Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes.

The subcellular localization of granulocyte collagenase, elastase and chymotrypsin-like cationic protein was determined using velocity centrifugation of cytoplasmic granules of human polymorphonuclear leukocytes. The proteases were assayed by immunochemical and enzymatic methods. Measurements of lactoferrin and myeloperoxidase distinguish exactly between constituents of specific and azurophil granules. Collagenase, elastase and chymotrypsin-like cationic proteins showed an almost identical sharp and unimodal distribution. They co-sedimented with myeloperoxidase demonstrating that these enzymes are localized exclusively in the azurophil granules.     

Demonstration of ricin-binding sites on the outer face of azurophil and specific granules of rabbit polymorphonuclear leukocytes

The presence of carbohydrate residues on the outer surface of PMN granules has been demonstrated by the use of ricin-conjugated ferritin. The binding of the lectin was inhibited by alpha-lactose. No difference in the binding densities of azurophil or specific granules was observed.     

Soluble Fas ligand is chemotactic for human neutrophilic polymorphonuclear leukocytes

It has been recently shown that Fas ligand (FasL) expression on islet beta grafts results in neutrophilic infiltration and graft rejection. In this study, we show that human recombinant soluble FasL is endowed with potent chemotactic properties toward human neutrophilic polymorphonuclear leukocytes (neutrophils) at concentrations incapable of inducing cell apoptosis. Furthermore, neutrophils exposed to soluble FasL did not display detectable change of intracellular Ca2+ and did not undergo superoxide production or exocytosis of primary and secondary granules. Our results show that FasL is a potent chemoattractant for human neutrophils without evoking their secretory responses. This finding suggests a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.     

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.     

Ultrastructure of neutrophilic granulopoiesis in the bone marrow of patients with chronic myeloid leukemia (CML). A morphometric study with special emphasis on azurophil (primary) and specific (secondary) granules

In seven patients with chronic myeloid leukemia (CML) and ultrastructural and morphometric study was performed on neutrophilic granulopoiesis in bone marrow trephine biopsies. Bone marrow specimens from five patients without hematological abnormalities served as controls. In stable phases of CML, abnormalities of the maturing granulocytic lineage were most conspicuously expressed by an infrequently occurring nuclear disfiguration (blebs and disturbed bridging of segments). Morphometric evaluation included the numbers of azurphil (primary) and specific (secondary) granules, the cisternal length of the endoplasmic reticulum and the area of the mitochondrial profiles. These variables could be determined in early and late myeloblasts, promyelocytes, metamyelocytes, band cells and mature polymorphonuclear granulocytes. Statistical analysis with regard to control specimens demonstrated no significant differences in the total amount of neutrophil granules or of the other cell organelles.     

Collection and phagocytic evaluation of uterine neutrophilic leukocytes

The study of phagocytosis in the bovine uterus requires the collection of large numbers of uterine leukocytes. A method of stimulating polymorphonuclear neutrophil (PMN) invasion into the bovine uterus with oyster glycogen was tested. Following glycogen treatment, uterine aspirates contained large numbers (10(6)-10(9)) of viable uterine PMN. In Experiment 1, six cows were treated at one-week intervals by intrauterine infusion with 0.1, 1.0 and 10.0% oyster glycogen, respectively. Uterine lumens were lavaged at 0, 12, 24, 48, 72 and 168 hours post-infusion with each concentration of glycogen. The mean peak response for PMN was at 12 hours post-infusion of glycogen. In Experiment 2, uterine and peripheral blood PMN were collected from each of 12 cows. Phagocytosis was measured as the percent of (32)Phosphorus-labeled Staphylococcus aureus ingested by PMN during a one-hour incubation. Mean (+/- standard deviation) percent phagocytosis was not statistically different between blood (53.6 +/- 11.0%) and uterine (43.8 +/- 13.0%) PMN. This indicates that phagocytic activity was similar for PMN collected either from the uterine lumen or peripheral blood.     

Effect of botulinum D toxin on human neutrophilic leukocytes and localization of its substrates

Botulinum D toxin has been shown to ADP-ribosylate 22-kD proteins in neutrophilic leukocytes, but the function of these GTP-binding proteins remains unknown. In analogy to small GTP-binding proteins like SEC4 to YPT1, it has been suggested that botulinum D toxin substrates might be involved in secretory process of myeloid cells. Three main findings lead to the opposite conclusion. First of all, in human neutrophils, botulinum D toxin does not modify the release of azurophilic and specific granules induced by a chemoattractant (a formylpeptide) or a phorbol ester. Second, botulinum D toxin ADP-ribosylates 24 to 26-kD proteins that are only present in plasma membranes of human neutrophils. The membrane location of these substrates differs largely from that of the GTP-binding proteins involved in exocytosis and located in granules. Finally, since the same quantity of the toxin substrates is present in neutrophils as in their precursors, HL60 cells (which are devoid of specific granules and characterized by immature azurophilic granules and NADPH oxidase), it is unlikely that endogenous botulinum D toxin substrates are directly involved in the secretory responses of neutrophils.     

Secretory activity of neutrophilic leukocytes in inflammatory periodontal diseases

Effects of various antigens on the secretory and diagnostically important functions of neutrophilic leukocytes were studied in peripheral venous blood samples of 106 patients with inflammatory periodontal diseases without any internal pathologies. Their neutrophils, stimulated by phagocytosable particles, were found to secrete increased amounts of alkaline phosphatase, β-glucuronidase, and lysozyme. This phenomenon suggests a general activation of lysosomal enzyme secretion and shows that the destruction of periodontal tissues may be caused by these proteases.     

Enterococcus faecalis: specific and non-specific interactions with human polymorphonuclear leukocytes

In previous studies we have demonstrated that the ability of Enterococcus faecalis to adhere to and to be internalized in human urinary tract epithelial cells, Girardi Heart cells and human polymorphonuclear leukocytes (PMNs), was dependent on whether the strain had been isolated from urinary tract infections (UTI) or endocarditis (EN) respectively. These properties were further modified by growth of the organism in human serum. In the present report, using competition assays we show that adhesins containing a D-glucose moiety play a role in mediating the interactions between human PMNs and E. faecalis strains isolated from UTI and grown in brain-heart infusion broth (BHIB). On the other hand, adhesins containing both D-glucose and D-galactose moieties were involved in the interactions between PMNs and serum grown UTI isolates or EN isolates grown in either BHIB or human serum. Moreover, the impairment in the association between both UTI and EN strains after growth in serum appears to be at least partially related to a decrease in enterococcal surface hydrophobicity.     

Lipid raft proteome of the human neutrophil azurophil granule

Detergent-resistant membrane domains (DRMs) are present in the membranes of azurophil granules in human neutrophils (Feuk-Lagerstedt et al., J. Leukoc. Biol. 2002, 72, 970). Using a proteomic approach, we have now identified 106 proteins in a DRM preparation from these granule membranes. Among these proteins were the lipid raft structural proteins flotillin-1 and -2, cytoskeletal proteins such as actin, vimentin and tubulin, and membrane fusion promoting proteins like annexins and dysferlin. Our results suggest that the azurophil granule membrane, in similarity to the plasma membrane, is an elaborate structure that takes part in intracellular signaling and functions other than the mere delivery of bactericidal effector molecules to the phagosome.     

Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl₂ with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl₂, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.     

Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes

We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co- localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.     

Demonstration of specific secretory granules for human chorionic gonadotropin in placenta

Existence of secretory granules and exocytosis during secretion of human chorionic gonadotropin (hCG) in human placenta has been a point of controversy. Using two methods, the highly sensitive avidin-biotin complex (ABC) method and the protein A-gold technique, for immunochemical identification of beta-hCG on electron microscopic sections, we have examined placentas at 8-10 weeks gestation and at term for the presence of secretory granules. First-trimester placentas demonstrated plentiful syncytiotrophoblast cytoplasmic granules, some undergoing exocytosis, when stained using specific beta-hCG antiserum in the ABC and protein A-gold methods. Term placentas did not show positive reaction product. The data demonstrate that the classic secretory granule-exocytosis pathway mediates placental hCG secretion. However, clear morphological differences exist between placenta granules and hormone secretory granules observed in pituitary, consistent with known functional differences between these organs. This methodology will be useful for further studies of the secretory pathways for placental peptides.