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.     

Functional activity of polymorphonuclear leukocytes in isoprenaline-induced cardiomyopathy in rats

The article studies the dynamics of changes of the parameters of the functional activity using chemiluminescent and turbidimetric methods as well as blood plasma myocardium and liver homogenates in isoprenaline cardiomyopathy in rats. Mechanisms of pathogenesis of isoprenaline-induced cardiomyopathy in rats is being discussed.     

Phorbol myristate acetate enhances adenylate cyclase activity in human polymorphonuclear leukocytes

The putative role of protein phosphorylation in modulating adenylate cyclase activity in polymorphonuclear neutrophil membranes was assessed using phorbol myristate acetate (PMA) to stimulate the activity of protein kinase C. PMA was demonstrated to enhance the adenylate cyclase activity stimulated by isoproterenol.     

Neutral endopeptidase activity in the interaction of N-formyl-L-methionyl-L-leucyl-L-phenylalanine with human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) hydrolyze the synthetic chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) at nanomolar concentrations in an autocatalytic-like manner that deviates from classical Michaelis-Menten kinetics [Yuli, I. & Snyderman, R. (1986) J. Biol. Chem. 261, 4902-4908]. By using inhibitors of distinct classes of endoproteases, this particular fMet-Leu-Phe degradation was attributed exclusively to an exoplasmic metalloendoprotease that matches the ubiquitous neutral endopeptidase (NEP). Membrane-bound NEP hydrolyzes non-chemotactic substrates according to a classic Michaelis-Menten mechanism. By competitive inhibition with non-chemotactic substrates, fMet-Leu-Phe was found to interact with membrane NEP through a single active site, in a non-cooperative mode with an apparent Km in the order of 1 mM. The discrepancy between the ordinary hydrolysis of the micromolar and millimolar concentrations of fMet-Leu-Phe, reported by others, and the particular degradation of the nanomolar fMet-Leu-Phe, could not be accounted for by any coherent correlation between NEP activity/inhibition and modulation of fMet-Leu-Phe binding to its receptor, and/or induction of fMet-Leu-Phe-receptor-mediated inflammatory responses. Based on these and previously reported results, a novel model is proposed in which the fMet-Leu-Phe-induced inflammatory stimulation of PMN involves both NEP and the fMet-Leu-Phe receptor. By this model, NEP and the fMet-Leu-Phe receptor are distinct membrane entities which can form dynamic binary and tertiary complexes; thus accounting for the unusual kinetic features of fMet-Leu-Phe degradation, as well as the two receptor states. The complex of NEP and the fMet-Leu-Phe receptor might be conceived as a chemotactic-perception mechanism that combines the high affinity of the receptor and the rapid turnover of NEP.     

Oxidative activity of human polymorphonuclear leukocytes stimulated by the long-chain phosphatidic acids

It has already been suggested that phosphatidic acids (PAs) play an important role in the regulation of signaling pathways involved in the production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs). The present study was performed to elucidate the effects of extracellularly added PA-- 1,2-distearoyl- (DSPA) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphate (SAPA)--on the ROS production and on the elastase release by human PMNs. ROS production was monitored by luminol-amplified chemiluminescence and the elastase activity was measured in the supernatant of the PA-stimulated human PMNs by colorimetric assay. Obtained effects were compared with those of cells stimulated by either a chemotactic tripeptide, phorbol ester or calcium ionophore. Our results show that long-chain PAs at concentrations higher than 3 x 10(-5) mol/l stimulate the ROS production by human PMNs, whereas they were ineffective in promoting the elastase release. The chemiluminescence pattern of the SAPA-stimulated cells exhibited a biphasic curve, whereas cell stimulation with DSPA resulted in a monophasic chemiluminescence curve. Stimulation of the ROS production by PAs in dependence of the fatty acid composition required the activity of protein kinases.     

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.     

Functional immaturity in neonatal polymorphonuclear leukocytes of rhesus monkeys

Abstract: Despite major advances in the management and care of critically ill and low-birthweight human and nonhuman primate infants over the past two decades, infection remains a major source of neonatal morbidity and mortality. Although the causes of enhanced susceptibility and dissemination of neonatal infections are incompletely defined in the literature, substantial evidence from this and other laboratories has implied that functional abnormalities of neonatal polymorphonuclear leukocytes (PMNs) may be a major contributor. Increased understanding of the functional characteristics of neonatal PMNs should, therefore, provide significant insight into the pathogenesis and possible therapy of infections in neonates. Our laboratory has been actively involved in evaluating the functional competence of PMNs in neonatal human and nonhuman primates. This report describes a study in which we have confirmed and characterized the functional compromises in neonatal PMNs of rhesus monkeys, including deficiencies in chemotaxis, membrane deformability, phagocytosis, and killing.     

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.     

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.     

Investigations of the biological activity of leukotriene A4 in human polymorphonuclear leukocytes

An investigation was undertaken to compare the responses of human neutrophils to the epoxide leukotriene A4 with those elicited by its stable product leukotriene B4 under identical IN VITRO conditions. LTA4 evokes neutrophil responses similar in nature to those induced by LTB4 but at much higher concentrations. Evidence suggests that LTA4 is important primarily for its role as an intermediate rather than for inherent activity.     

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.     

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.     

Regulation of 5-lipoxygenase activity by the glutathione status in human polymorphonuclear leukocytes

The influence of the glutathione status of human polymorphonuclear leukocytes (PMN) on 5-lipoxygenase activity was studied by treating cells with increasing concentrations of 1-chloro-2,4-dinitrobenzene (Dnp-Cl) or azodicarboxylic acid bis(dimethylamide) (Diamide). Subsequent incubation with arachidonate resulted in an up to tenfold-stimulated formation of 5-hydroxyeicosatetraenoic acid, leukotriene B4, leukotriene B4 isomers and omega-hydroxyleukotriene B4. Higher concentrations of the GSH reagents were inhibitory. At maximal stimulation by Dnp-Cl, 5-hydroperoxyeicosatetraenoic acid started to be built up at the expense of 5-HETE at glutathione levels which were diminished by about 50% compared to resting cells. No increase in cytosolic Ca2+ could be measured under these conditions by the fura-2 method. In PMN homogenates Dnp-Cl and Diamide were without effect and even caused inhibition when 5-lipoxygenase was stimulated by Ca2+ and ATP. 15-Lipoxygenase was either unchanged in the case of Diamide, or even increased after pretreatment with Dnp-Cl. The results allow us to conclude that 5-lipoxygenase activity in intact PMN is regulated not only by Ca2+ but in a complex manner also by the glutathione redox status. Conditions of oxidative stress increase the activity which may reflect the in vivo situation under phagocytosis and oxidative burst.     

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.     

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.     

Evidence that bacteria induce translocation of protein kinase C activity in human polymorphonuclear leukocytes

Particulate fraction associated protein kinase activity was studied in human polymorphonuclear leukocytes stimulated by bacteria. Staphylococcus aureus was found to increase particulate fraction associated protein kinase C activity in a time and concentration dependent manner. The increase comprised both the phospholipid dependent and independent kinase activity and was augmented by addition of serum. Similar observations were done using Staphylococcus epidermidis and Klebsiellae pneumoniae. However, Escherichia coli only increased the phospholipid independent kinase activity in the particulate fraction, which suggests the presence of protease activity.