of gelatinase granules from human neutrophils is studied. It is found that cytosol induces Ca²⁺-dependent aggregation of granules. The aggregation-inducing capacity of cytosol considerably decreases in the presence of monoclonal antibodies to annexin I, a transmitter of Ca²⁺-dependent aggregation of gelatinase granules probably involved in their secretion. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 403–405, April, 1999     

Ultrastructural localization of lysozyme in human neutrophils by immunogold

The subcellular localization of lysozyme (LZ) has been investigated by immunogold electron microscopic cytochemistry in human neutrophils from bone marrow and blood. Intact cells or subcellular granule fractions were fixed in glutaraldehyde and embedded in glycol methacrylate. Thin sections were incubated with monospecific antibodies followed by antiglobulins coupled to colloidal gold. LZ was detected within both elliptical and spherical primary granules in bone marrow neutrophil promyelocytes. In myelocytes and more mature neutrophils immunolabeling for LZ was observed within the primary granules, although fainter than in promyelocytes. However secondary granules from bone marrow and blood neutrophils were not consistently labeled by gold particles. Immunogold staining was then performed on sections of subcellular fractions of secondary granules: immunogold staining of lactoferrin demonstrated 95% of secondary granules in this fraction. Labeling for LZ of this granule fraction was intense, and except that the few primary granules were also labeled, looked similar to that of lactoferrin. In conclusion, this study is the first to utilize electron microscopic cytochemistry to show that LZ is present in both the primary and secondary granules of blood and bone marrow neutrophils. This technique has the advantage of allowing LZ distribution to be studied within a single organelle and/or in relation to the rest of the cell structure.     

Ultrastructural localization of lactoferrin and iron-binding protein in human neutrophils and rabbit heterophils.

Lactoferrin in marrow and blood granulocytes from rabbits and humans was stained with an immunoferritin method. Iron-binding protein(s) was localized by the staining of granulocytes with acid ferrocyanide after saturation of the iron-binding protein with iron. The latter was most readily accomplished by treatment of the glutaraldehyde-fixed cell suspension with 1% saponin, followed by treatment with an iron-nitrilotriacetate (Fe-NTA 3mM:4mM) solution, adjusted to pH 7.0 with NaHCO3. The affinity of purified lactoferrin and transferrin for radioiron after such treatment was minimally diminished. Both immunoferritin and iron-binding methods heavily stained osmiophiliuc (phospholipid-containing) mature primary granules in late promyelocytes, myelocytes, and polymorphonuclear cells. Early promyelocytes containing abundant immature primary granules lacked immunoferritin or iron staining. Trypsin digestion of rabbit marrow cells considerably diminished the cytochemically demonstrable iron-binding capability of the mature primary granules. Specimens sequentially stained for peroxidase and immunostained for lactoferrin or cytochemically stained for iron-binding protein confirmed that lactoferrin and iron-binding protein were in peroxidase-positive primary granules. Some peroxidase positive granules appeared to lack staining for lactoferrin and iron-binding proteining protein, and all secondary granules uniformly lacked staining. Treatment of human neutrophils with phorbol myristate acetate demonstrated early release of granules containing iron-binding protein with subsequent agglutination of neutrophils and attachment of iron-binding protein to the cell surface. In summary, this study distinguishes at least two subpopulations of primary granules and identifies lactoferrin and an iron-binding protein(s) in a subpopulation of peroxidase-positive primary granules in rabbit heterophils and human neutrophils.     

Prodefensins are matrix proteins of specific granules in human neutrophils

Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptide (HNP)-1-3 are synthesized as 94 amino acide (aa) preproHNPs, which are converted to 75 aa proHNPs by cotranslational removal of a 19 aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29-30 aa HNPs. In contrast, proHNPs produced by more mature myeloid cells are not subjected to proteolytic cleavage and undergo a high degree of constitutive exocytosis. The proHNPs are devoid of antimicrobial potential, and the significance of their secretion is unknown. To investigate whether mature neutrophils contain proHNPs, we developed antibodies against proHNP-1 by DNA immunization of rabbits. In addition, antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. These antibodies allowed detection of proHNPs in homogenates of peripheral blood neutrophils. The proHNPs were isolated by affinity chromatography, and their identity was confirmed by mass spectrometry and N-terminal aa sequence analysis. Finally, the neutrophil proHNPs were identified as novel matrix proteins of specific granules by subcellular fractionation experiments, release studies, and immunoelectron microscopy. Thus, human neutrophils not only store large amounts of mature defensins in azurophil granules but also contain a more easily mobilized reservoir of unprocessed prodefensins in specific granules.     

Ultrastructural diversity in secretory granules of human major salivary glands

Acinar cells of human major salivary glands, fixed and processed for electron microscopy under identical conditions, exhibit secretory granules endowed with distinctive ultrastructural features.     

NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils.

In addition to the extracellular production of O2- by NADPH oxidase in neutrophils stimulated by soluble stimuli, the intracellular formation of oxygen reactive species has been described. Cytochrome b559, the redox component of the NADPH oxidase complex, is mainly associated with specific granule membrane in resting neutrophils. We examined whether these granules could be a site for intracellular production of O2-. Phorbol myristate acetate (PMA)-stimulated neutrophils were fractionated by differential centrifugation, and generation of O2- was detected in both the granule and the plasma membrane-enriched fractions, but more in the granules. Translocation of p47phox and p67phox, two cytosolic components of the NADPH oxidase, was also quantitatively more important in the granules than in the plasma membrane fraction. After separation of the specific from the azurophil granules, p47phox and p67phox were found to be present only in the specific granules of PMA-activated cells. As a control, the production of O2- was studied in retinoic acid-differentiated NB4 cells that lack specific granules. During stimulation of NB4 cells with PMA, only the plasma membrane-enriched fraction was the site of O2- production. Together, these results indicate that NADPH oxidase can be functionally assembled in specific granules.     

Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes.

The ultrastructural localization of the CD68 antigen, a 110-kd intracellular glycoprotein associated with myeloid cells and with monocytes/macrophages, was investigated in human neutrophil granulocytes by postembedding immunogold staining, using monoclonal antibody KP1. The antigen was found in the primary granules of neutrophils, although not all primary granules were labeled. It was absent from the plasma membrane. In monocytes, it was also detected within cytoplasmic granules, colocalized with lysozyme and myeloperoxidase. This observation confirms and completes results obtained by immunofluorescence and other light-microscopic methods. Moreover this study shows that the CD68 epitope recognized by antibody KP1 is able to resist fixation and embedment and therefore emphasizes the value of using KP1 as a marker for this macrophage-associated molecule.     

Vicinal glycol-staining identifies secondary granules in human normal and chédiak-higashi neutrophils

Neutrophil secondary granules contain large amounts of glycoprotein. We evaluated periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of these granules after α-amylase digestion to assess their content of vicinal glycol-containing glycoconjugates and the usefulness of this stain as a positive stain for secondary granules. Using this method, early stages of secondary granule genesis were observed prior to completion of primary granule genesis in myelocytes. Immature secondary granules appeared round to ovoid, but irregular in outline and demonstrated strong staining of the limiting membrane and matrix material. In mature granules, matrix staining was unaltered; however, membrane staining was decreased. Some immature primary granules in promyelocytes demonstrated strong PA-TCH-SP reactivity which was masked in mature primary granules of band and segmented neutrophils. The Golgi apparatus showed progressively increasing PA-TCH-SP reactivity toward its mature surface which was often convex in promyelocytes and myelocytes and concave in segmented neutrophils. The Chédiak-Higashi secondary granules were cytochemically and morphologically similar to those of normal individuals and were not statistically decreased in number when compared to controls. They were only rarely observed contacting or fusing with giant granules which had consumed all primary granules leaving an easily detected population of secondary granules. Thus the α-amylase-PA-TCH-SP method demonstrates a large amount of unmasked vicinal glycol-containing glycoconjugates in neutrophil secondary granules, which allows their differentiation from primary granules.     

High-resolution localization of lactoferrin in human neutrophils: labeling of secondary granules and cell heterogeneity

We studied by electron microscopy the topography of the iron-binding protein lactoferrin in human peripheral blood neutrophils by immunogold labeling of thin-sectioned cells. We show that fixation in 0.3% glutaraldehyde, supplemented by 2% formaldehyde, and combined with embedding in the hydrophilic resin LR White results in excellent preservation of the lactoferrin antigenicity and of the neutrophil microanatomy. Most of the immunogold marking was seen on the specific (secondary) granules of polymorphonuclear leukocytes, thus strengthening prevailing views on the subcellular distribution of lactoferrin in neutrophils. We document that a minority (8-10%) of neutrophils showed significant amounts of lactoferrin associated with a flocculent, protein-like material seen inside large cytoplasmic vacuoles. We show that circulating neutrophils comprise 6-10% of cells that are virtually devoid of lactoferrin labeling. This finding provides specific cytochemical support for the concept that human polymorphonuclear leukocytes are made up of a heterogeneous population of cells.     

Regulation of superoxide responses of human neutrophils by adenine compounds. Independence of requirement for cytoplasmic granules

Recent evidence suggests that the adenine compounds ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), and adenosine have important regulatory effects on O2- responses of human neutrophils stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP). Because of evidence that receptors on neutrophils may be modified by granule fusion events, we assessed the extent to which these adenine compounds affect fMLP and CR3 (C3bi) receptors on neutrophils and whether cytoplasmic granules are required for the ability of the adenine compounds to modify O2- responses in neutrophils stimulated with fMLP. Incubation of neutrophils with ATP gamma S or adenosine led to a decrease in numbers of fMLP receptors (17 and 9.2%, respectively) but no change in receptor affinity (Kd). Paradoxically, ATP gamma S caused an increase in CR3 receptors (Mo1, CD-11b antigen), suggesting that fMLP and CR3 receptors may be under separate control. The ability of the adenine compounds to modify O2- responses in fMLP-stimulated cells was equivalent in both neutrophils and cytoplasts, suggesting that the regulatory effect of ATP, ATP gamma S, and adenosine do not require the presence of cytoplasmic granules. ATP gamma S caused enhancement of O2- responses of neutrophils to phorbol 12-myristate 13-acetate, raising the possibility that ATP gamma S may be affecting late events in the signal transduction pathway.     

Ultrastructural evidence that the granules of human natural killer cell clones store membrane in a nonbilayer phase.

Electron-microscopic examination of five LGL clones, JT3, JTB18, CNK6, CNK7, and CNK10, expressing natural killer activity and T11 and NKH1 phenotype, showed that three of the clones, JT3, CNK6, and CNK7, had crystalline structures in their densest granules. These structures generally consisted of hexagonally packed lattices with a 6.9-nm point-to-point spacing. JTB18 and CNK10 had no structures in their granules. The attack of one clone, JT3, on two resistant target tumor cell lines, KG1 and Laz509, was also examined under three conditions. First, JT3 cells and targets were incubated together. There was little adherence, degranulation, or killing. Second, cells were incubated with anti-T11(2) and T11(3), antibodies against the E-rosette receptor/antigen complex, which activate resting T cells and enhance cytolytic activity of NK clones and CTL. JT3 cells adhered to the targets, formed zones of apposition between NK and target cell membranes, polarized, and degranulated into the space between the two cells, killing the targets. Third, cells were incubated with both anti-T11(2/3) and anti-LFA-1, an antibody that inhibits adherence. The JT3 cells did not form zones of apposition with the targets, but degranulated in discrete areas on their own surface. In all cases, discharged crystalline granules transformed to sheets of membrane and vesicles. These studies suggest that phospholipids are packed in hexagonal lattices in the granules of the resting cells and transform to bilayer structures during exocytosis. The crystalline nature of the granule may immobilize lytic molecules and protect the resting cell from lysis. Further, the vesicles may serve to transport the lytic molecules from the effector to the target cell. Anti-LFA-1 does not inhibit target recognition or exocytosis, but instead blocks membrane interactions of the effector cell with its target.     

Granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation.

Granulophysin, a protein described in platelet dense granule membranes, has been shown to be similar or identical to CD63, a lysosomal membrane protein. We have previously shown granulophysin to be present in neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochemistry. Granulophysin expression on the surface membrane of the neutrophil is increased following stimulation of the cells, demonstrated by flow cytometry and fine structural immunocytochemistry. A similar pattern is shown for an anti-CD63 antibody. Incubation of activated neutrophils with D545, a monoclonal antibody to granulophysin, blocks subsequent binding of anti-CD63 antibodies to the cell surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cytometry and immunoblotting. Our results support the homology of CD63 and granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granule membrane marker of neutrophils.     

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.     

Nucleotide responses of human neutrophils.

Since human polymorphonuclear neutrophils (PMN) exposed to ATP or its poorly hydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), respond with increases in intracellular calcium and enhanced O2- responses to the chemotactic peptide N'-formyl-Met-Leu-Phe (fMLP), we systematically evaluated responses of PMN to various nucleotides. The P2X and P2Y receptor agonists, 2-methylthioadenosine triphosphate and beta, gamma-methyleneadenosine triphosphate, failed to induce increases in intracellular calcium and did not desensitize PMN to increases in intracellular calcium induced by ATP gamma S. Since it has been suggested that P2Z receptor occupancy with the ATP4- species caused nonselective increases in cell permeability, the ability of ATP to induce increases in intracellular calcium was evaluated in the presence and absence of extracellular Ca2+ and Mg2+. In the presence of these cations, 5-fold greater concentrations of ATP were required. The effects of ATP4- were not associated with changes in cell membrane permeability. This suggests that ATP4- is the active species but that its effect on PMN is not linked to a nonselective increase in permeability of the cell membrane. With respect to responses of PMN to purine and pyrimidine nucleotides as defined by increases in intracellular calcium, the rank order of potency for the nucleotides was ATP = UTP greater than ATP gamma S greater than or equal to ITP greater than GTP greater than or equal to CTP. These responses were blocked by pretreatment of PMN with pertussis toxin. Prior exposure of PMN to ATP gamma S blocked cellular responses (calcium increases) to these nucleotides but not to fMLP. Likewise, exposure of PMN to any nucleotides blocked subsequent cellular responses to ATP gamma S but not to fMLP. These data support the concept that nucleotide responses of PMN utilize either a common receptor or a common signal transduction pathway involving a guanine nucleotide binding protein in events leading to elevations in intracellular calcium. Nucleotide interaction with PMN does not follow the established pattern of responses associated with P2X or P2Y purinergic receptor occupancy.     

Potency of bactericidal proteins purified from the large granules of bovine neutrophils.

The novel population of large granules of bovine neutrophils, which is the cell store of bactericidal activity independent of O2 derivatives, was extracted with an acid medium. Several fractions were resolved from the extract by ion-exchange chromatography (with carboxymethyl-cellulose) and gel filtration (with Sephadex G-50). Some of these fractions contained only a very limited number of major components, as detected by polyacrylamide gel electrophoresis. The purified bactericidal proteins exhibited their activity at 0.1 to 10 micrograms/0.3 ml of assay mixture containing 1 X 10(6) to 2 X 10(6) CFU of Staphylococcus aureus or Escherichia coli in media with physiological concentrations of Na+, K+, Mg2+, and Ca2+. Two fractions, containing polypeptides with apparent molecular weights ranging from 28,000 to less than 12,000, caused rather selective and rapid (5 to 20 min) killing of S. aureus. Their action was accompanied by significant binding to the gram-positive bacteria of some low (less than 12,000)-molecular-weight components. Other Sephadex G-50 fractions, containing the first emerging proteins with relatively high molecular weights, were more active on E. coli than on S. aureus. With the gram-negative bacteria there was a 10-min delay in the onset of bactericidal activity, which thereafter developed very fast. On the basis of the in vitro potency of the large-granule bactericidal proteins, we suggest that even partial discharge of granule content into the phagosomes may supply the phagocytic vacuoles of bovine neutrophils with a very efficient nonoxidative bactericidal system acting on both gram-positive and gram-negative microorganisms.     

Immunocytochemical identification of azurophilic and specific granule markers in the giant granules of Chediak-Higashi neutrophils.

We used immunofluorescent microscopy to characterize the abnormal granules in neutrophils from five patients with Chediak-Higashi disease. Monospecific antiserums to the azurophilic markers myeloperoxidase, elastase, cathepsin G and lysozyme, and to the specific granule markers lactoferrin and lysozyme, were labeled with fluorescein and rhodamine and were used to demonstrate two antigens in the same cell simultaneously. The abnormal granules in Chediak-Higashi neutrophils contained both azurophilic and specific granule markers. Normal-appearing lactoferrin-positive granules were also present, but normal azurophilic granules were not seen. Analysis of bone-marrow samples from two of these patients suggested that the abnormal granules were formed during granulocyte maturation by the progressive aggregation and fusion of normally formed azurophilic and specific granules. These results are consistent with a membrane abnormality or a defect of microtubular function leading to inappropriate granule fusion, and suggest that the granular abnormality is more generalized than previously appreciated.