Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function

During periods of ischemia and vascular injury, factors are released which recruit monocytes and polymorphonuclear leukocytes (PMNs) to the site of injury by promoting adherence to the endothelium and transmigration across the endothelial cell (EC) layer. During coronary artery stenosis, we have shown that the endothelium-derived, cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are elevated. Therefore, we examined if the EETs could stimulate PMN adherence to cultured ECs. Pretreatment of ECs with EETs for either 30 min or 4 hr did not alter the adherence of 51Cr-labelled PMNs to ECs while phorbol myristate acetate (PMA) produced a 4-fold increase in PMN adherence. The combination of EETs and PMA did not significantly augment or diminish PMA-induced PMN adherence to ECs. When ECs and 51Cr-labelled PMNs were coincubated, treatment with EETs alone did not alter PMN adherence. However, when EETs and PMA were added together during the coincubation of ECs and 51Cr-labelled PMNs, the EETs produced a concentration-related decrease in PMN adherence. Microscopic analysis of the culture media bathing the cells revealed aggregates of the labeled PMNs. We examined the effects of the EETs on PMN aggregation. 8,9-EET (10, 50, and 100 microM) increased PMN aggregation (7 +/- 3, 35 +/- 10, and 65 +/- 11%) and intracellular calcium by 1.7 +/- 0.5, 4.7 +/- 1.4, and 6.8 +/- 2.3-fold above basal. 5,6-, 11,2- and 14,15-EETs also stimulated aggregation. FMLP stimulated the production of superoxide; however, 8,9-EET did not. These observations indicate that the decrease in PMN adherence observed in the coincubation experiment is the result of EET-induced PMN aggregation. Given the increase in EET production during coronary artery stenosis, these data may provide insight into their potential biological significance during myocardial ischemia and vascular injury.     

Protease inhibitors antagonize the activation of polymorphonuclear leukocyte oxygen consumption.

We report that the burst of oxygen consumption, as well as the resultant production of O₂^?? and H₂O₂, occurring in activated human polymorphonuclear leukocytes is inhibited by various compounds which have in common the ability to antagonize the effects of proteolytic enzymes. This effect of protease inhibitors was observed with a variety of stimuli, both phagocytic and non-phagocytic, used to activate O₂^?? production in human polymorphonuclear leukocytes. Inhibition was also noted in rat polymorphonuclear leukocytes and alveolar macrophages. The results indicate that proteolysis may be involved in activating the burst of oxygen consumption following stimulation of phagocytic cells.     

Effects of 60 Hz magnetic field exposure on polymorphonuclear leukocyte activation

We have investigated the effects of a sinusoidal 60 Hz magnetic field on free radical (superoxide anion) production, degranulation (beta-glucuronidase and lysozyme release) and viability in human neutrophils (PMNs). Experiments were performed blindly in very controlled conditions to examine the effects of a magnetic field in resting PMNs and in PMNs stimulated with a tumor promoter: phorbol 12-myristate 13-acetate (PMA). Exposure of unstimulated human PMNs to a 60 Hz magnetic field did not affect the functions examined. In contrast, exposure of PMNs to a 22 milliTesla (mT), 60 Hz magnetic field induced significant increases in superoxide anion (O2-) production (26.5%) and in beta-glucuronidase release (53%) when the cells were incubated with a suboptimal stimulating dose of PMA. Release of lysozyme and lactate dehydrogenase was unchanged by the magnetic field, whether the cells were stimulated or not. A 60 Hz magnetic field did not have any effect on O2- generation by a cell-free system xanthine/xanthine oxidase, suggesting that a magnetic field could upregulate common cellular events (signal transduction) leading to O2- generation and beta-glucuronidase release. In conclusion, exposure of PMNs to a 22 mT, 60 Hz magnetic field potentiates the effect of PMA on O2- generation and beta-glucuronidase release. This effect could be the result of an alteration in the intracellular signaling.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Abstract Porphyromonas gingivalis culture supernate was found to induce hemotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of aglutination induced by the secretagogues PMA and FMLP. Lipopolysaccaharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors of PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity fot P. gingivalis cells but when PMN were treated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutinatin or enhanced adherence could also lead to decreased phagocytic capacity of the adherant or agglutinated cells.     

Effects of Porphyromonas gingivalis culture products on human polymorphonuclear leukocyte function

Porphyromonas gingivalis culture supernate was found to induce homotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of agglutination induced by the secretagogues PMA and FMLP. Lipopolysaccharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors on PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity for P. gingivalis cells but when PMN were pretreated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutination or enhanced adherence could also lead to decreased phagocytic capacity of the adherent or agglutinated cells.     

Effects of various inhibitors of arachidonic acid oxygenation on macrophage superoxide release and tumoricidal activity

Macrophages release a variety of arachidonic acid metabolites after treatment with various membrane triggers or particulate stimuli. We examined the role of phospholipase and lipoxygenase inhibitors in the modulation of superoxide production and tumor cytolysis by murine macrophages. Superoxide was induced by the soluble stimulus, phorbol myristate acetate (PMA), and the particulate stimulus, opsonized zymosan, and was measured by the reduction of ferricytochrome c with the use of a micro ELISA reader. Macrophage-mediated tumor cytolysis was induced by hybridoma-derived, macrophage-activating factor (MAF) and was quantitated by 51Cr release from P815 target cells. In both assays, 72-hr peptone-elicited macrophages were used. Dexamethasone, and to a lesser degree hydrocortisone, inhibited superoxide release and MAF-induced tumor cytolysis. Inhibition in the superoxide assay required pretreatment with corticosteroid. Only the gold compound, auranofin, inhibited superoxide when given simultaneously with stimulant. Other phospholipase inhibitors, including mepacrine and 4-bromophenacyl bromide, and several lipoxygenase inhibitors, including BW755c, nordihydroguaiaretic acid (NDGA), and 5,8,11,14-eicosatetraynoic acid (ETYA), failed to modulate either macrophage response at nontoxic concentrations. At the concentrations tested in the tumoricidal and superoxide assays, mepacrine and 4-bromophenacyl bromide inhibited the release of 14C-arachidonic acid from macrophages stimulated with opsonized zymosan. Our data strongly suggest that corticosteroids suppress macrophage superoxide production and tumoricidal function by a nonphospholipase-dependent mechanism.     

Induction of polymorphonuclear leukocyte response by human cytomegalovirus

Neutrophils are important in the defense against bacterial infections, by ingesting and killing invading microorganisms. Because of the higher incidence of bacterial infections in patients with active human cytomegalovirus (HCMV) infections, we hypothesized that HCMV-infected neutrophils were inefficient in eliminating the bacteria. Therefore, we mock infected or infected neutrophils with HCMV by contact with HCMV-infected human pulmonary artery endothelial cells. We found that HCMV infection without N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation increased the surface expression of CD11b to the same extent as fMLP stimulation of mock infected cells. Also, HCMV-infected neutrophils became more efficient in phagocytosing serum opsonized yeast particles than mock infected cells. Furthermore, we observed an increase in intracellular free calcium and chemiluminescence in HCMV-infected cells, in response to fMLP compared to fMLP-treated mock cells. We also found that apoptosis was significantly inhibited in HCMV-infected neutrophils. In conclusion, our results suggest that neutrophils become more effective in performing their effector functions when infected with HCMV. Thus, the higher incidence of bacterial infections in HCMV patients might not be due directly to a dysfunction in the neutrophils. Instead, the fact that apoptosis is inhibited may cause over-reactive neutrophils to remain in the tissues, where they will start leaking their contents, damaging the tissues and contributing to inflammatory processes.     

Protection of crystal-induced polymorphonuclear leukocyte membranolysis by phosphocitrate

The protection afforded by phosphocitrate, a phosphorylated polycarboxylic acid, against crystal-induced membrane damage to polymorphonuclear leukocytes was studied in vitro. Membranolysis was assessed by nitro blue tetrazolium salt reduction, lactate dehydrogenase release, and scanning electron microscopy. Phosphocitrate protected strongly against hydroxyapatite crystal-induced damage, an action attributable to crystal surface binding of phosphocitrate rather than to the membrane. The ability of phosphocitrate to prevent hydroxyapatite crystallization, together with its membrane protective effect against preformed crystals, would suggest that the compound might have a useful future role against crystal-induced arthropathies.     

Modulation of polymorphonuclear leukocyte locomotion by synthetic amphiphiles

The capacity of synthetic amphiphiles, poly(ethyleneglycol) 6000 (PEG) esterified with saturated fatty acids (C₂–C₁₈), to modify polymorphonuclear leukocyte (PMNL) locomotion has been investigated. It was noticed that PEG-myristate (M-PEG; C₁₄) stimulated the random locomotion of PMNL populations in concentrations up to about 1 g/L. The esters with shorter aliphatic chains had negligible effects, whereas those with longer chains, PEG-palmitate (P-PEG; C₁₆) and PEG-stearate (S-PEG; C₁₈) reduced the locomotion, irrespectively of concentration. The ability of the PMNL to be stimulated by an attractant liberated from normal human serum was slightly impaired by M-PEG, but not by P-PEG. The response to M-PEG of individual PMNL was heterogeneous in that some cells were stimulated and others were inhibited. However, the average result was a reduction of the motility. This indicates that methods used for the study of the locomotion of cell populations may not always reflect the average behavior of the whole population. It was also concluded that the different effects of M-PEG and P-PEG owed to dissimilar effects on the membrane structure of the PMNL since (1) M-PEG perturbated the PMNL membrane more than P-PEG, as assayed by the release of superoxide anion (0 ₂ ⁻ , although the binding was smaller, and (2) M-PEG and P-PEG increased and decreased the membrane fluidity, respectively, as measured with fluorescent bleaching and recovery after bleaching of labeled PMNL. The results indicate a subtle coupling between membrane structure and PMNL locomotion.     

Inhibition of polymorphonuclear leukocyte functions by fluorinated nitrobenzenes

The bifunctional fluorinated nitrobenzenes, 1,5-difluoro-2,4-dinitrobenzene (DFDNB) and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (DFDNDPS), and the monofunctional 1-fluoro-2,4-dinitrobenzene (FDNB) inhibit chemotaxis, phagocytosis, exocytosis and the respiratory burst of rabbit polymorphonuclear leukocytes. Inhibition occurs in the micromolar concentration range; the bifunctional compounds are stronger inhibitory than the monofunctional one. The inhibitory effect can be counteracted by sulfhydryl compounds and not with amino-group containing compounds. The results suggest that an interaction with vulnerable sulfhydryl groups, located in a hydrophobic surrounding, is the basis of the inhibitory effect of the fluorinated nitrobenzenes.     

Modulation of rat polymorphonuclear leukocyte 5-lipoxygenase activity by 5-HPETE and NADH-dependent flavin inhibition

The effect of nicotinamide and flavin coenzymes on the 5-lipoxygenase activity has been determined in cell-free extracts from rat polymorphonuclear leukocytes. 5-lipoxygenase was assayed in the presence of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which caused a 3 to 4-fold stimulation in the maximal conversion of radiolabeled arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) and 5,12-dihydroxyeicosatetraenoic acid (5,12-di-HETE). Addition of FMN or FAD to the assay mixture had little effect on the 5-lipoxygenase activity and caused inhibition only at high concentrations (IC50 greater than 100 microM). NADH markedly potentiated the inhibition of lipoxygenase by flavins with a 100-fold decrease in the FMN concentration required to inhibit the enzyme (IC50 approximately equal to 2 microM). Similar effects were observed for FAD although this flavin derivative was slightly less potent than FMN (IC50 congruent to 10 microM). NADH could be substituted by NADPH but not by NAD or NADP, indicating that the inhibition was not due to the production of the oxidized forms of these co-factors. These results show that the 5-lipoxygenase activity is stimulated by 5-HPETE and inhibited by flavin-dependent redox transformations.     

Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.     

Impairment of human polymorphonuclear leukocyte chemotaxis by 2,5-hexanedione

The effect of n-hexane metabolites on human polymorphonuclear leukocyte chemotaxis and luminol-dependent chemiluminescence was investigated. No effect was detected when 2-hexanol, 2-hexanone and -valerolactone were used, 2,5-hexanedione at 75 g/ml inhibited chemotaxis and a direct correlation between increasing the xenobiotic concentration and the degree of inhibition was found. Chemotactic peptide-induced chemiluminescence was not affected by 2,5-hexanedione. In order to clarify the phenomenon, plasma membrane fluidity was investigated by fluorescence polarization of the fluorescent probe trimethylammonium diphenylhexatriene. 2,5-hexanedione increased the membrane fluidity, while the other n-hexane metabolites did not change the degree of flourescence polarization. Results suggest that the cellular functions modulated by membrane-cytoskeletal organization are affected by 2,5-hexanedione also at the low concentrations.Abbreviations 2,5-Hxdn 2,5-hexanedione - 2-Hxl 2-hexanol - 2-Hxn 2-hexanone - PMN polymorphonuclear leukocyte - TMA DPH trimethylammonium diphenylhexatriene - -V1 -valerolactone     

Oxygenation of phosphatidylcholine by human polymorphonuclear leukocyte 15-lipoxygenase

A cell-free human polymorphonuclear leukocyte preparation containing both 15- and 5-lipoxygenase activities was found to oxygenate phosphatidylcholine at carbon-15 of the arachidonic acid moiety. No oxygenation at carbon-5 was found. Under similar incubation conditions, soybean and rabbit reticulocyte 15-lipoxygenases also oxygenated phosphatidylcholine, whereas rat basophilic leukemia cell 5-lipoxygenase, rabbit platelet 12-lipoxygenase and rat liver cytochrome P-450 preparations did not. Our results suggest that the oxygenation of phospholipids may be a unique property of the 15-lipoxygenases.     

Experimental study on filtrability of polymorphonuclear leukocyte suspensions

Filtrability of a suspension of polymorphonuclear leukocytes (PMNs) was examined in a Nuclepore membrane filtration system utilizing a gradually reduced pressure difference with or without an additional negative pressure. The filtration process was continuously recorded using a TV-video system for data analysis. The PMN content in the filtrate was directly measured. The pressure-flow relation was analyzed in terms of the relative resistance of the PMN suspension to that of the suspending medium. The relative resistance of the PMN suspension increased with an increase in the filtered volume until it approached infinity at the level of low pressure difference (2.8 - 0 cmH2O). The remarkable increase in flow resistance was closely associated with the plugging of PMNs in the membrane pores. At high pressure differences (12.8 - 10 cmH2O, 7.8 - 5 cmH2O), the relative resistance increased up to finite values, as the filtered volume increased. The variation in the relative resistance was greatly dependent upon the pressure difference or the flow condition. The amount of filtered cell fraction increased with an increase of additional pressure, indicating that the relative resistance was changed according to the rate of PMN plugging and dislodging in the pores of the membrane.     

Alterations of polymorphonuclear leukocyte surface charge by stimulated lymphocyte supernatants

The effects of human lymphokines on the surface charge density of human polymorphonuclear (PMN) leukocytes have been determined using the laser Doppler technique of electrophoretic light scattering. Unfractionated antigen (streptokinase-streptodornase or candida)-stimulated lymphocyte supernatants were found to decrease the mode electrophoretic mobility by 14%. In order to identify the responsible factor, we subjected supernatants from concanavalin A-stimulated lymphocytes to gel filtration on Sephadex G-100 columns and assayed the fractions for their ability to alter PMN electrophoretic mobilities. Two distinct species in the molecular weight ranges of 30–60K and 10–20K, respectively, were found to decrease the electrophoretic mobilities of PMN leukocytes. We have observed no effect of leukocyte inhibitory factor (LIF) on the electrophoretic mobility distribution of PMN leukocytes over a varying period of time (0–8 hr) and over a range of 2- to 10-fold supernatant concentration. Pretreatment of PMN leukocytes with neuraminidase substantially reduced their electrophoretic mobility; the addition of LIF to these pretreated cells did not alter their electrophoretic mobility distribution further. The latter finding is particularly significant in view of the fact that neuraminidase pretreatment of the target cells is known to potentiate LIF activity. We conclude that the mechanism of the inhibition of leukocyte migration by LIF does not involve an alteration of the leukocyte surface charge density.     

Inhibition of polymorphonuclear leukocyte oxidative metabolism by exogenous phospholipase C

We studied the effects of exogenous, purified phospholipase C (PLC) on neutrophil oxidative metabolism, lysosomal enzyme release and aggregation. We found that PLC inhibited O2- and H2O2 generation and oxygen consumption, but did not alter glucose oxidation via the hexose monophosphate shunt. In contrast, we found a striking stimulation of aggregation and release of the lysosomal enzymes lysozyme and beta-glucuronidase. In experiments designed to further characterize the mechanism of the PLC effect on membrane activation we studied the effect of PLC on intracellular calcium concentration [Ca2+]i and found that PLC did not interfere with the fMLP-mediated rise in [Ca2+]i, suggesting that its inhibitory effect on the respiratory burst does not involve inhibition of early signal transduction events. In addition, we found that PLC alone results in mobilization of intracellular Ca2+ stores, consistent with its stimulatory effect on aggregation and lysosomal enzyme release.     

Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor.

Incubation of human polymorphonuclear leukocytes with colchicine and fluorescein-concanavalin A leads to the formation of a polarized cap of fluorescence not seen if cells are incubated with fluorescein-Con A along. When cells are preincubated with a chemotactic factor before colchicine treatment, the capping is inhibited in a dose-related manner. Studies with alpha-methylmannoside indicate that the caps represent extracellular fluorescein-Con A and are not areas of Con A internalization. Experiments utilizing an irreversible inhibitor of serine esterases suggest that a chemotactic factor-activated enzyme is involved in the inhibition of cap formation in the human neutrophil.     

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.     

Effects of oxygen radicals on the conformation of sulfhydryl groups on human polymorphonuclear leukocyte membranes.

The healthy intact polymorphonuclear leukocytes (PMNs) were labeled with 4-maleimide-TEMPO spin labeling compound (MAL) to study the effects of oxygen radicals produced by phorbol myristate acetate (PMA)-stimulated PMNs on the conformation of sulfhydryl (SH) groups of PMN membrane proteins. The lipid peroxidation induced by PMA-stimulated PMNs was detected by evaluating the formation of malonaldehyde (MDA) with the thiobarbituric acid (TBA) test. From the experiments of luminol-dependent chemiluminescence (CL) and fluorometry, it was found that Chinese herbs schizandrin B (Sin B) and quercetin (Q) possessed scavenging properties for oxygen radicals produced during the PMN respiratory burst. These two herbs can also inhibit the conformation changes in SH binding sites on the PMN membrane proteins caused by oxygen radicals produced by the PMNs themselves. They also decreased the amount of MDA, which was a final product formed during lipid peroxidation.