Chemiluminescence response of beta-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with beta-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O-2 dismutation to H(2)O(2), supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca(++) pump channels and calmodulin are involved in a metabolic energy-dependent pathway.     

Chemiluminescence response of β-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with β-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O−2 dismutation to H₂O₂, supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca⁺⁺ pump channels and calmodulin are involved in a metabolic energy-dependent pathway.     

Attenuation of glyceraldehyde-3-phosphate dehydrogenase activity and ATP levels in rat brain synaptosomes by acrylamide

The effect of neurotoxin acrylamide (AC) on energy metabolism has been studied in a purified preparation of the synaptosomes. The synaptosomes were prepared by the flotation technique in a discontinuous Ficoll/sucrose gradient. The purity of the synaptosomes was checked by electron microscopy and by assaying the activity of marker enzymes. By these criterias, free mitochondrial contamination in the synaptosomes was found to be >2%. Incubation of the synaptosomes with different concentrations of AC (2.5, 5.0, and 10mM) produced a concentration-dependent inhibition (15, 35, and 60%, respectively) of glyceraldehyde-3-phosphate dehydrogenase activity. Acrylamide also produced a time-dependent decrease of ATP concentrations in the synaptosomes; about 25% loss of ATP was seen within 1h, while about 60% ATP was lost after 120 min incubation with 10 mM AC. The effect of known inhibitors of glycolysis-iodoacetic acid (IAA), and of oxidative phophorylation-rotenone and antimycin A, was also studied on ATP synthesis by the synaptosomes. IAA was found to be the most potent inhibitor of ATP synthesis, while both rotenone and antimycin A were equally effective in blocking ATP synthesis in the synaptosomes. These studies show that the synaptosome might be used as a suitablein vitro model to study the effect of neurotoxin such as AC on neuronal energy metabolism.Special issue dedicated to Dr. Sidney Ochs.     

Spontaneous cytotoxic activity of eosinophilic granule cells separated from the normal peritoneal cavity of Dicentrarchus labrax

In this study the spontaneous in vitro cytotoxic activity to tumour cell lines, (K562), by unstimulated sea bass (Dicentrarchus labrax) leukocytes was examined by trypan blue exclusion test and lactate dehydrogenase release assay. A high anti-tumour cell line activity of resident peritoneal leukocytes was found at an effector to target ratio (E:T) of 25:1 after incubation for 2 h at 18 degrees C. Rabbit and sheep erythrocytes were not lysed. A low activity was displayed by head kidney and spleen cell populations whereas blood leukocytes revealed no significant activity. The effect of E:T ratio on cytotoxicity as well as microscopy observations suggested that the cytotoxic reaction required effector-target cell contact. Eosinophilic granule cells, isolated on a Percoll density gradient from a peritoneal wash, appeared to be responsible for the in vitro cytotoxic activity.     

The latex particle adherence (LPA) assay for detection of leukocytes with adhesive surface properties.

A new, simple, and rapid in vitro assay has been developed for identification of adherent and nonadherent leukocytes. The assay is based on adherence of latex (polystyrene) particles to the cell surface. Using the latex particle adherence (LPA) assay, the percentage of adhesive leukocytes has been determined in human peripheral blood mononuclear preparations and in the lymph nodes, thymus, bursa of Fabricius, spleen, and bone marrow of mouse, chicken, and rat origin. The highest proportion of LPA-positive cells was found in peritoneal exudate, bone marrow, and spleen, the lowest proportion, in thymus and bursa of Fabricius. LPA-Positive cells in human peripheral blood mononuclear preparations were identified as surface immunoglobulin-positive lymphocytes nonrosetting with sheep red blood cells. LPA-Positive cells in peritoneal exudate were identified as macrophages. Incubation of leukocyte suspensions on polystyrene petri dishes or nylon wool columns reduces substantially the percentage of LPA-positive cells in the nonadherent fraction. The LPA assay seems to be a method of choice for establishing the relationship between adhesiveness of the cell surface and other cell membrane markers on a single-cell level.     

DYNAMICS OF LEUKOCYTE MIGRATION INTO THE MOUSE ASCITES TUMOR

There is a marked increase in the number of peritoneal leukocytes (lymphocytes, monocytes and granulocytes) during the growth of Ehrlich ascites tumor in mice. No local proliferation (as indicated by a labeling at 1 hr following a single ³H-TdR injection) was observed in the normal peritoneal leukocytes or those in the ascites tumor, except for a very minor labeling of some tumor macrophages. Kinetics of peritoneal leukocytes was studied with a series of twelve injections of ³H-thymidine (20 μCi every 8 hr) in normal mice as well as mice injected with 10⁶ tumor cells i.p. 2 hr after the last ³H-TdR injection. Animals were sacrificed at intervals up to 6 days. Granulocyte labeling in the blood as well as peritoneal space was near 100% in both groups of animals at all the intervals. Temporal changes in the labeling of lymphocytes (from 10% at 0 day to 22% at day 6), and monocytes (from 20% at 0 day to 57% at day 6) were identical in the blood and peritoneal space of normal animals, indicating a free exchange of cells between these compartments. Higher labeling indices than those in the controls were attained in the blood of tumor-bearing hosts (viz 40% for lymphocytes and 80% for monocytes at 6 days) suggesting an increased turnover of these cells in the circulation. In addition, peritoneal mononuclear cells of tumor-bearing mice showed even a higher labeling than those in the blood (viz 65% for lymphocytes and 92% for monocytes at 6 days) indicating a selective migration and/or retention of newly formed cells within the tumor, in contrast to a random migration into the normal peritoneal cavity. Furthermore, an identical labeling of macrophages to that of monocytes within the tumor indicated a short monocyte-macrophage transition. The preferential accumulation of young mononuclear cells into the tumor may be of functional importance.     

Cell-type specificity of l-leucyl l-leucine methyl ester

l-Leucyl l-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent which is converted to a membranolytic compound by dipeptidyl peptidase I and kills human leukocytes such as CD8+ T cells and monocytes but not B cells. The reagent has also been used in mice on the assumption that the cell-type specificity to murine leukocytes is the same as that to human leukocytes. During study on the effect of LeuLeuOMe on antigen-driven IL-2 production using murine splenocytes as antigen-presenting cells, however, we noticed that murine B cells were sensitive to LeuLeuOMe. We therefore examined the cell-type specificity using murine splenocytes and peritoneal macrophages. Flow cytometric analysis revealed that the most sensitive cells to LeuLeuOMe were CD8+ cells and that CD19+ cells (B cells) were as sensitive as CD3+ cells (T cells). Murine splenic B cells, which were either positively or negatively sorted with a cell sorter, were also sensitive to LeuLeuOMe, whereas human peripheral blood B cells, which were positively sorted, were not. Peritoneal macrophages were the most insensitive to LeuLeuOMe. Thus, this study demonstrated that the cell-type specificity to murine leukocytes is different from that to human leukocytes.     

Dietary alpha-linked galacto-oligosaccharide suppresses ovalbumin-induced allergic peritonitis in BALB/c mice

To determine whether alpha-linked galacto-oligosaccharide (alpha-GOS) prevents allergic peritonitis, BALB/c mice were fed a synthetic diet with and without alpha-GOS supplementation for 7 d, and were then subcutaneously immunized with ovalbumin on days 0 and 7. The mice were challenged by intraperitoneal injection with ovalbumin on day 14, followed by peritoneal lavage on day 15. The total number of peritoneal exudate cells was significantly lower in the mice fed the alpha-GOS diet than in those fed the control diet. Peritoneal lavage fluid from mice fed the alpha-GOS diet not only had less potency to attract peripheral blood leukocytes and peritoneal exudate cells ex vivo, but also had lower concentrations of monocyte chemoattractant protein-1 (MCP-1) and eotaxin. Preincubation of the cells with alpha-GOS failed to affect the migration to peritoneal lavage fluid. We propose that dietary alpha-GOS reduces cell infiltration in allergic peritonitis by reducing antigen-induced elicitation of MCP-1 and eotaxin in mice.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

ATP level in synaptosomes--a determining factor of the functional activity of ion channels induced by alpha-latrotoxin

The paper deals with characteristics of relationship between synaptosomal calcium permeability induced by alpha-latrotoxin and cytosolic concentration of ATP. It is shown that reagents decreasing the ATP level in the synaptosomes (monoiodoacetate, papaverine) inactivate the toxin-induced ionic fluxes and, on the contrary, reagents increasing the ATP level in synaptosomes enhance the toxin-induced calcium influx. The treatment of synaptosomes with inhibitors of phosphodiesterase of cAMP and cAMP-dependent protein kinase has no effect on the alpha-latrotoxin-induced calcium influx.     

Trypanosoma musculi: population dynamics of erythrocytes and leukocytes during the course of murine infections

Cells of the hemocytic and lymphoreticular series located in the blood, bone marrow, spleen, and peritoneal space have been analyzed throughout the course of Trypanosoma musculi infections of intact and splenectomized C3H female mice. Following an early (within 2 days after trypanosome inoculation intraperitoneally) shift of leukocytes from the blood to the peritoneal space, there occurred a more gradual, prolonged infusion of leukocytes into the peritoneal space, the primary site of infection, that continued until the infection was terminated. There was intense cytogeneractive activity in the spleen that included erythrocytes, lymphocytes, myelocytes, and megakaryocytes. The marrow became primarily a site of monocytopoiesis and, to some extent, of lymphopoiesis. During the first 8 days (approximately) of infection, there was a decline in mature erythrocytes in the blood (the well-known anemia) and development of a profound thrombocytopenia. In splenectomized mice, the depletion of these elements continued unabated until the mice died; the marrow of infected, splenectomized mice failed to provide these elements, as was also the case in intact mice. In the peritoneal space, the intense battle between leukocytes and trypanosomes was reflected in a gradual, impressive rise in the number of dead and fatigued cells and, late in infection, in the development of ascites. Both of these abnormal conditions disappeared shortly after cure of the infection. We conclude that infections of mice with T. musculi result in dedication of the entire lymphoreticular system to the generation of cells that are exported to the peritoneal space to combat the major infection the occurs in that locale. This is consistent with the evidence that the belated immune elimination of T. musculi is a cell-mediated (probably antibody-dependent) process. The disruption of the normal histoarchitecture, the shift in the normal proportions of cells and in cells of different degrees of maturity, and probably, a block imposed on precursor cell maturation, account to a large extent for the well-known failure of immune responses commonly associated with trypanosome infections.     

Early production of a chemotactic factor to T lymphocytes by peritoneal macrophages

Supernatant fluids (SNF) were obtained from peritoneal exudate adherent cells stimulated in vitro with sheep red blood cells (SRBC) or BCG, and SNF collected at 6 and 24 hr were able to induce the migratory responses of rat leukocytes from the spleen and peripheral blood. The production of these SNF was dependent on protein active synthesis upon in vitro antigenic stimulation. The chemotactic activity from 6-hr SNF was inhibited by using several proteolytic enzymes and temperatures. We found the macrophages to be the producer cell of this activity, while the T cells were the target cells. The chemotactic activity from 6-hr SNF was found not to be due to IL-1. Six-hour chemotactic activity has not been reported previously.     

Forssman glycolipid, an antigenic marker for a major subpopulation of macrophages from murine spleen and peripheral lymph nodes

Three hybridoma clones were isolated after hybridization of a mouse myeloma line with splenocytes from rats immunized with Forssman glycosphingolipid (Fo). Two of these clones produced Fo-specific monoclonal antibodies (MAB) of the IgM class, one MAB of the IgG2c class. In complement-dependent depletion experiments and immunofluorescence studies on the nature of Fo-positive leukocytes in CBA/J mice the following results were obtained: whereas blood monocytes, polymorphonuclear leukocytes, and lymphocytes were Fo negative, 5 to 10% of suspended spleen cells were positive. The majority of these were macrophage-like, glass- and nylon-adherent, nonspecific esterase-positive phagocytizing cells carrying Ia and globoside markers. These cells participated as accessory cells in the mixed lymphocyte culture reaction. In cell suspensions from axillary and inguinal lymph nodes, 2% were Fo positive. They were enriched up to 70% in the glass-adherent, esterase-positive population from this source. In contrast, no Fo-positive cells were detected in mesenteric lymph nodes, and less than 0.1% of the resident peritoneal macrophages bore this marker. The percentage of Fo-positive cells increased to 1% in thioglycollate-elicited peritoneal cells. Immunostaining of cryosections of lung and liver tissue showed alveolar macrophages and Kupffer cells, respectively, to be Fo negative.     

Changes in the chromatin structure of lymphoid cells under the influence of low-intensity extremely high-frequency electromagnetic radiation against the background of inflammatory process

Using the alkaline single cell gel electrophoresis technique (comet assay), changes in chromatin structure of peripheral blood leukocytes and peritoneal neutrophils have been studied in mice exposed to low-intensity extremely high-frequency electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, 20 min at 1 h after induction of inflammation) against the background of the systemic inflammatory process. It was revealed that the exposure of mice with the developing inflammation leads to a pronounced decrease in the level of DNA damage to peripheral blood leukocytes and peritoneal neutrophils. It is supposed that the changes in the chromatin structure of lymphoid cells have a genoprotective character in the inflammatory process and can underlie the mechanisms of realization of antiinflammatory effects of the electromagnetic radiation.     

A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages

Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages.     

An alternate mechanism for regulation of leukotriene production in leukocytes: studies with an anti-inflammatory drug, sodium diclofenac

Sodium diclofenac, a potent cyclooxygenase inhibitor, was recently shown to inhibit arachidonic acid conversion to leukotriene products in human leukocytes. This activity was confirmed by radioimmunoassay in calcium ionophore A 23187-stimulated leukocytes isolated from the rat peritoneal cavity and human peripheral blood. Studies with rat peritoneal leukocytes revealed that this effect was not mediated by inhibition of 5-lipoxygenase or phospholipase A2, but rather through modulation of arachidonic acid uptake and release. The potency of this effect was dependent upon cell type; macrophages being more sensitive to the drug than neutrophils. In leukocytes treated with sodium diclofenac, arachidonic acid released from phospholipids in response to A 23187 challenge was reincorporated into triacylglycerols. The drug enhanced the spontaneous uptake of arachidonic acid into the cellular triacylglycerol pool and, in this manner, decreased the availability of intracellular arachidonic acid. Therefore, sodium diclofenac, in addition to inhibition of cyclooxygenase, regulates leukotriene production of inflammatory cells by a mechanism mediated in part through the redistribution of arachidonic acid in lipid pools.     

Changes in the chromatin structure of lymphoid cells under the influence of low-intensity extremely high-frequency electromagnetic radiation against the background of inflammatory process

Using the alkaline single cell gel electrophoresis technique (comet assay), changes in chromatin structure of peripheral blood leukocytes and peritoneal neutrophils have been studied in mice exposed to low-intensity extremely high-frequency electromagnetic radiation (42.2 GHz, 0.1 mW/cm², 20 min at 1 h after induction of inflammation) against the background of the systemic inflammatory process. It was revealed that the exposure of mice with the developing inflammation leads to a pronounced decrease in the level of DNA damage to peripheral blood leukocytes and peritoneal neutrophils. It is supposed that the changes in the chromatin structure of lymphoid cells have a genoprotective character in the inflammatory process and can underlie the mechanisms of realization of antiinflammatory effects of the electromagnetic radiation.     

An indirect bioluminescence method for the quantitative measurement of polymorphonuclear cell chemotaxis

A method is presented which allows the quantification of the effects of chemotactic factors on polymorphonuclear leukocytes on the basis of a sensitive ATP measurement using bioluminescence. The assay measures those cells which have migrated through a commercial 3 μm filter system (Transwell?). The assay was tested under standardized conditions with different chemotactic agents (leukotriene B4 [LTB4], N-formyl-methionyl-leucyl-phenylalanine [FMLP], N-formyl-methionyl-leucyl-phenylalanine-methyl ester [M-FMLP]). Under appropriate conditions the migration of PMN-cells is time-dependent and linear for 60 minutes. Spontaneous migration of PMN cells is simultaneously quantified in a simple way, and the value obtained allows a determination of the actual chemotactic stiuation of the PMN cells. In healthy humans the spontaneous migration varied between 4.2% and 14.4% of the total number of PMN cells. An optimal chemotactic activity was detected at 10^?8/mol/I for FMLP and 10^?7 mol/l for M-FMLP in PMN leukocytes, which correlates with literature values. It was also found that in contrast to EDTA blood, heparinized blood lowers the ATP level of PMN cells (by about 50%) and therefore heparinized blood is not recommended for chemotactic experiments. This assay is a simple tool for quantification of the spontaneous migration, and the chemotactic response to specific factors and their inhibitors in particular for pharmacological experiments. In contrast to the ‘classical’ chemotactic assays this method also permits the simultaneous testing of the influence of chemotactic substances on cellular ATP levels.     

RESPIRATION IN VITRO OF SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

Abstract— —(1) The respiratory properties of synaptosomes and mitochondria from mammalian cerebral cortex are compared.
(2) Synaptosome showed high and linear respiration with glucose and pyruvate as substrates in Krebs-Ringer media. Mitochondria showed such respiration only with pyruvate as substrate in media lacking Na and high in K and phosphate.
(3) Exposure of synaptosomes to hypotonic media caused loss of lactate dehydrogenase (LDH) and protein, and respiration diminished and became non-linear.
(4) Both ATP and phosphocreatine were synthesised by synaptosomes with glucose as substrate. ATP was synthesised by mitochondria in the presence of pyruvate.
(5) Synaptosome but not mitochondria showed some capacity for active accumulation of potassium.
(6) Both mitochondria and synaptosomes respired with glutamate as substrate. Glutamate caused 80 per cent loss of ATP and phosphocreatine in synaptosomes but did not diminish the level of mitochondrial ATP.     

多形核白细胞上嘌呤受体的初步研究

ATP和ADP能激活多型核白细胞引起细胞内［Ca²⁺］_i的明显升高,AMP则无此作用.多型核白细胞对ATP和ADP具有不同的浓度依赖性.当细胞外的钙离子被螯合后,ATP和ADP仍能引起细胞内游离钙浓度的升高.结果表明多形核白细胞存在着对ATP和ADP敏感的P2型嘌呤受体,并且属于P2型受体中的P2Y亚类.