Influence of platelet-activating factor,its cell analogs,and antagonist on the production of superoxide radicals by blood leukocytes of healthy and hypercholesterolemic individuals

The influence of the phospholipid platelet-activating factor (PAF), its cell analogs, and lipid PAF antagonist on the production of superoxide radicals by leukocytes isolated from the blood of healthy and hypercholesterolemia IIA individuals was studied. It was found that endogenous superoxide production level in the leukocytes of hypercholesterolemic individuals more than 4-5 times higher than in the leukocytes of healthy individuals. Exogenous PAF stimulates the superoxide production in the leukocytes of healthy individuals but significantly inhibits the superoxide production in the leukocytes of hypercholesterolemic individuals. The compounds 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-PAF) and 1-alk-1"-enyl-2-acetyl-sn-glycero-3-phosphocholine (1-alkenyl-PAF) only slightly inhibited the endogenous superoxide production in the leukocytes of hypercholesterolemia individuals. However, pretreatment of leukocytes by 1-alkenyl-PAF or PAF-antagonist (1-O-alk-1"-enyl-2-(2"-acetoxybenzoyl)-sn-glycero-3-phosphocholine) results in a 50% inhibition of the PAF-induced superoxide production by leukocytes of healthy individuals. This PAF-antagonist alone or in combination with PAF induces a substantial (65-70%) inhibition of superoxide production in the leukocytes of hypercholesterolemic individuals. It is concluded that superoxide production by leukocytes of healthy individuals and especially by leukocytes of hypercholesterolemic individuals is process that depends on PAF or PAF-like lipids.     

Evidence for bactericidal activity of polymorphonuclear leukocytes without phagocytosis.

The relationship between phagocytosis and bactericidal action of polymorphonuclear leukocytes was examined by comparing the functions of cytochalasin D-treated leukocytes with those of the control. Measurement of phagocytotic and bacterial DNA-degrading activities using Escherichia coli prelabeled with [3H]thymidine revealed that phagocytosis and bacterial DNA degradation were inhibited by treatment with cytochalasin D to about 50 and 10% of the control, respectively. Nevertheless, the bactericidal activity of the cytochalasin D-treated leukocytes was almost the same as that of the control leukocytes; almost all the bacteria were phagocytized by the latter leukocytes. Under the same experimental conditions, the production and release of superoxide anions and hydrogen peroxide, which are both known to be involved in the bactericidal action of the leukocytes, were markedly increased by cytochalasin D. Release of several lysosomal hydrolases was also increased markedly by cytochalasin D treatment, except for myeloperoxidase. However, lactate dehydrogenase, a typical cytosolic marker, was not released by the same treatment. Thus, it is unlikely that the increase in the release of the above-mentioned bactericidal factors was due to decomposition of the leukocytes. These results indicate that the site of bactericidal action of cytochalasin D-treated leukocytes is not necessarily intracellular but may be around the external surface of the cells.     

Oxidative stress-related alteration of the copy number of mitochondrial DNA in human leukocytes

The role of oxidative stress in the regulation of the copy number of mitochondrial DNA (mtDNA) in human leukocytes is unclear. In this study, we investigated the redox factors in plasma that may contribute to the alteration of mtDNA copy number in human leukocytes. A total of 156 healthy subjects of 25-80 years of age who exhibited no significant difference in the distribution of subpopulations of leukocytes in blood were recruited. Small-molecular-weight antioxidants and thiobarbituric acid reactive substances (TBARS) in plasma and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4,977bp deletion of mtDNA in leukocytes were determined. The mtDNA copy number in leukocytes was determined by real-time PCR. The results showed that the copy number of mtDNA in leukocytes was changed with age in a biphasic manner that fits in a positively quadratic regression model (P = 0.001). Retinol (P = 0.005), non-protein thiols (P = 0.001) and ferritin (P = 0.004) in plasma and total glutathione in erythrocytes (P = 0.046) were the significant redox factors that correlated with the mtDNA copy number in leukocytes in a positive manner. By contrast, alpha-tocopherol levels in plasma (P = 0.001) and erythrocytes (P = 0.033) were negatively correlated with the mtDNA copy number in leukocytes. Three oxidative indices including the incidence of 4,977 bp deletion of mtDNA (P = 0.016) and 8-OHdG content in leukocytes (P = 0.003) and TBARS in plasma (P = 0.001) were all positively correlated with the copy number of mtDNA in leukocytes. Taken these findings together, we suggest that the copy number of mtDNA in leukocytes is affected by oxidative stress in blood circulation elicited by the alteration of plasma antioxidants/prooxidants and oxidative damage to DNA.     

Rapid flow cytometric studies of Borrelia burgdorferi phagocytosis by human polymorphonuclear leukocytes

The interactions between a strain of Borrelia burgdorferi and human polymorphonuclear leukocytes were studied by flow cytometry in the presence of specific or non-specific opsonizing factors. The capacity of the borrelias to stimulate leukocyte metabolism was also investigated. The results indicated that a low phagocytosis by isolated purified polymorphonuclear leukocytes did occur in the presence or absence of specific antibodies. Within whole blood the percentages of phagocytosting leukocytes increased in the presence of non-specific opsonizing factors. No stimulation of the oxidative metabolism stimulated by Borrelia was observed and PMA or zymosan stimulation of leukocytes was inhibited by the spirochaetes.     

Reduced chemotaxis of polymorphonuclear leukocytes in sera from patients with rheumatoid arthritis

The chemotaxis of normal leukocytes washed and suspended in Hanks solution is inhibited by addition of serum from patients with definite rheumatoid arthritis as was shown by experiments in which an extract from Proteus mirabilis served as attractant. From a suspension of normal leukocytes to which normal serum had been added, significantly more leukocytes actively moved through a millipore filter toward the attractant than from a suspension of similar leukocytes in serum from patients.     

Stimulation of free radical generation in human leukocytes by various agents including tumor necrosis factor is a calmodulin dependent process

The mechanism(s) involved in the generation of free radicals in human leukocytes by phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMP), lipopolysaccharide (LPS), arachidonic acid (AA), and recombinant-tumor necrosis factor-1-alpha (r-TNF-1 alpha) was investigated. Calmodulin antagonists, chlorpromazine and trifluoperazine, inhibited free radical generation in human leukocytes by these stimulants. Dexamethosone, an inhibitor of phospholipase A2, could also block free radical generation in human leukocytes induced by r-TNF 1 alpha. PMA, FMP, LPS and TNF can activate phospholipase A2 and induce the release of AA from the cell membrane lipid pool. AA induced free radical generation in human leukocytes can be inhibited by calmodulin antagonists. Hence, it is likely that calmodulin dependent events play a crucial role in the generation of free radicals by human leukocytes in response to various stimulants including TNF.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Meconium-stained amniotic fluid activates polymorphonuclear leukocytes ultrastructural and enzyme-cytochemical evidence

The purpose of the present study was to demonstrate morphological evidence that meconium-stained amniotic fluid activates polymorphonuclear leukocytes. We used Boyden chamber techniques to determine the chemotactic activity of meconium-stained amniotic fluid for leukocytes, and ultrastructural enzyme-cytochemistry for peroxidase and alkaline phosphatase to characterize the leukocyte features induced by contact with meconium-stained amniotic fluid. Amniotic fluid with meconium staining enhanced migration of leukocytes. These leukocytes exhibited more numerous cytoplasmic processes and more prominent phagosomes compared to peripheral blood leukocytes. Peroxidase and alkaline phosphatase activity were evident on the phagosomal membranes. Our results indicate that meconium-stained amniotic fluid activates or stimulates polymorphonuclear leukocytes. Meconium-stained amniotic fluid induced leukocyte activation might play important roles in the pathophysiology of initiation of term labor or of the meconium aspiration syndrome.     

Human blood platelets, PMN leukocytes and their interactions in vitro. Responses to selective and non-selective stimuli.

Using simultaneous recording of aggregation and chemiluminescence, responses of human polymorphonuclear leukocytes, blood platelets and their mixture were investigated after stimulation by specific as well as non-specific stimuli for each cell. In our experimental settings, aggregation of platelets and PMN leukocytes was increased in the following order of stimuli: PMA相似文献   

Deletion of the mouse meprin beta metalloprotease gene diminishes the ability of leukocytes to disseminate through extracellular matrix

Meprins are metalloendopeptidases expressed by leukocytes in the lamina propria of the human inflamed bowel, that degrade extracellular matrix proteins in vitro implicating them in leukocyte transmigration events. The aims of these studies were to 1) examine the expression of meprins in the mouse mesenteric lymph node, 2) determine whether macrophages express meprins, and 3) determine whether deletion of the meprin beta gene (Mep-1beta) mitigated the ability of leukocytes to disseminate through extracellular matrix in vitro. These studies show that meprin alpha and beta are expressed in leukocytes of the mouse mesenteric lymph node, and meprin alpha, but not beta, decreased during intestinal inflammation. Deletion of Mep-1beta gene decreased the ability of leukocytes to migrate through matrigel compared with wild-type leukocytes. Meprin beta, but not alpha, was detected in cortical and medullary macrophages of the lymph node. Thus overall, meprin beta is expressed by leukocytes in the draining lymph node of the intestine, regardless of the inflammatory status of the animal, and is likely to contribute to leukocyte transmigration events important to intestinal immune responses. Thus, the expression of meprins by leukocytes of the intestinal immune system may have important implications for diseases such as inflammatory bowel diseases, which are aggravated by leukocyte infiltration.     

Rapid flow cytometric studies of Borrelia burgdorferi phagocytosis by human polymorphonuclear leukocytes

The interactions between a strain of Borrelia burgdorferi and human polymorphonuclear leukocytes were studied by flow cytometry in the presence of specific or non-specific opsonizing factors. The capacity of the borrelias to stimulate leukocyte metabolism was also investigated. The results indicated that a low phagocytosis by isolated purified polymorphonuclear leukocytes did occur in the presence or absence of specific antibodies. Within whole blood the percentages of phagocytosting leukocytes increased in the presence of non-specific opsonizing factors. No stimulation of the oxidative metabolism stimulated by Borrelia was observed and PMA or zymosan stimulation of leukocytes was inhibited by the spirochaetes.     

Restoration of antineoplastic reactivity of the leukocytes after preincubation]

Specific antitumor sensitization in patients with carcinoma of the stomach was revealed by means of the leukocyte migration inhibition test. After 24-hour preincubation at 4 degrees C in a serum-free medium the areactive leukocytes from patients with carcinoma of the stomach (stages III--IV) acquired the ability to react specifically to allogeneic antigens of the tumor of the same localization. Preincubation did not influence the inhibition of migration of leukocytes in non-tumor patients by stomach carcinoma antigens. The supernatant of preincubated leukocytes of tumor patients contained substances that inhibited migration of indicator leukocytes.     

CD44 mediated Hyaluronan adhesion of Toxoplasma gondii-infected leukocytes

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans and animals. Ingested parasites cross the intestinal epithelium, invade leukocytes and are then disseminated to peripheral organs. However, the mechanism of extravasation of the infected leukocytes remains poorly understood. In this study, we demonstrate that T. gondii-invaded human and mouse leukocytes express higher level of CD44, a ligand of hyaluronan (HA), and its expression on myeloid and non-myeloid leukocytes causes T. gondii-invaded human and mouse leukocyte to adhere to HA more effectively than non-invaded leukocytes. The specific adherence of parasite-invaded leukocytes was inhibited by anti CD44 antibody. Leukocytes of CD44 knockout mice did not show parasite-invaded leukocyte specific adhesion. Our results indicate that parasite-invaded leukocytes, regardless of whether myeloid or not, gain higher ability to adhere to HA than non-invaded leukocytes, via upregulation of CD44 expression and/or selective invasion to CD44 highly expressing cells. The difference in ability to adhere to HA between parasite-invaded cells and non-invaded neighboring cells might facilitate effective delivery of parasite-invaded leukocytes to the HA-producing endothelial cell surface and/or HA-rich extra cellular matrix.     

Effect of redox-modified fibrinogen on blood leukocyte functions

The influence of oxidized fibrinogen on the intensity of luminol-dependent chemilumin escence of blood leukocytes, stimulated by opsonized zymosan was studied. It was shown that the introduction of fibrinogen modified by UV-irradiation in to a suspension of cells resulted in a significant increase in the intensity of the luminol-dependent chemiluminescence of leukocytes. It was suggested that oxidized fibrinogen can influence blood leukocytes, enhancing their functional activity.     

Robert Feulgen Lecture 1998

Attachment of leukocytes to the blood vessel wall initiates leukocyte extravasation. This enables leukocytes to migrate to and accumulate at sites of tissue injury or infection where they execute host-defense mechanisms. A series of vascular cell adhesion molecules on leukocytes and on endothelial cells mediate leukocyte attachment to the endothelium in a stepwise process. A large panel of about 40 known human chemokines is able to specifically activate certain leukocytes and attract them to migrate across the endothelial barrier and within tissue. The specific combination of molecular signals provided by the diversity of cytokines, adhesion molecules, and chemokines regulates the specificity and selectivity of the recruitment of certain subpopulations of leukocytes in vivo. This review will focus on selectins and chemokines which initiate the cell contact and regulate activation and chemoattraction of leukocytes. Accepted: 20 May 1999     

Angiopeptin,a somatostatin-14 analogue,decreases adhesiveness of rat Leukocytes to unstimulated and IL-1β-activated rat heart endothelial cells

We have previously demonstrated that somatostatin-14 and its octapeptide analogue, angiopeptin, decrease the ability of rat heart endothelial cells to bind leukocytes [Leszczynski, et al., Reg. Pept. 43 (1993) 131–140]. Here, we examined whether exposure of leukocytes to angiopeptin modifies their adhesiveness to the unstimulated and to IL-1β-activated endothelium. Monolayers of unstimulated endothelial cells bind 274 ± 12 leukocytes/mm². Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 μM) reduced significantly (p < 0.05) adhesion of leukocytes from 274 ± 12 to 188 ± 10, 185 ± 8 and 172 ± 3 cells/mm², respectively. Stimulation of endothelial cells with IL-1β (100U/ml) for 24 hours increased endothelial adhesiveness from 274 +- 12 to 381 ± 17 adhering leukocytes/mm². Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1μM) reduced significantly (p < 0.05) binding of leukocytes to IL-1β-activated endothelium from 381 ± 17 to 237 ± 8, 254 ± 11 and 248 ± 13 cells/mm², respectively. Angiopeptin had no effect on the expression of lymphocyte function-associated molecule-1 (LFA-1; CDlla/CD18) by leukocytes, as assessed by flow cytometry. This suggests that angiopeptin modulates adhesive properties of leukocytes by (1) altering the expression of other than LFA-1 adhesion molecule(s) and/or (2) modulating the affinity of adhesion molecule(s) expressed by leukocytes. In conclusion, our results demonstrate that angiopeptin reduces leukocyte adhesiveness to unstimulated and to IL-1β-activated endothelium. It suggests that angiopeptin may suppress immune response via modulation of the leukocyte-endothelial interaction.     

Molecular events during leukocyte diapedesis

The recruitment of leukocytes from the circulation into tissues requires leukocyte migration through the vascular endothelium. The mechanisms by which leukocytes attach and firmly adhere to the endothelial cell surface have been studied in detail. However, much less is known about the last step in this process, the diapedesis of leukocytes through the vascular endothelium. This minireview focuses on the interactions between leukocyte and endothelial cell adhesion molecules that are important during leukocyte extravasation. In the past few years a series of endothelial cell surface and adhesion molecules have been identified that are located at endothelial cell contacts and found to participate in leukocyte diapedesis. These junctional cell adhesion molecules are believed to have an active role in controlling the opening and closure of endothelial cell contacts to allow the passage of leukocytes between adjacent endothelial cells. Alternatively, leukocytes can cross the endothelium at nonjunctional locations, with leukocytes migrating through a single endothelial cell. Further work is clearly needed to understand, in greater detail, the molecular mechanisms that allow leukocytes to cross the endothelium via either the paracellular or the transcellular pathway.     

The effect of ACTH administration on the phagocytic and bactericidal activities of human polymorphonuclear leukocytes.

Metabolic and bactericial activities of leukocytes obtained from 5 normal volunteers receiving 1 mg of synthetic B1-24 ACTH intra-muscularly daily for 7 days, were studied. Bactericidal activity and phagocytosis induced hexosemonophosphate shunt activity of leukocytes were found to be depressed following ACTH administration. However, glycolytic activity, which provides the necessary energy for particle uptake by leukocytes, was not altered. These findings indicate that a moderately prolonged exposure to elevated levels of plasma cortisol does not affect the phagocytic activity of leukocytes (as indicated by glycolytic activity) but significantly impairs their ability to destroy the ingested bacteria. It is suggested that the impaired bactericidal activity of leukocytes reported in children suffering from protein-calorie malnutrition may partly be due to elevated plasma cortisol levels seen in them.     

Peculiarities of specific binding of serotonin by blood leukocytes, peritoneal cells and synaptosomes of mice

The properties of serotonin-active sites were studied on peritoneal cells, blood leukocytes and synaptosomes of mice (CBA line). Treatment of cell suspensions with EDTA, ouabain, strophanthin, 2,4-dinitrophenol, dithiothreitol and trypsin demonstrated that serotonin binding by peritoneal cells and leukocytes depends on bivalent ions and K+, Na+-pump operation, requires energy and intact disulfide bonds and is determined by a protein structure. ATP and ADP were found to inhibit amine adsorption by peritoneal cells. These cells specifically bind ATP 10 times more intensively than leukocytes. The data obtained are suggestive of differences in the composition of serotonin-active structures of blood leukocytes, peritoneal cells and synaptosomes.     

Mitogen responsiveness after exposure of influenza virus-infected human mononuclear leukocytes to continuous or pulse-modulated radiofrequency radiation

Data are available regarding interactions of radiofrequency radiation (RFR) with normal human mononuclear leukocytes. However, no data have emerged regarding effects of RFR on human leukocytes already challenged by a commonly encountered alternate agent, such as a virus. Therefore, in these studies, uninfected (control) and in vitro influenza virus-infected human mononuclear leukocytes were exposed to 2450 MHz RFR as continuous waves or pulse-modulated at 60 or 16 Hz, at a specific absorption rate of 4 mW/ml. Such exposures produced no significant effects on leukocyte viability or on mitogen-stimulated DNA synthesis by either uninfected or influenza virus-infected leukocytes when compared to sham-RFR-exposed of cells.