Morten H. Nielsen

Human neutrophil granules and secretory vesicles

Abstract: The traditional classification of neutrophil granules as peroxidase‐positive (azurophil, or primary) and peroxidase‐negative (specific or secondary) has proven to be too simple to explain the differential exocytosis of granule proteins and incorporation of granule membrane into the plasma membrane which is an important aspect of neutrophil activation. Combined subcellular fractionation and immunoelectron microscopy has revealed heterogeneity among both peroxidase‐positive and peroxidase‐negative granules with regard to their content, mobilization and time of formation. Peroxidase‐negative granules may be classified according to their content of lactoferrin and gelatinase: 15% of peroxidase‐negative granules contain lactoferrin, but no gelatinase. 60% contain both lactoferrin and gelatinase. The term specific or secondary granule should be reserved for these two subsets. In addition, 25% of peroxidase‐negative granules contain gelatinase but no lactoferrin. These should be termed gelatinase granules or tertiary granules. Gelatinase granules are formed later than specific granules and mobilized more readily. In addition, a distinct, highly mobilizable intracellular compartment, the secretory vesicle, has now been recognized as an important store of surface membrane‐bound receptors. This compartment is formed in band cells and segmented cells by endocytosis. This heterogeneity among the neutrophil granules is of functional significance, and may also be reflected in the dysmaturation which is an important feature of myeloproliferative and myelodysplastic disorders.

Genetic instability of cell lines derived from a single human small cell carcinoma of the lung

Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations with different DNA content appeared. By cloning, permanent cell lines were established from the new subpopulations, whereas the original population stopped growing. The cloned cell lines were characterized by morphology, chromosomes analysis, electron microscopy and plating efficiency; the stability of the DNA content was examined regularly by flow cytometric DNA analysis and instability was found in one of the cloned cell lines. Chromosome analysis showed that the cloned cell lines consisted of more than one population after 17 in vitro passages. Both cloned cell lines produced tumors in nude mice. Genetic instability was demonstrated in these mouse-grown tumors as well. Development of resistance to antineoplastic treatment may be due to heterogeneity in sensitivity among subpopulations in a tumor. Isolation of populations with different DNA contents allows the study of interaction between subpopulations and the observations provide evidence in support of the hypothesis of clonal evolution.

Different ultrastructural morphology of Pneumocystis carinii derived from mice, rats, and rabbits

Pneumocystis carinii (PC) is a fungus present in the lungs of many mammal species. Even though studies of the genome, the isoenzymes, and the antigens have proved some host‐species‐linked heterogeneity, the existence of distinct Pneumocystis species or subspecies has still not been accepted. Comparative studies of the ultrastructural morphology of pneumocysts derived from several host species may support evidence of host‐species‐linked heterogeneity. We have compared the ultrastructural morphology of pneumocysts derived from mice, rats, and rabbits. The density of membrane‐limited electron‐dense cytoplasmic granules was found to be higher in mouse‐derived pneumocysts than in rabbit‐derived pneumocysts, and furthermore the average diameter of the granules from mouse pneumocysts was larger than that of granules from rabbit‐derived pneumocysts. The average diameter of the filopodia of mouse‐derived pneumocysts was smaller than that of filopodia from rat‐derived pneumocysts, which was smaller than that of filopodia from rabbit‐derived pneumocysts. Globular electron‐dense bulbous dilatations at the tip of the filopodia were described for the first time and they were only found on filopodia of mouse‐derived pneumocysts. These distinct host‐species‐linked morphological differences of pneumocysts from mouse, rat, and rabbit may support previous biochemical data indicating the existence of different Pneumocystis species or subspecies.

Isolation and culture of human endometrial cells

1Herlev Hospital, Department of Obstetrics and Gynaecology, University of Copenhagen, DK 2730 Herlev, Denmark; Chromosome Laboratory, Section of Clinical Genetics, Department of Obstetrics and Gynaecology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK 2100 Copenhagen, Denmark; Frederiksberg Hospital, Department of Obstetrics and Gynaecology, University of Copenhagen, DK 2000 Frederiksberg, Denmark; ^Department of Obstetrics and Gynaecology, University of Göteborg, Sahlgrenska University Hospital, Sweden; and 5Herlev Hospital, Department of Pathology, University of Copenhagen, DK 2730 Herlev, Denmark

Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane.

Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures; some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.

Localization of NCAM on NCAM-B-expressing cells with inhibited migration in collagen

The extracellular matrix is a key element in neuronal development and tumour invasion, providing a substratum which sustains the adhesion and migration of cells. In order to study interactions between the neural cell adhesion molecule (NCAM) and collagen, we transfected mouse L cells with cDNA encoding the human transmembrane NCAM isoform of 140 kDa (NCAM‐B). An L‐cell/collagen type I system was used to study the influence of NCAM expression on in vitro invasion. We here report that migration of NCAM‐expressing cells in collagen was inhibited compared to that of NCAM‐negative cells transfected with the empty vector. Immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold electron microscopy using anti‐human NCAM antibodies demonstrated a heterogeneous distribution of NCAM on the plasma membrane of transfected L cells grown on collagen. NCAM was preferentially located at the surface of broad cytoplasmic protrusions and slender extensions, some of which were facing the collagen. This was in contrast to the homogeneous surface distribution of NCAM on cells grown on plastic. These data suggest that NCAM and collagen type I interact, and that this might lead to the migration inhibition of NCAM‐expressing cells.

Chromosomal breakage, endomitosis, endoreduplication, and hypersensitivity toward radiomimetric and alkylating agents: A possible new autosomal recessive mutation in a girl with craniosynostosis and microcephaly

A high frequency of spontaneous chromosomal breakage, endomitosis, endoreduplication and hypersensitivity toward both the alkylating agent Trenimon and the radiomimetric drug bleomycin was observed in phytohemagglutinin-stimulated peripheral lymphocytes from a girl with craniosynostosis, microcephaly, ptosis, bird-like facies, and moderate mental retardation. We also observed abnormal chromosomal spiralization and some aspects of abnormal cellular division. Several fruitless attempts were made to establish a cell line. The parents were consanguineous, supporting the existence of a new, rare, autosomal, recessive condition in man. The mutation might involve a gene involved in DNA repair and/or regulation of the mitotic cycle.

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.

Morphology of Pneumocystis carinii and activation of the plasmalemmal vesicular system in alveolar epithelial cells of the host: an ultrastructural study

Lungs from rats with dexamethasone‐induced Pneumocystis carinii pneumonia were examined. The ultrastructure of Pneumocystis carinii and their zone of attachment on type I alveolar epithelial cells are described. An activation of the plasmalemmal vesicular system of type I alveolar epithelial cells was observed and is described here for the first time. The significance of this observation is discussed.

Immunoelectron microscopy of the receptor for urokinase plasminogen activator and cathepsin D in the human breast cancer cell line MDA‐MB‐231

Receptors for urokinase‐type plasminogen activator (uPAR) are present on the surface of many cell types and appear to be the key determinant controlling extracellular proteolysis catalyzed by the urokinase‐type plasminogen activator (uPA). Receptor‐bound uPA may be inhibited by the specific inhibitors PAI‐1 and PAI‐2, and the complex thus formed may subsequently be internalized and degraded in lysosomes. Biochemical evidence has recently indicated that also uPAR is internalized with the uPA/uPAI complex. We report here the subcellular localization of uPAR and cathepsin D in the MDA‐MB‐231 human breast cancer cell line studied by immuno‐electron microscopy of ultrathin cryosections using single or double immunostaining techniques. Cell surface uPAR was preferentially localized at cell‐cell junctions; cytoplasmic uPAR was inside large vesicles of different morphology and in flat Golgi saccules. A number of vesicles also contained cathepsin D. The uPAR was exclusively membrane‐bound at the cell surface and in cytoplasmic vesicles without cathepsin D. In lysosomal vesicles with both cathepsin D and u‐PAR, uPAR was probably degraded as it was observed in the luminal contents.