Phagocytic capacity of cytokineplasts from human blood polymorphonuclear leukocytes

Cytokineplasts (CKP) are membrane-bounded anucleate cytoplasmic fragments, induced from polymorphonuclear leukocytes (PMN) by the brief application of heat; derived from the cortical cytoplasm that gathers at the leading front of migrating PMN; and endowed with many of the motile properties of the parent cell. In this study we examine their phagocytic capacity by quantitative methods. CKP ingest Staphylococcus aureus and Serratia marcescens somewhat less avidly than do the corresponding intact PMN, yet rather impressively when one considers how restricted a portion of the parent cell they represent. Under the conditions employed, CKP killed about half as many of the bacteria presented to them as did their parent PMN. Thus, despite a heat-associated loss of demonstrable respiratory burst oxidase activity and a paucity of cytoplasmic granules, the organelle-depleted CKP deals with bacteria in a way that mimics its parent PMN.     

Alkaline phosphatase activity in human polymorphonuclear leukocytes

Synopsis Alkaline phosphatase has been localized ultracytochemically in PMN of man with normal and elevated levels of this enzyme. Contrary to guinea-pig PMN, no activity appears to be present in the specific granules. Instead, the plasma membrane and the membrane of the endocytic vacuoles show a strong staining. However, the demonstration of this activity depends on the preparatory procedure employed for PMN isolation. the use of dextran and Ficoll-Hypaque in the isolation procedure induces a marked increase in alkaline phosphatase staining of the PMN plasma membrane. Strongly increased activity at this site has been found in PMN from cancer patients. In most of them, additional staining has been observed in atypical vesicles and sometimes in the Golgi apparatus. These findings are discussed in the light of some previously reported controversial biochemical and cytochemical data on the distribution of alkaline phosphatase in human PMN.     

Separation of granule subpopulations in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes were isolated, disrupted by sonification and the nuclei and unbroken cells removed by centrifugation. The supernatant was applied on top of an optimised discontinuous Percoll gradient. After centrifugation we found nine gradient bands of distinct density. Both the nine bands and the whole fractionated gradient material were assayed for granule marker enzymes. Granule fractions of distinct density, enclosing different enzyme concentrations demonstrated the existence of granule subpopulations. There were three subpopulations of azurophil granules, about four subpopulations of specific granules, one granule fraction perhaps representing the C-particles, and a fraction of plasma membrane vesicles.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Effects of microcystins on human polymorphonuclear leukocytes

Microcystins (MCs) are cyclic heptapeptides produced by cyanobacteria present in water contaminated reservoirs. Reported toxic effects for microcystins are liver injury and tumour promotion. In this study, we evaluated the effects of two MCs, MC-LR and [Asp(3)]-MC-LR, on human neutrophil (PMN). We observed that even at concentrations lower than that recommended by World Health Organization for chronic exposure (0.1 nM), MCs affect human PMN. Both MCs have chemotactic activity, induce the production of reactive oxygen species, and increase phagocytosis of Candida albicans. MC-LR also increased C. albicans killing. The effect of MCs on PMN provides support for a damage process mediated by PMN and oxidative stress, and may explain liver injury and tumour promotion associated to long-term MCs exposures.     

Arachidonic acid-induced chemiluminescence of human polymorphonuclear leukocytes

When human polymorphonuclear leukocytes were incubated with arachidonic acid, a rapid light emission was observed which reached a maximum within 2 min. The magnitude of chemiluminescence depended on the number of polymorphonuclear leukocytes and the concentration of arachidonic acid. The light emission was inhibited by about 40% or 70% by 100 μM 3-amino-1-(m-(trifluromethyl)-phenyl)-2-pyrazoline (BW755C) or 100 μM nordihydroguaiaretic acid as lipoxygenase inhibitors. In contrast, 100 μM indomethacin, a cyclooxygenase inhibitor, had no effect. These results suggested a pivotal role of the lipoxygenase pathway rather than the cyclooxygenase pathway in the light emission.     

Assay method for myeloperoxidase in human polymorphonuclear leukocytes

A simple assay method for measuring myeloperoxidase (MPO) has been developed. MPO is found in polymorphonuclear leukocytes and is important as a bactericidal agent in the presence of H2O2 and halide ions. This improved assay method is based on work of Andrews and Krinsky using tetramethylbenzidine (TMB) a noncarcinogenic substrate. By assaying MPO under optimal conditions of TMB at 1.6 mM, H2O2 concentration of 0.3 mM, pH 5.4, and incubation temperature of 37 degrees C, sensitivity of MPO measurements increased eightfold in comparison with the original TMB method. A method has been established to determine absorbance at 655 nm of the reaction mixture by incubation for 3 min and then stopping the reaction by the addition of pH 3.0 buffer. An attempt was also made to raise the sensitivity by using 3,3'-dimethyoxybenzidine (DMB), a carcinogenic substrate. The improved TMB method was 34 times more sensitive than the DMB method.     

Phosphorylase kinase from human polymorphonuclear leukocytes.

Phosphorylase kinase from human polymorphonuclear leukocytes was investigated in a gel filtered crude preparation (17,000 x g supernatant). It was found to exist in two forms, one (the phosphorylated form) more active than the other (the dephosphorylated form). Interconversion between the two forms was carried out by a cyclic AMP dependent protein kinase and phosphoprotein phosphatase, respectively. The ratio of activity measured at pH 8.0 and 6.0 was 0.36 for the non-activated and 0.83 for the activated form, which is in contrast to the behaviour of phosphorylase kinase from muscle. Km app for the substrate phosphorylase b was 650 U/ml and 85 U/ml for the non-activated and activated form, respectively, whereas Km app for ATP was 0.03 mM and identical for the two forms. The non-activated form of phosphorylase kinase was activated by Ca2+ in the range 10(-7)--5 . 10(-6) M, which may have physiological importance, whereas the activated form was insensitive to variations in Ca2+ concentration between 10(-9) and 10(-3) M.     

Tumor promoter-induced centrosome splitting in human polymorphonuclear leukocytes

Human neutrophilic polymorphonuclear leukocytes (neutrophils) adhering to a substratum undergo a dramatic change in cellular morphology when treated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). Within a few minutes of TPA treatment, the cells cease locomotion and spread symmetrically on the substratum. Concomitantly, TPA initiates centrosome splitting in a manner similar to that induced by treatment of randomly locomoting cells with a chemotactic factor. The two centrioles of a centrosome separate by a distance of several micrometers, and each of the solitary centrioles is surrounded by an aster of microtubules. Some cells also establish a third, centriole-free aster of microtubules. TPA treatment increases the total number of microtubules associated with the centrosome(s) and also increases overall polymer length. The frequency of centrosome splitting is enhanced transiently by treatment with the synthetic chemotactic peptide f-Met-Leu-Phe. Centrosome splitting is interpreted in terms of an interaction between the cell periphery and the microtubule system. Possible cellular mechanisms of this unusual phenomenon are considered.     

Calcium-induced lysozyme secretion from human polymorphonuclear leukocytes

Calcium ions, in the absence of other stimuli, are capable of provoking the release by exocytosis of the granule-associated enzyme, lysozyme, from human polymorphonuclear leukocytes. Calcium-induced extrusion of lysozyme occurs in a concentration, time and temperature-dependent fashion. It is enhanced in the presence of extracellular inorganic phosphate and the ionophore, A-23187, and is not accompanied by the release from cells of cytoplasmic or lysosomal marker enzymes.     

Facilitated transport of 3-O-methyl-D-glucose in human polymorphonuclear leukocytes

Transport of the nonmetabolizable hexose analogue 3-O-methyl-D-glucose (30MG) was measured in human polymorphonuclear leukocytes at 37 degrees C, pH 7.4. 3OMG at very low concentration (0.05 mM) equilibrated with the intracellular water with a rate constant of about 0.08 s-1. Transport of 3OMG in the presence of 20 microM cytochalasin B and transport of L-glucose were insignificant. Countertransport of 14C-labelled 3OMG was demonstrated. Exchange of 3OMG between the extracellular and intracellular water showed saturation with a Km of about 4 mM. Thus, the transport of 3OMG is mediated almost exclusively by facilitated diffusion.     

Regulation of phosphatidic acid phosphohydrolase activity during stimulation of human polymorphonuclear leukocytes.

Phosphatidic acid phosphohydrolase (PPH) activity was determined in human polymorphonuclear leukocytes (PMNs) by measuring the hydrolysis of [32P]phosphatidic acid (PA) added to cell sonicates. Enzyme activity was localized primarily to a soluble fraction. Soluble and particulate activities required magnesium and were inhibited by calcium, N-ethylmaleimide, sphingosine, and propranolol. The activity in unstimulated PMNs was 0.64 +/- 0.11 nmol of PA hydrolyzed.mg protein-1.min-1 in particulate and 4.20 +/- 0.42 in soluble fractions. Stimulation of PMNs with 1 microM f-Met-Leu-Phe (FMLP) for 10 min caused a slight decrease in soluble activity and a small increase in the activity of particulate fractions. Preincubation with 10 microM cytochalasin B for 5 min before FMLP stimulation markedly enhanced both of these changes. The effect of FMLP plus cytochalasin B was rapid (less than 10 s), whereas the calcium ionophore A23187 (1 microM) and phorbol myristate acetate (100 ng/ml) caused slower and smaller changes in enzyme activity. These results indicate that after chemoattractant stimulation; PPH activity decreases in the soluble fraction and increases in the particulate fraction suggesting that PPH may participate in signal transduction in the PMN.     

ESR studies on oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes

Oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes (PMN) stimulated with phorbol myristate acetate (PMA) was studied with spin probe oxymetry and using the transition metal ion CrOX (potassium trioxalatochromate) as a widening agent. The experimental results demonstrated that during the respiratory burst of PMN stimulated with PMA, oxygen consumption was found mainly in the intercellular medium but no change of oxygen concentration was found in the intracellular medium.     

Mechanism of arachidonic acid release in human polymorphonuclear leukocytes

Previous studies have demonstrated that [³H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A₂ activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [¹⁴C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A₂ or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [¹⁴C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [¹⁴C]stearate, replaced [¹⁴C]arachidonate, very little [¹⁴C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [¹⁴C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.     

Immediate-early antigen expression and modulation of apoptosis after in vitro infection of polymorphonuclear leukocytes by human cytomegalovirus

Polymorphonuclear leukocytes (PMNL) are a major carrier of human cytomegalovirus (CMV) in viremic immunodepressed patients. We transmitted infectious virions and viral components to PMNL by coculturing these cells with infected human embryonic lung fibroblasts (HELF) or human umbilical vein endothelial cells (HUVEC). Quantitative time-course analysis of viral DNA and protein expression in PMNL, after functional separation from infected donor cells, indicated the initiation of viral cycling, with immediate-early protein expression. No viral replication or early or late gene expression was observed, but infected PMNL were able to infect naive fibroblasts more than 48 h after the end of co-culture. PMNL apoptosis was significantly delayed during co-culture with infected or uninfected HUVEC, and this phenomenon did not require contact between the two cell populations. The increased production of IL-8 in the same culture conditions that protect PMNL from apoptosis, associated with the reversion of this protection by inhibiting or depleting this factor in the culture media, targets this cytokine as a likely candidate for this protective effect. These data suggest that PMNL play a key role in virus dissemination in vivo, through their interactions with infected endothelial cells.     

Inhibition of phospholipid methyltransferase during zymosan induced secretion of platelet-activating factor in human polymorphonuclear leukocytes

Phagocytosis of zymosan particles coated with complement induces a time and dose dependent inhibition of the enzyme phospholipid methyltransferase in human polymorphonuclear cells. The extent of phospholipid methyltransferase inhibition induced by various concentrations of zymosan strongly correlates with the secretory process: liberation of platelet-activating factor (PAF) and β-glucuronidase. Zymosan also decreases the incorporation of ³H-methyl group into phospholipids in cells pre-labeled with (³H-methyl)-methionine. Finally, preincubation of cells with 3-deaza-adenosine and homocysteine thiolactone, inhibitors of phospholipid methyltransferase, decrease the incorporation of ³H-methyl group into phospholipids in cells pre-labeled with (³H-methyl)-methionine and modulate the release of PAF. These results suggest that phospholipid methylation plays an important role during the transduction of the secretory signal triggered by zymosan in human polymorphonuclear cells.