Surface thermodynamics of normal and pathological human granulocytes

Surface tensions of normal and pathological granulocytes were determined by (1) adhesion to solid substrates of different surface tensions while suspended in liquid media of different surface tensions, and by (2) measurement of cell-liquid-vapor contact angles obtained with sessile drops of saline water on cell monolayers. The results obtained by the two different methods were in close conformation with one another. With the cell adhesion emthod some residual leukocyte adhesion still persists even under conditions where there no longer is a van der Waals attraction between cells and solid substrate. At low ionic strength and by the abolishment of all multivalent cations through the admixture of EDTA, that residual cell adhesion virtually disappears (with normal as well as with pathological granulocytes), indicating that the earlier residual cell adhesion did indeed arise from electrostatic interactions mediated by multivalent cations (probably Ca²⁺). Comparison of the capacities for engulfment and the surface thermodynamics data of normal and pathological granulocytes obtained in this study leads to the novel observation that the phagocytic episode from half to complete engulfment of bacterial particles by granulocytes appears to be the crucial step from the thermodynamic point of view.     

Galvanotaxis of human granulocytes

The galvanotactic response of human granulocytes was investigated theoretically and experimentally. The basic results are: (i) The granulocytes move towards the anode. (ii) The directed movement has been quantified by two different polar order parameters-the McCutcheon index and the average of cos . (iii) The polar order parameters are a function of the applied electric field (= dose-response curve). (iv) The inverse of the galvanotactic constant of migrating cells (analogous to the Michaelis-Menten constant) has a value of-0.2±0.03 V/mm. (v) The galvanotactic response of granulocytes is a non-cooperative process with a cooperativity coefficient of 1±0.2. (vi) The galvanotactic constant is a function of pH. (vii) The protein essential for the galvanotactic response is very likely a G-protein.     

Different binding capacities of influenza A and Sendai viruses to gangliosides from human granulocytes

The structures of gangliosides from human granulocytes were elucidated by fast atom bombardment mass spectrometry and by gas chromatography/mass spectrometry as their partially methylated alditol acetates. In human granulocytes besides G_M3 (II³Neu5Ac-LacCer), neolacto-series gangliosides (IV³Neu5Ac-nLcOse₄Cer, IV⁶Neu5Ac-nLcOse₄Cer and VI³Neu5Ac-nLcOse₆Cer) containing C_24:1, and to some extent C_22:0; and C_16:0 fatty acid in their respective ceramide portions, were identified as major components. In this study we demonstrate that gangliosides from human granulocytes, the second most abundant cells in peripheral blood, can serve as receptors for influenza viruses A/PR/8/34 (H1N1), A/X-31 (H3N2), and a parainfluenza virus Sendai virus (HNF1, Z-strain). Viruses were found to exhibit specific adhesion to terminal Neu5Ac2-3Gal and/or Neu5Ac2-6Gal sequences as well as depending on the chain length of ganglioside carbohydrate backbones from human granulocytes, these important effector cells which represent the first line of defence in immunologically mediated reactions. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GC/EIMS, gas chromatography/electron impact mass spectrometry; GSL(s) glycosphingolipids; HPTLC, high performance thin-layer chromatography; Neu5Ac,N-acetylneuraminic acid [26], PFU, plaque forming unit. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations, and the ganglioside nomenclature system of Svennerholm was used. LacCer or lactosylceramide, Gal1-4Glc1-1Cer gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; lacto-N-tetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4-Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse₆Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal 1-4-Glc1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer; G_T1b, IV³Neu5Ac, II³(Neu5Ac)₂-GgOse₄Cer; G_Q1b, IV³(Neu5Ac)₂, II³(Neu5Ac)₂-GgOse₄Cer; sialyllacto-N-tetraosylceramide, IV³Neu5Ac/IV⁶Neu5Ac-nLcOse₄Cer; sialyllacto-N-norhexaosylceramide or i-active ganglioside, VI³Neu5Ac-nLcOse₆Cer.     

Excretory-secretory product of Paragonimus westermani newly excysted metacercariae inhibits superoxide production of granulocytes stimulated with IgG

It is well known that the cysteine proteases in excretory-secretory product (ESP) of Paragonimus westermani newly excysted metacercariae (PwNEM) are capable of degrading IgG in vitro. Recent evidence suggests that the IgG-coated surface, such as found on parasites, is one of the most effective physiologic stimuli for granulocyte activation. Therefore, this study was designed to investigate the effect of excretory-secretory product (ESP) of PwNEM on superoxide production of granulocytes stimulated with IgG. The 96-well plates were coated with human IgG (0, 10, 30, 100 micrograms/ml) in the absence or presence of ESP. When granulocytes were incubated in the wells coated with human IgG in the presence of ESP, the level of superoxide production of granulocytes was reduced to about 90% when compared to the cells incubated in the wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production of granulocytes was concentration-dependent. These results suggest that ESP secreted by PwNEM may be important in the control of effector functions of granulocytes stimulated with IgG in human paragonimiasis.     

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes

Phorbol ester treatment of granulocytes triggers release of superoxide (O) and a concomitant burst of DNA strand breaks. The relationship between the amount of O and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O generated over a 40-min period by 1 × 10⁶ granulocytes/mL, a discontinuous “10-min pulse” method employing cytochrome c was used; 140 nmol O per 1 × 10⁶ cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1–10 mM) and staurosporine (2–10 nM) both inhibited O production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O. Zinc chloride (50–200 μM) inhibited both O and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O production more effectively than it inhibited DNA breaks. O dismutes to H₂O₂, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H₂O₂ had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O or H₂O₂ in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. J. Cell. Biochem. 66:219–228, 1997. © 1997 Wiley-Liss Inc.     

Increased adherence of human granulocytes to herpes simplex virus type 1 infected endothelial cells

Summary We studied the interaction of human polymorphonuclear leukocytes (PMNs) with umbilical vein endothelial cells infected with herpes simplex virus (HSV) type 1. PMNs labeled with⁵¹Cr were added to endothelial monolayers at varying times after infection and their adherence assessed 1 h later. Granulocyte adherence (GA) to uninfected cells averaged 26.5±1.9%. Increased adherence began 6 h postinfection and rose to a maximum at 20 to 24 h. HSV-1 glycoproteins seemed to mediate the increase in GA: tunicamycin treatment of infected monolayers for 18 h abolished the increased GA as did incubation of infected cells with F(ab')₂ fragments prepared from human antiserum containing HSV-1 antibody. Supported by grants R01-AA-06029 and T32-AA07233 from the National Institute of Alcohol Abuse and Alcoholism, and R01-HL-28220 from the National Heart, Lung, and Blood Institute.     

Application of PIXE analysis to the study of the regional distribution of trace elements in normal human brain

A particle-induced X-ray emission (PIXE) analysis method is presented, which allows measurement of eight elements (i.e., K, Ca, Mn, Fe, Cu, Zn, Se, and Rb) in human brain samples of only a few mg dry weight. The precision and accuracy of the method were investigated by analyzing animal brain matter with both PIXE and instrumental neutron activation analysis (INAA). The method was applied to measure the 8 elements in 46 different regions of 3 human brains. The sections analyzed originated from either the left or the right cerebral hemisphere, brain stem, and cerebellum. For one of the brains, sections were also analyzed from 26 corresponding regions of both hemispheres. For all elements, similar concentrations were found in the corresponding areas of the left and right sides of the brain. The concentrations (in μg/g dry weight) of the elements K, Fe, Cu, Zn, Se, and Rb were consistently higher in cortical structures than in white matter. Deep nuclei and brain stem, which have a mixed composition, showed intermediate values for K, Zn, Se, and Rb. A hierarchical cluster analysis indicated that the various brain regions clustered into two large groups, one comprising gray and mixed matter regions and the other, white and mixed matter brain areas.     

Zinc and copper in human cerebrospinal fluid

Zinc and copper have been estimated in CSF of 14 normal volunteers, nine men and five women. Zinc was analyzed by limited-aspiration flame atomic absorption spectrophotometry using a deuterium continuum light source. Copper was analyzed in 0.1% HNO₃ by flameless atomic absorption spectrophotometry with a graphite cuvette on a flameless atomizer. Recovery of added zinc varied less than 5% and that of the added copper varied less than 8%. CSF zinc was 31.5±19.8 μg/L (mean ± 1 SD); CSF copper, 7.5±3.1 μ/L. Values obtained for CSF zinc are about 1/2 those we and others obtained previously, the decrease related almost exclusively to removal of interference by the CSF matrix, which produced spuriously elevated values without use of the deuterium light source. Values obtained for CSF copper were approximately one-tenth those we and others had obtained previously. The decrease related, in part, to the removal of matrix effects, but also to improvement of the signal-to-noise ratio present in other techniques.     

Ultrastructural,cytochemical and functional studies on the eosinophilic granulocytes of larval lampreys

Summary Blood from larval lampreys (ammocoetes) contains a small number of eosinophilic granulocytes which are formed in the protospleen and kidney. Both immature and mature forms of this cell type are present in the blood and these are easily identified from other cell types due to the prominent eosinophilic granules that fill the cytoplasm. Ultrastructurally, these granules are electron-dense, largely unstructured, Golgi-derived and contain acid phosphatase but not peroxidase. Eosinophilic granulocytes ingest bacteria but fail to internalise colloidal carbon. The functional and phylogenetic significance of these cells is discussed.     

Individual variation of nucleoside triphosphate pyrophosphohydrolase activity in human erythrocytes,granulocytes, lymphocytes,and platelets

Analysis of the nucleoside triphosphate pyrophosphohydrolase specific activity of red cells obtained from a random Caucasian population indicated at least two subclasses. The specific activity of 18% of the population ranged from undetectable activity to 27.5 nmol ITP cleaved/20 min/mg hemoglobin. The remainder of the population had higher activity, 27.5–125 nmoles ITP cleaved/20 min/mg hemoglobin. The variation of NTPH activity evident in the red cells of an individual is reflected in granulocytes, lymphocytes, and platelets of that individual. Erythrocyte activity ranges from 0.7 to 21 units (nmol of ITP cleaved in 20 min)/10⁷ cells, granulocytes have 17–201 units/10⁷ cells, lymphocytes have 91–462 units/10⁷ cells, and platelets have 1.1–7.1 units/10⁷ platelets. These cell differences are discussed with respect to the hypothesis that NTPH prevents incorporation of ITP or dITP into nucleic acids.This work was supported by funds allocated by the Agricultural Experiment Station, Michigan State University. Michigan Agricultural Experiment Station Journal No. 8727.     

Analysis of coated pit recycling on human fibroblasts

The recycling to the cell surface of previously internalized coated pits has been proposed as a likely mechanism for the rapid regeneration of coated pits on human fibroblast surfaces at 37°C (1). We present a general mathematical model of coated pit recycling for the case when the coat cycles as a single unit, and use it to analyze certain time and temperature dependent data obtained by Anderson et al. (1) and Vermeer et al. (2). We show how recycling can account for these data and how this type of data can be used to distinguish between different possible recycling mechanisms. We show that these data are inconsistent with a two compartment model where coat material simply shuttles back and forth between coated pits and short-lived coated vesicles. From these data we estimate for human fibroblasts at 37°C: that the time for a coated pit to be replenished through recycling after it is lost through internalization is greater than 3.5 min; and that at any moment 53% or less of the cell’s clathrin that is involved in coated pit recycling is on the cell surface.     

The presence of blood group and lymphocyte antigens on porcine granulocytes

Suspensions of highly viable (< 95 %) granulocytes minimally contaminated by other cell types were isolated from the peripheral blood of pigs by a single centrifu-gation with low molecular weight dextran and after preferential lysis of erythrocytes by hypotonic shock. A complement-dependent cytotoxic test showed the presence of antigens of the SLA major histocompatibility complex, the SLB leucocyte system and the A and E blood group systems on the granulocytes. Some SLA typing reagents against class I (SD) antigens did not react with granulocytes, however, or yielded dubious reactions. The findings showed that the reactivity of SLA sera resembles the reactivity of human HLA sera. The results also show that compatibility in the SLA, SLB, A and E systems will have to be taken into account when preparing alloimmune sera for the determination of granulocyte-specific antigens of pigs.     

Gravity sedimentation analysis of human blood leukocytes

A new method of sedimentation analysis of human blood leukocytes is described. Platelets, lymphocytes, monocytes, and polymorphonuclear cells isolated from normal human peripheral blood have been analyzed alone and in mixture by gravity sedimentation employing a computerized scanning instrument. All four classes could be clearly resolved from each other exhibiting sedimentation velocities of 0.6 ± 0.00, 1.04 ± 0.11, 1.27 ± 0.15 and 1.89 ± 0.21 · 10^?4 cm/s, respectively, at 37°C in a 2.5–6.25% Ficoll gradient in Medium 199. Less than 10⁶ cells can be used for analysis. Possible applications of the method are discussed.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

Conversion of albumin into a transmitter of the ultra long-range interaction of human erythrocytes

Previous experiments in our laboratory and elsewhere have shown that extended macromolecules of high molecular weight are needed in the suspending medium to transmit the quantum-mechanical coherent interaction of human erythrocytes. Albumin, a globular protein, does not transmit the interaction at physiological concentrations. Albumin can be converted into a transmitter by preheating a solution of albumin to 62°C for 20 min. Such treatment unravels the tertiary structure of albumin and allows it to polymerize.     

Intracellular localization of N-formyl chemotactic receptor and Mg dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm⁻³. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes

Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83 000 × g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40–60% were achieved with a 20–70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g·cm^?3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimanted with specific granules (g9 = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg²⁺ -ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg²⁺ -ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.     

Absolute volume of differentiating cells in the epithelium of the human hard palate

Summary In previous studies the differentiation of the epithelium in the human hard palate has been described stereologically using parameters expressed per unit tissue volume. Since single epithelial cells represent the true biological units of this tissue, it became necessary to estimate the absolute size of such cells in order to transform density data into absolute data. Therefore, in the present study, a stereological method (originally developed for myocyte volume determination) was tested in terms of its applicability to stratified epithelia; the absolute size of differentiating epithelial cells was determined in the epithelium of the human hard palate. The results suggest that (1) rather precise determination of epithelial cell size is possible by using the modified myocyte volume determination, and (2) the average cell volumes are 926 ± 148, 4,111 ± 1,619, 4,394 ± 551 m³ for the stratum basale, the upper stratum spinosum and the stratum granulosum, respectively. The results are discussed with respect to methodology and to differentiation phenomena in the epithelium of the human hard palate.