Role of Ca2+ in activation of reactive oxygen species production in polymorphonuclear leukocytes during tumour growth in rats.

The role of Ca(2+) ions in PMA-induced generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) was studied during Zajdela hepatoma growth in the peritoneal cavity of rats. In PMNL from control healthy animals, a manifold Ca(2+)-induced enhancement of ROS generation and its significant reduction in the presence of Ca(2+) binding agent (BAPTA-AM) were observed. In contrast, ROS generation by PMNL from tumour-carrying animals dramatically increased in Ca(2+)-free medium, being practically insensitive to the agents, which can increase or decrease intracellular Ca(2+) levels. Free cytosolic Ca(2+) ([Ca(2+)](i)) in control PMNL was found to be relatively low ( approximately 250 nmol/L), rising slowly after Ca(2+) addition and further to two-fold in the presence of Ca(2+) and ionomycin in the incubating medium. Tumour growth in animals was accompanied with a significant [Ca(2+)](i) elevation. In Ca(2+)-free medium, [Ca(2+)](i) elevation was up to 480 nmol/L in tPMNL with the additions of Ca(+) and ionomycin as well as EGTA and ionomycin being able to increase [Ca(2+)](i) to 700-900 nmol/L onward. It was concluded that a higher Ca(2+) permeability of the plasma membrane and higher Ca(2+) accumulation in intracellular pools of PMNL was developed at the advanced stages of malignant disease. These results indicate the primed state of circulating PMNL and the independence of PMA-induced ROS generation at intra- and extracellular Ca(2+) levels at the advanced stages of tumour growth in animals.     

Effects of a nitric oxide donor, the substratum of NO-synthase, and an inhibitor of NO-synthase on the ROS-generation activity of phagocytes in the course of ascites tumor growth

Experiments in vitro were performed to investigate the effects of the nitric oxide donor (SNP), the substratum of NO-synthase (L-arginine), and the inhibitor of NO-synthase (nitroarginine) on the ROS-generating activity of blood plasma polymorphonuclear leucocytes and ascitic fluid macrophages isolated at different times of tumor (Zaidel hepatoma) growth in animal organism. It was found that in the initial period of tumor growth the nitric oxide donor at a concentration of 8 x 10(-5) M reduced the potential ROS-generating activity of macrophages by 38.5 +/- 9.0% and that of polymorphic-nuclear leucocytes of plasma by 27.6 +/- 7.0 %. However, the dynamics of this process during tumor growth was conservative: variations in the production of ROS by phagocytes were 10 +/- 3.0%. L-arginine induced a decrease in the ROS-generating activity of granulocytes and mononucleares by 25-30%. This fact points to an inducible inhibiting effect of NO-synthase on the ROS-generating activity of NADPH-oxidase in the course of tumor growth. The inhibitor of NO-synthase, nitroarginine, produced a monotonous increase in the ROS-generating activity of phagocytes isolated from the tumor at different periods of its growth. The use NO-synthase inhibitors for increasing ROS levels in the region of tumor growth may favor the suppression of tumor cell growth in vivo.     

Cytotoxic action of polymorphonuclear leukocytes on tumor and normal cells during ascite tumor development in vitro and in vivo

A vast number of studies, including the authors' own research, support the important role polymorphonuclear leukocytes (PMNL) in the development of ascite tumors. The method of luminol-dependent chemiluminescence (CL) was used to show the presence of two functionally different PMNL pools in a tumor-bearing organism: 1) "primed" PMNL, which circulate in the blood stream, and 2) "activated" PMNL, which are accumulated in the tumor zone and are capable of spontaneous CL. The purpose of the present investigation was to compare cytotoxic effects of primed and activated PMNL on tumor cells (ascite Ehrlich carcinoma (AEC), ascite Zajdel hepatoma) upon co-cultivation, as well as on normal cells of the organism, erythrocytes in vitro and in vivo. Upon stimulation with phorbol myristate acetate (PMA), PMNL effectively damaged AEC cells within the first 24 h until PMNL apoptosis occurred. Upon further co-cultivation, the tumor cells grew in number, which suggest the participation of PMNL in tumor protection. When stimulated with PMNL, pools suppressed tumor growth in vitro, since in this case the cytotoxicity was due to both reactive oxygen species and proteolytic enzymes. As it has been shown earlier by the authors, the functional potential of PMNL increases many times during tumor growth, and we suggested that not only tumor but also normal cells could be damaged. In this connection, we have studied the cytotoxic effect of primed and activated PMNL on rat erythrocytes in vitro on their co-cultivation. On stimulation with PMA, the rate of lysis of erythrocytes by primed PMNL increase many times compared to the norm. The fMLP-stimulated cytotoxity was 1.5-2.0 times higher than in the norm. Activated PMNL without stimulation are capable of producing only a partial lysis of erythrocytes (5-7 %). In order to assess the cytotoxic action of PMNL on erythrocytes in vivo, the hemoglobin content in erythrocytes and blood plasm of rats was measured in the course of tumor growth. The hemoglobin content in erythocytes during growth tumor decreased from 135 +/- 10 to 85 +/- 5 g/l, whereas in the blood plasm the hemoglobin content gradually increased by almost two times. The results enable us to suggest that one of death causes of tumor-bearing organisms may be the cytotoxic action of PMNL on normal cells of the organism caused by hyperproduction of ROS.     

Influence of nitric oxide donor,NO-synthase substrate,and inhibitor on leucocyte ROS-generating activity during ascites tumor growth

The influence of nitric oxide (NO) donor, NO-synthase substrate (L-arginine), and inhibitor (nitroarginine) on the reactive oxygen species (ROS)-generating activity of blood plasma polymorphonuclear leucocytes and ascitic fluid macrophages was studied during tumor growth in animal organisms. It was found that, in the initial period of tumor growth, 8 × 10⁻⁵ M sodium nitroprusside (SNP), which is an NO donor, reduced the potential ROS-generating activity of macrophages by 38.5 ± 9% and plasma polymorphicnuclear leucocytes by 27.6 ± 7%. However, the dynamics of this process during the tumor growth was conservative and variations in ROS production by phagocytes were 10 ± 3%. L-arginine induced a decrease in the ROS-generating activity of granulocytes and mononucleares by 25–30%. The results point to inducible inhibition effect of NO-synthase on the ROS-generating activity of NADPH-oxidase in the course of tumor growth. Nitroarginine, an inhibitor of NO-synthase, produced stable increase in the ROS-generating activity of phagocytes isolated from the tumor at different periods of its growth. The use NO-synthase inhibitors to increase the ROS level in the area of tumor growth may favor the suppression of tumor-cell growth in vivo.     

Monocyte-derived soluble protein confers 5-lipoxygenase activity Ca2+-dependent

5-Lipoxygenase (5-LO) is a Ca2+-stimulated enzyme that initializes the formation of proinflammatory leukotrienes from arachidonic acid (AA). In this report, we demonstrate that a soluble protein of the monocytic cell line Mono Mac 6 confers 5-LO activity Ca2+-dependent in vitro. Thus, in broken cell preparations of human polymorphonuclear leukocytes (PMNL) and rat basophilic leukemia (RBL)-1 cells, 5-LO converted AA (>20 microM) in the absence of Ca2+, whereas Ca2+ was absolutely required for 5-LO activity in broken cell preparations of MM6 cells. 5-LO partially purified from MM6 cells was substantially active in the absence of Ca2+. Recombination experiments revealed that the cytosolic fraction of MM6 cells contains a factor that suppresses the activity of partially purified 5-LO from PMNL, RBL-1, and MM6 cells in the absence but not in the presence of Ca2+. Further characterization showed that this factor is a 80-100 kDa heat-sensitive protein.     

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.     

Inhibitors of actin polymerisation stimulate arachidonic acid release and 5-lipoxygenase activation by upregulation of Ca2+ mobilisation in polymorphonuclear leukocytes involving Src family kinases

Here, we show that actin polymerisation inhibitors such as latrunculin B (LB), and to a minor extent also cytochalasin D (Cyt D), enhance the release of arachidonic acid (AA) as well as nuclear translocation of 5-lipoxygenase (5-LO) and 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL), challenged with thapsigargin (TG) or N-formyl-methionyl-leucyl-phenylalanine. The concentration-dependent effects of LB (EC50 approximately 200 nM) declined with prolonged preincubation (>3 min) prior TG and were barely detectable when PMNL were stimulated with Ca2+-ionophores. Investigation of the stimulatory mechanisms revealed that LB (or Cyt D) elicits Ca2+ mobilisation and potentiates stimulus-induced elevation of intracellular Ca2+, regardless of the nature of the stimulus. LB caused rapid but only moderate activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)2. The selective Src family kinase inhibitors PP2 and SU6656 blocked LB- or Cyt D-mediated Ca2+ mobilisation and suppressed the upregulatory effects on AA release and 5-LO product synthesis, without affecting AA metabolism evoked by ionophore alone. We conclude that in PMNL, inhibitors of actin polymerisation cause enhancement of intracellular Ca2+ levels through Src family kinase signaling, thereby facilitating stimulus-induced release of AA and 5-LO product formation.     

Correlation of quantitative human cytomegalovirus pp65-, p72- and p150-antigenemia, viremia and circulating endothelial giant cells with clinical symptoms and antiviral treatment in immunocompromised patients

Human cytomegalovirus (HCMV) replication in peripheral blood polymorphonuclear (PMNL) and mononuclear (MNL) leukocytes was investigated by quantitative determination of pp65-, p72- and p150-antigenemia and viremia in 7 (4 heart or heart-lung transplanted and 3 AIDS) immunosuppressed patients. These parameters were correlated with appearance of clinical symptoms and with their disappearance following antiviral treatment. Onset and progression of HCMV infection was associated to increasing levels of pp65-, p72- and p150-antigenemia and viremia, and a significant correlation was found between antigenemia and viremia in both PMNL and MNL. pp65-antigenemia showed absolute levels higher than p72- and p150-antigenemia both in PMNL and MNL, but PMNL showed figures consistently higher than MNL for all 3 viral proteins. levels of p150-antigenemia and viremia > 100 were associated to clinical symptoms in patients with peak of infection within 40 days after transplantation. In addition, number of HCMV-infected circulating giant cells (CGC) progressively increased in the presence of an organ syndrome. Antiviral treatment with either foscarnet or ganciclovir induced rapid disappearance of p150-positive PMNL and MNL as well as CGC, followed by disappearance of p72-positive leukocytes within a few days. pp65-positive cells were the last to disappear. Reported data suggest that viral replication may occur not only in MNL, but also in PMNL. Interaction between HCMV-infected circulating leukocytes and CGC may represent one of the major pathogenetic pathways for the development and dissemination of HCMV infection in immunocompromised patients.     

Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity

Continuous exposure of polymorphonuclear leukocytes (PMNLs) to circulatory hemodynamics points to fluid flow as a biophysical regulator of their activity. Specifically, fluid flow-derived shear stresses deactivate leukocytes via actions on the conformational activities of proteins on the cell surface. Because membrane properties affect activities of membrane-bound proteins, we hypothesized that changes in the physical properties of cell membranes influence PMNL sensitivity to fluid shear stress. For this purpose, we modified PMNL membranes and showed that the cellular mechanosensitivity to shear was impaired whether we increased, reduced, or disrupted the organization of cholesterol within the lipid bilayer. Notably, PMNLs with enriched membrane cholesterol exhibited attenuated pseudopod retraction responses to shear that were recovered by select concentrations of benzyl alcohol (a membrane fluidizer). In fact, PMNL responses to shear positively correlated (R(2) = 0.96; P < 0.0001) with cholesterol-related membrane fluidity. Moreover, in low-density lipoprotein receptor-deficient (LDLr(-/-)) mice fed a high-fat diet (a hypercholesterolemia model), PMNL shear-responses correlated (R(2) = 0.5; P < 0.01) with blood concentrations of unesterified (i.e., free) cholesterol. In this regard, the shear-responses of PMNLs gradually diminished and eventually reversed as free cholesterol levels in blood increased during 8 wk of the high-fat diet. Collectively, our results provided evidence that cholesterol is an important component of the PMNL mechanotransducing capacity and elevated membrane cholesterol impairs PMNL shear-responses at least partially through its impact on membrane fluidity. This cholesterol-linked perturbation may contribute to dysregulated PMNL activity (e.g., chronic inflammation) related to hypercholesterolemia and causal for cardiovascular pathologies (e.g., atherosclerosis).     

Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection

Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2-dependent oxidative product formation, the intracellular oxidation of 2',7'-dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely between patients (range 0 to 80%). This variable subpopulation may explain the variability of mean responsiveness of the PMNL of patients reported previously. Moreover, the marked increase in oxidative metabolic capability of the primed PMNL may be a significant component of the host response to acute infection. It could also contribute to the damage to host tissues such as pulmonary vascular endothelium during bacteremia.     

Effect of calcium on corticosteroid secretion by isolated frog interrenal gland

The direct effect of extracellular calcium concentrations on corticosteroidogenesis has been examined in the frog, using a perifusion system technique. The release of corticosterone and aldosterone in the effluent medium was monitored by specific radioimmunoassays. Increasing concentrations of Ca2+ (from 2 to 15 mM) gave rise to a dose-related stimulation of corticosteroid release, whereas the increment of either Na+ or K+ concentrations did not modify steroid production. Iterative administration of a moderate concentration of calcium (6 mM) led to a reproducible stimulation of steroid secretion whereas the same dose infused during 6 h induced a transient rise in corticosteroid secretion followed by a plateau. The direct effect of Ca2+ on steroidogenesis was confirmed by the dose-dependent stimulation of steroid secretion induced by the calcium ionophore A 23187. Perifusion with a calcium-free medium or blockade of Ca2+ channels by 4 mM Co2+ both resulted in a significant decrease in steroid production. Conversely, the administration of verapamil (up to 10(-4) M) did not affect steroidogenesis. These results provide evidence that extracellular calcium ions are required for basal production of corticosteroids in amphibians and that Ca2+ influx does not occur through voltage-dependent channels. Since, in the frog, blood Ca2+ concentrations vary in a rather large range, these results suggest that circulating Ca2+ levels may regulate corticosteroid production in these animals.     

Synergism between protein kinase C activator and fatty acids in stimulating superoxide anion production in guinea pig polymorphonuclear leukocytes

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with various fatty acids elicited superoxide anion (O2-) production and an increase in intracellular Ca2+ [( Ca2+]i). Both responses, however, were seldom observed when PMNL were treated at lower concentrations. But, simultaneous addition of 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, caused an increase in O2- production even at the lower concentrations of fatty acids. In contrast to the synergism in O2- production, [Ca2+]i remained at almost the basal level irrespective of the presence of OAG. Among saturated fatty acids, those with carbon numbers of 14 to 18 were most effective in stimulating O2- production in combination with OAG. Unsaturated fatty acids with a carbon number of 18 were almost equally effective irrespective of the number of double bonds.     

Metabolism of arachidonic acid in neutrophils from alloxan-diabetic rabbits

The production of 5-lipoxygenase products from arachidonic acid was investigated in polymorphonuclear leukocytes (PMNL) isolated from non-diabetic and alloxan-induced diabetic rabbits: (i) production of 5-hydroxyeicosatetraenoic acid, leukotriene B4, and the two 6-trans-leukotriene B4 isomers were significantly decreased in the PMNL of diabetic rabbits when compared to non-diabetic rabbits; (ii) production of LTB4 and 5-HETE from diabetic PMNL required the addition of Ca2+ and A23187 to a greater degree than control incubations; and (iii) the availability of substrate in the PMNL of diabetics was not a limiting factor for 5-lipoxygenase product formation. Alternative pathways of arachidonic acid metabolism were also evaluated: the recovery of exogenous leukotriene B4 and 5-hydroxyeicosatetraenoic acid were identical using PMNL from control and diabetic rabbits and peptido-leukotrienes were not detected by radioimmunoassay. The data suggest that the activity of 5-lipoxygenase and the production of 5-hydroperoxyeicosatetraenoic acid in the diabetic PMNL may be limiting factors since the formation of leukotriene B4, leukotriene B4 isomers, and 5-hydroxyeicosatetraenoic acid are depressed in PMNL of diabetic rabbits. Alternative pathways do not account for the conversion of arachidonic acid to other products nor are the elimination pathways for LTB4 and 5-HETE different. Decreased formation of 5-hydroxyeicosatetraenoic acid and leukotriene B4 could predispose diabetic subjects to infection due to a decrease in mediators leading to the local accumulation of PMNL in the inflammatory response.     

Initiation of peroxidation of liposome membrane lipids by activated polymorphonuclear leukocytes

The influence of active oxygen forms produced by zymosan-stimulated polymorphonuclear leukocytes (PMNL) on the initiation of liposome lipid peroxidation has been studied. It has been shown, by measuring the concentration of TBA-active products, that lipid peroxidation induced by PMNL stimulation occurs only in the presence of Fe-ADP. This fact demonstrates that OH'-radicals are responsible for the initiation of lipid peroxidation. Superoxide dismutase and catalase almost completely inhibited PMNL-stimulated peroxidation. The results obtained suggest that active oxygen forms that appear during PMNL stimulation can migrate at a considerable distance from the place of their origin, initiating peroxidation of cell membrane lipids and lipoproteins in the presence of Fe ions, which seems to underlie bacteriocidal and cytotoxic action of phagocytes.     

Calcium-independent induction of cytocidal activity of polymorphonuclear leukocytes by phorbol myristate acetate-like tumor promoters

Tumor promoters were tested for the ability to induce cytocidal activity of polymorphonuclear leukocytes (PMNs), and the extracellular calcium-dependency of their PMN cytotoxicities were examined in comparison with that by some immunomodulators. Immunomodulators such as linear beta-1, 3-D-glucan (TAK) induced potent cytocidal activity of PMNs. The induction was dependent on extracellular Ca2+. Tumor promoters such as phorbol 12-myristate 13-acetate (PMA) and its derivatives, teleocidin which is structurally unrelated to PMA, and croton oil as an example of mixture also induced potent PMN cytotoxicities. In the latter cases, however, the induction was not dependent on extracellular Ca2+. The ability of these tumor promoters to induce PMN cytotoxicity correlated well with their skin-tumor promoting activities. These results indicate that inductions by PMA-like tumor promoters are distinguishable from those by TAK-like immunomodulators in not being Ca2+-dependent. The application of Ca2+-independent PMN cytotoxicity to detect PMA-like tumor promoters is discussed.     

Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein

Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.     

Induction of 5-lipoxygenase activation in polymorphonuclear leukocytes by 1-oleoyl-2-acetylglycerol

1,2-Diacylglycerols (DAGs) can prime polymorphonuclear leukocytes (PMNL) for enhanced release of arachidonic acid (AA) and generation of 5-lipoxygenase (5-LO) products upon subsequent agonist stimulation. Here, we demonstrate that in isolated human PMNL, 1-oleoyl-2-acetylglycerol (OAG) functions as a direct agonist stimulating 5-LO product formation (up to 42-fold). OAG caused no release of endogenous AA, but in the presence of exogenous AA, the magnitude of 5-LO product synthesis induced by OAG was comparable to that obtained with the Ca(2+)-ionophore A23187. Interestingly, OAG-induced 5-LO product synthesis was not connected with increased 5-LO nuclear membrane association. Examination of diverse glycerides revealed that the sn-2-acetyl-group is important, thus, also 1-O-hexadecyl-2-acetylglycerol (EAG) stimulated 5-LO product formation (up to 8-fold).Treatment of PMNL with OAG did not alter the mobilization of Ca(2+) but removal of intracellular Ca(2+) abolished the upregulatory OAG effects. Notably, the PKC activator phorbol-myristate-acetate hardly increased 5-LO product synthesis and PKC inhibitors failed to suppress the effects of OAG. Although OAG rapidly activated p38 MAPK and p42/44(MAPK), which can stimulate 5-LO for product synthesis, specific inhibitors of these kinases could not prevent 5-LO activation by OAG. Together, OAG acts as a direct agonist for 5-LO product synthesis in PMNL stimulating 5-LO by novel undefined mechanisms.     

Partial dependence of human peripheral blood leukocyte binding to high molecular weight fucoidan on divalent cations

L-selectin (CD62L) is the principal leukocyte adhesion molecule for the high endothelial venules of peripheral lymph nodes. This adhesion has an absolute requirement for calcium ions. Nevertheless, some studies have shown carbohydrate adhesion receptor interactions on lymphocytes and neutrophils, including the L-selectin molecule, that are Ca-independent. In the present study fucoidan, a reportedly Ca2+ independent ligand of L-selectin, and Mabs to human CD62L were coupled to magnetic polystyrene beads (MPB), as a model of leukocyte-surface interactions, and the efficiency of human leukocyte separation was investigated. 30% of Ficoll-purified human mononuclear cells and 75% of dextran-purified human leukocytes (DPHL) were specifically bound by fucoidan-modified MPB in the presence of Ca2+; 55% of dextran-purified leukocytes were specifically bound in the absence of Ca2+. The specific binding was inhibited by an excess of free fucoidan. The data obtained show the presence of Ca-independent adhesion determinants, specific to fucoidan on human leukocytes. No significant specific binding of leukocytes to fucoidan-modified MPB was found after the incubation with fresh human Ca(2+)-depleted whole blood. More than 90% of DPHL were specifically bound to MPB modified with Mabs to human CD62L irrespective of Ca2+ presence. The same degree of separation was achieved after the incubation with fresh human Ca(2+)-depleted-whole blood with anti-CD62L modified beads.     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (Tc) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB4 can induce human Tc to exert suppressor cell activity, and incubation of lymphocytes with LTB4 for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40-90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O2 X-) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 +/- AA for icosanoid release; phorbol myristate acetate for O2 X-production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca2+-dependent K+permeability in PMNL was measured by the cell K+ release induced by A 23187. Tc and Tc subsets were determined using monoclonal antibodies (OKT3+, OKT4+ and OKT8+). A biphasic sequential release of the different substances (leukocytic icosanoids and O2 X-) was monitored: increase (approximately 36-48 h after Tl) and decrease (greater than or equal to 72 h after Tl). The increase in AA stimulation was more transient than that of O2 X -. The decline in the release of AA metabolites and O2 X-production was associated with the anergic phase (decrease OKT4+/OKT8+ ratio) and with the clinical outcome of the patients. The decrease in LTB4 and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca2+-dependent K+ permeability increased early up to 2 or 3 times normal. In order to go further with the mechanism of inhibition of LTB4 and O X-release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB4 secretion. A less important inhibition was observed with O2 X-release (-32%) and Ca2+-dependent K+ permeability (-30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): "suppressive factor(s) of membrane activation "SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromatography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O X 2-release.     

Effect of glutaraldehyde on NADPH oxidase system of guinea pig polymorphonuclear leukocytes

In an attempt to elucidate properties and activation mechanisms of the NADPH oxidase system, which is known to be responsible for the production of superoxide anion (O2-) in cell membranes of polymorphonuclear leukocytes (PMNL), intact guinea pig PMNL were treated with glutaraldehyde, a protein crosslinking reagent, before or after stimulation with phorbol 12-myristate 13-acetate (PMA). Then, PMNL were disrupted and NADPH oxidase activity was measured. After the treatment of resting PMNL with glutaraldehyde, NADPH oxidase was no longer activated by PMA. On the other hand, the NADPH oxidase activity enhanced by PMA in advance was markedly retained by the glutaraldehyde treatment of such PMA-stimulated PMNL as compared to that in untreated cells. Similar retention by glutaraldehyde of the stimulated NADPH oxidase activity was observed in PMNL stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP) and cytochalasin D. Furthermore, the oxidase activity of glutaraldehyde-treated PMNL was stable during incubation at 37 degrees C, the half life of the oxidase activity of the treated PMNL being more than 90 min whereas that of the untreated PMNL is about 15 min. This ability of the glutaraldehyde treatment to retain the activity was also observed against inactivation by high concentrations of NaCl and by positively charged alkylamine.