Effect of interferon on human neutrophilic granulocytes

Summary The in vitro influence of interferon (IFN) on various functions of human neutrophilic granulocytes was investigated. It was observed that the attachment and engulfment of opsonized yeast particles by human neutrophilic granulocytes were enhanced after preincubation in vitro with IFN for 30 min. The same result was obtained whether the particles were opsonized with fresh normal serum (complement) or with specific antibodies. However, after incubation of the granulocytes with IFN for 3 h the phagocytosis rate was somewhat decreased. Nitroblue tetrazolium (NBT) reduction by resting granulocytes was slightly, although not significantly, increased by preincubation with IFN for 30 min, but their NBT reduction during phagocytosis of E. coli was significantly increased. No major effects of preincubation with IFN were observed on spontaneous or random migration of granulocytes.     

Effect of verapamil and nifedipine on the adhesion, migration and phagocytosis of human neutrophilic granulocytes

The calcium antagonists Verapamil and Nifedipin have a different effect on adherence, migration and phagocytosis of human neutrophilic granulocytes. Whereas Verapamil (10(-4) mol/1) will inhibit the leukocyte function, Nifedipin is ineffective. The leukocyte function cannot be expected to be impaired by using Nifedipin and Verapamil in therapeutic doses. The differences in efficiency and their possible causes are discussed.     

Effect of amrinone and trapidil on adherence, migration and phagocytic behavior of neutrophilic granulocytes of the human

Amrinone and trapidile belong to a group of positive inotropic and simultaneously vasodilating substances, whose effects are based on an inhibition of phosphodiesterase (PDE), thus enriching intracellular c-AMP. In neutrophilic granulocytes these PDE-antogonists will produce an inhibition of adherence and phagocytosis, whereas the migration was not affected. The lower leukocyte effective concentration of 10 mmol/l can be achieved by a therapeutical application, so that a slight inhibition of the leukocyte function has to be accounted for as a side effect.     

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.     

Ultrastructural and morphometric characteristics of neutrophilic granulocytes of blood

Ultrastructural and morphometric characteristics of neutrophilic granulocytes (NG) of blood were investigated. It was found that every cell size fraction distinguished by its own morphologic type of NG and its specified quantitative and qualitative characteristic of primary granules. There is a close interrelation between morphometric and ultrastructural characteristics, and such parameters as perimeter, area of NG, and area of primary granules can be considered as criteria of the NG functional state.     

Confocal Raman microspectroscopy of the activation of single neutrophilic granulocytes

Confocal Raman micro-spectroscopy has been applied to investigate the activation process of single, living neutrophilic granulocytes. Both resting cells as well as activated cells were measured. The activation of cells was performed with phorbol-12-myristate-13-acetate activator and Escherichia Coli bacteria. Raman microspectroscopy combines a high spatial resolution inside a single, living cell with detailed material information. Using this approach it can be concluded that activation of the cells with phorbol-12-myristate-13-acetate causes a change in the redox state of cytochrome b₅₅₈. This protein is a part of the NADPH-oxidase complex that neutrophilic granulocytes employ to generate O₂ ^–, superoxide anion. Additionally a change in the redox state of myeloperoxidase can be observed. Myeloperoxidase is known to react with O₂ ^–. Activation of the cells with bacteria gives rise to corresponding changes in the Raman spectra. From this single cell study it can be concluded that the enzymes cytochrome b₅₅₈ and myeloperoxidase are present inside the cytoplasm of the living cell, while participating in the redox processes. Activation causes an intra-cellular release of oxygen metabolites. Activation with bacteria of neutrophilic granulocytes from a patient with chronic granulomatous disease, that contain no cytochrome b₅₅₈, led to typical changes in the redox state of myeloperoxidase. This indicates that in the bacterium/neutrophilic granulocyte system oxygen metabolites are generated that are capable of reacting with MPO. Received: 1 September 1998 / Revised version: 20 February 1998 / Accepted: 22 February 1998     

Estimation of kinetic parameters of neutrophilic, eosinophilic, and basophilic granulocytes in human blood.

Two hematologically normal patients with glioblastoma and six patients with chronic lymphocytic leukemia received continuous 3H-thymidine infusions for 3--10 days. In autoradiographs of blood cell smears taken for 25 days or more after the beginning of 3H-thymidine administration the labeling index and the labeling intensity of granulocytes were determined. A sufficiently high labeling intensity, i.e. a sufficiently long autoradiographic exposure time was found to be critical for obtaining valid and reproducible results. On the basis of certain assumptions discussed in detail, complete labeling of cells with 3H-thymidine followed by autoradiographic evaluation and mathematical analysis of the labeling patterns seems to be a suitable method for estimation of kinetic parameters of postmitotic granulocytes in vivo. The mean intramedullary maturation and storage time was observed to be 115 +/- 7 h or neutrophils, 103 +/- 4 h for eosinophils and 103 +/- 11 h for basophils. The mean relative inflow rate into the blood (or relative turnover rate in the blood) was found to be 4.2 +/- 0.4/h for neutrophils, 4.0 +/- 0.4%/h for eosinophils and 1.2 +/- 0.3%/h for basophils. The mean blood transit time (or blood sojourn time) was estimated to be 25 +/- 2 h or neutrophils, 26 +/- 3 h for eosinophils and 89 +/- 21 h for basophils. Accordingly the half lifes (T 1/2) of granulocytes in the blood were 17.3 +/- 1.4 h for neutrophils, 18.0 +/- 2.1 for eosinophils and 62 +/- 15 h for basophils. Under the quasi steady state conditions of this study the kinetics of granulocytes in the present CLL patients appeared to be normal, despite a marked lymphocytic infiltration of the bone marrow. The apparent discrepancy between these findings and the data obtained with autotransfusion of DFP-labeled granulocytes is discussed.     

Degree of structural homology of lactoferrins from milk and neutrophilic granulocytes

Some physico-chemical properties of human and pig lactoferrins from milk and neutrophilic granulocytes were compared. It was shown that the lactoferrins from different cell and tissue sources of the same species (humans or pigs) are identical in terms of electrophoretic mobility, molecular weight, iron-binding capacity, absorbance spectra, amino acid and sugar compositions and peptide maps. Human and pig lactoferrins show a high degree of structural homology (approximately 50%), but are immunochemically different.     

Carp neutrophilic granulocytes form extracellular traps via ROS-dependent and independent pathways

Neutrophil extracellular traps (NETs) have recently been described as an important innate defense mechanism that leads to immobilization and killing of invading pathogens. NETs have been identified in several species, but the mechanisms involved in NET formation and their role in infection have not been well determined yet. Here we show that upon in vitro stimulation with different immunostimulants of bacterial, fungal or viral origin, carp neutrophilic granulocytes rapidly release NET structures. We analyzed the composition of these structures and the kinetics of their formation by confocal microscopy, by quantifying the levels of extracellular DNA and the release of enzymes originating from neutrophilic granules: myeloperoxidase, neutrophil elastase and matrix metalloproteinase 9 (MMP-9). Profiles of NET release by carp neutrophils as well as their enzyme composition are stimulus- and time-dependent. This study moreover provides evidence for a stimulus-dependent selective requirement of reactive oxygen species in the process of NET formation. Collectively the results support an evolutionary conserved and strictly regulated mechanism of NET formation in teleost fish.     

The human neutrophilic myelocyte

Summary The developmental changes in the neutrophilic myelocyte from normal human bone marrow have been analyzed by means of phase contrast and electron microscopy. This developmental stage is characterized principally by the elaboration of secondary (specific) granules. In addition, there is a modest decrease in cell size, a decrease in the number and mean size of primary (azurophil) granules, a decrease in the number of polysomes, free ribosomes and mitochondria, a depletion of rough endoplasmic reticulum, an increase in cytoplasmic glycogen, an increase in chromatin aggregations and a loss of nucleoli, and the formation of a markedly indented nucleus. The myelocyte stage has been subdivided into three arbitrary phases based upon morphological and functional characteristics which relate to the onset, active production and cessation of secondary granulogenesis.Supported by Grant No. AM-HE-12084-13 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Dr. Arthur Sagone who performed the bone marrow aspirations and to Anita Topson, Barbara Jordan and Marjorie Griffith for their technical assistance.     

Characteristics of RNA and protein synthesis in the polymorphonuclear neutrophilic granulocytes of wounds

RNA and protein synthesis was comparatively studied in blood and wound neutrophils by electron microscopic autoradiography. It has been shown that the penetration of neutrophils into the wound was associated with a significant rise in RNA and a decrease in protein synthesis.     

The human neutrophilic promyelocyte

Summary The developmental changes in the neutrophilic promyelocytes from normal human bone marrow have been analyzed by means of phase contrast and electron microscopy. This developmental phase is characterized by the elaboration of primary (azurophillysosomal) granules and the entire intracellular machinery is directed principally toward this goal. The promyelocyte stage has been subdivided into three arbitrary stages based upon morphological, histochemical and functional characteristics which relate to the onset, active production and cessation of primary granulogenesis.Supported by Grant No. AM-HE-12084-12 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Dr. Arthur Sagone who performed the bone marrow aspirations and to Marjorie Griffith, Anita Topson and Barbara Jordan for their technical assistance.