Human Peripheral Polymorphonuclear Leukocytes Research Articles

15-acetylthioxy-furodysinin lactone, isolated from a marine sponge [formula omitted], SP. is a potent agonist to human leukotriene B 4 receptor

A sesquiterpene thioacetate, 15-acetylthioxy-furodysinin (SK&F 105900) has been isolated from the sponge Dysidea SP. This compound can bind specifically to the human peripheral blood polymorphonuclear leukocyte (PMN) and to the differentiated human monocytic leukemic U-937 cell membrane leukotriene B 4 (LTB 4) receptors with high-affinity. This compound can also promote a concentration-dependent chemotaxis in PMNs and an intracellular calcium mobilization in U-937 cells that can be blocked by the LTB 4 receptor antagonist, LY-223982. Furthermore, the calcium mobilization induced by SK&F 105900 can specifically cross-desensitize with the LTB 4-induced calcium mobilization. These observations indicate that SK&F 105900 is a novel and specific high-affinity agonist that can bind to the LTB 4 receptors and activate the receptor-mediated signal transduction processes in human PMN and U-937 cells.

Nifedipine Inhibits Magnetic Field-Induced Lysosomal Degranulation

Human peripheral polymorphonuclear leukocytes were exposed to static magnetic fields of variable intensity (0-1.0 T). Following exposure for 30 minutes, the cells were centrifuged and the supernatant was assayed for release of lactate dehydrogenase, a marker of cytotoxicity. Up to 9% of the cells' total content of this lysosomal enzyme was released after exposure to a 1.0-T magnetic field. Significantly less degranulation occurred following pretreatment with 5 μM nifedipine, a calcium channel antagonist. Calcium channel antagonists may serve as magnetoprotective agents against magnetic field-induced changes in Ca2+-dependent cellular processes.

Orally administered tolfenamic acid inhibits leukotriene synthesis in isolated human peripheral polymorphonuclear leukocytes

Special interest has been focused on the development of dual inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism. In contrast to other classic NSAIDs, some fenamates in clinically achievable concentrations have been shown to inhibit synthesis of 5-lipoxygenase products in vitro. In the present work, we studied the effect of orally administered tolfenamic acid (600 mg) on Ca ionophore A 23187 -induced leukotriene synthesis in isolated human polymorphonuclear leukocytes. Leukotriene production was reduced in all 14 subjects studied, the mean inhibition of LTB4 synthesis being 16 +/- 3% and that of LTC4 33 +/ 7%. The inhibition correlated positively with serum acid concentrations. We suggest that inhibition of leukotriene synthesis is an additional mechanism of the anti-inflammatory, antimigraine and antidysmenorrhoeic effects of tolfenamic acid, and a possible explanation for its rare gastric and bronchoconstrictive side-effects.

Alpha 2HS glycoprotein is chemotactic for mononuclear phagocytes.

alpha 2HS glycoprotein is a normal constituent of plasma. It has a high affinity for hydroxyapatite and is concentrated in bone 100-fold greater than albumin, however its biological function in bone is not known. We have purified alpha 2HS from human plasma and examined it for evidence of chemotactic activity against human peripheral mononuclear cells, polymorphonuclear leucocytes and lymphocytes in Boyden chamber assays. We observed that the protein was chemotactic for mononuclear cells and that maximal cell migration occurred when its concentration in the lower compartment of Boyden chambers was 10(-10) M. Chemotaxis did not occur in the absence of a concentration gradient. In addition, cell migration was blocked when the cells were preincubated with 10(-10) M alpha 2HS or when the protein was preincubated with rabbit anti-human alpha 2HS glycoprotein IgG. Neither polymorphonuclear leucocytes, which are responsive to a wide range of chemoattractants, or peripheral lymphocytes exhibited directed migration to alpha 2HS in Boyden chamber assays. The glycoprotein appears therefore to act as a chemotaxin directed to monocyte recruitment, and we speculate that the protein may have an important role in monocyte recruitment to bone and possibly their subsequent fusion and differentiation into functional osteoclasts.

Lipids of human promyelocytic leukemia cell HL-60: increasing levels of ether-linked phospholipids during retinoic acid-induced differentiation.

Cells of the human promyelocytic leukemia cell line HL-60 as well as HL-60 granulocytes induced in vitro by retinoic acid were examined for lipid composition. One of our original aims was to clarify how human granulocyte (differentiated HL-60 cells) synthesized enough precursors of lipid mediators, such as prostaglandins and/or platelet activating factor. Comparison studies yielded the following results. 1) After granulocyte differentiation, total phospholipid of HL-60 cells decreased to about 70% of that of untreated cells, while the content of triglyceride increased to about 200% of the original level. 2) The subclass composition of ethanolamine-containing glycerophospholipid was greatly altered during differentiation; 1-alkenyl-2-acyl glycerophosphoethanolamine (GPE) increased to 166% of that in the untreated cells, while 1,2-diacyl GPE decreased to 46% of the original value. The resultant profile became very similar to that of human peripheral polymorphonuclear leukocytes. 3) During differentiation, the amount of arachidonic acid stored in both phospholipid and triglyceride of retinoic acid-treated HL-60 cells significantly increased. Its distribution was also modified; arachidonic acid in 1,2-diacyl GPE decreased to 63%, while those of 1-alkenyl-2-acyl GPE, choline-containing glycerophospholipids, and phosphatidylinositol increased to 169, 154, and 153%, respectively. These results suggested that the regulatory mechanism of lipid turnover in HL-60 cells was modified during retinoic acid-induced granulocyte differentiation. The alterations were not enough to explain fully the capability of differentiated HL-60 cells to produce lipid mediators upon stimulation, but they were probably one of the factors that regulate these reactions.

Studies on the activities of tannins and related compounds, X. Effects of caffeetannins and related compounds on arachidonate metabolism in human polymorphonuclear leukocytes.

As part of a series of biological examinations of various tannins and related compounds, the present paper reports the effects of caffeetannins and related compounds isolated from medicinal plants on arachidonate metabolism in human peripheral polymorphonuclear leukocytes (PMN-L). The formation of leukotriene B4 (LTB4) induced by calcium ionophore A 23187 (A 23187) in human PMN-L was inhibited by 3,5-, 4,5-, and 3,4-di-O-caffeoylquinic acid, caffeoylmalic acid, caffeoyltartric acid, rosmarinic acid, and caffeic acid. Rosmarinic acid strongly inhibited the formation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and LTB4 (5-lipoxygenase products) at concentrations of 10(-5)-10(-3) M. On the other hand, the formation of prostaglandin E2 (PGE2) was enhanced in a concentration-dependent fashion by caffeic acid, caffeoylmalic acid, caffeoyltartaric acid, and 3,4-di-O-caffeoylquinic acid. On the basis of experimental results, it seems likely that caffeoyl derivatives could be developed as therapeutic drugs for treatment of allergic inflammation such as asthma.

Effects of caffeoylglycosides on arachidonate metabolism in leukocytes.

The effects of caffeoylglycosides (forsythiaside, suspensaside, acteoside, and β-hydroxyacteoside), isolated from dried fruits of Forsythia species, on the formations of the 5-lipoxygenase products 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4), the 15-lipoxygenase product 15-hydroxy-5,8,11,13-eicosastetraenoic acid (15-HETE) and the cyclooxygenase products 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), thromboxane B2 and 6-keto-PGF1α in rat peritoneal cells and human peripheral polymorphonuclear leukocytes were studied. Forsythiaside, suspensaside, acteoside, and β-hydroxyacteoside were found to inhibit selectively the formation of the 5-lipoxygenase products 5-HETE and LTB4 in human peripheral polymorphonuclear leukocytes; their concentrations for 50% inhibition (IC50) of 5-HETE formation being 1.92, 49.0, 4.85, and 40.0 µM, and their IC50 values for LTB4 formation being 1.01, 8.85, 2.93, and 8.50 µM, respectively.

High-density and low-density capsule production byStreptococcus zooepidemicus

A group C streptococcus (Streptococcus zooepidemicus), isolated from the blood of a 79-year-old man who succumbed to infection, was examined for animal virulence, reactivity in an in vitro phagocytic assay, and the production of antiphagocytic structures. Initial examination of the clinical isolate by electron microscopy revealed the presence of high-density (HDC) and low-density (LDC) capsule variants. The HDC variant capsular material could be removed from the cell wall by hot acid extraction or obtained from the supernatant fluids when the organisms were grown in a defined medium. The capsular material was purified by ion-exchange chromatography on diethylaminoethyl cellulose and gel filtration on Sepharose 4B and was shown to be composed largely of a high molecular weight hyaluronic acid. Both the high-density and low-density capsule variants were shown to be mouse virulent with lethal dose 50 (LD50) values of 3.7×104 colony-forming units (CFU) and 3.3×105 CFU, respectively. Opsonophagocytic assays with human peripheral polymorphonuclear leukocytes demonstrated that only the LDC organism could be phagocytized and killed in the presence of white blood cells, specific antibody, and complement.

Augmentation of antibody-dependent cellular cytotoxicity of polymorphonuclear leukocytes by interferon-gamma: mechanism dependent on enhancement of Fc receptor expression and increased release of activated oxygens.

The effect of recombinant human interferon (IFN) -gamma (Met-Gln form), having the same amino acid sequence as that of natural human IFN-gamma except for the N-terminal Met residue, on antibody-dependent cellular cytotoxic (ADCC) activity of human peripheral polymorphonuclear leukocytes (PMNs) against anti-chicken red blood cell (CRBC) immunoglobulin G (IgG) -coated CRBC was investigated comparatively with those of natural human IFN-alpha and -beta in vitro. IFN-gamma dramatically induced the ADCC activity at all doses tested when examined at a 1/104 dilution rate of the IgG, while no significant induction was observed with IFN-alpha or-beta. The Fc receptor (FcR) analysis by flow cytometry using fluorescein isothiocyanate-labelled (FITC) -anti-CRBC IgG revealed that IFN-gamma but not IFN-alpha or-beta remarkably increased the number of PMNs bearing FcR. By applying an in vitro measurement system for activated oxygen species using luminol solution, it was demonstrated that activated oxygen species were actually released from PMNs in the ADCC reaction. Further, inhibition experiments using three oxygen scavengers (superoxide dismutase, catalase and dimethyl sulfoxide) demonstrated the involvement of superoxide anion and hydroxy radical as cytolytic mediators in the PMN-ADCC activity induced by IFN-gamma.These results indicate a much more potent PMN-activating ability of IFN-gamma than IFN-alpha or-beta and involvement of a mechanism dependent on the enhancement of FcR expression on the PMN surface and the increased release of superoxide anions and hydroxy radicals in the PMN-ADCC augmentation by IFN-gamma.

Chloride movements in human neutrophils. Diffusion, exchange, and active transport.

Chloride content and fluxes were measured in isolated resting human peripheral polymorphonuclear leukocytes. The intracellular Cl concentration of cells kept at 37 degrees C in 148 mM Cl media was approximately 80 meq/liter cell water, fourfold higher than expected for passive distribution at the cell's estimated membrane potential (approximately -53 mV). All intracellular Cl was rapidly exchangeable with external 36Cl. Cells lost Cl exponentially into Cl-free media, and reaccumulated it when Cl was restored to the bath; this reuptake was dependent on metabolism. One-way 36Cl fluxes in steady state cells were approximately 1.4 meq/liter X min. The bulk (approximately 70%) of these represented electrically silent Cl/Cl exchange mediated by a carrier insensitive to disulfonic stilbenes but blocked by the anion carrier inhibitor alpha-cyano-4-hydroxycinnamate (CHC). The remaining fluxes were characterized in some detail. About 20% of 36Cl influx behaved as active transport: it moved thermodynamically uphill and was absent in cells treated with 2-deoxy-D-glucose, displayed Michaelis-Menten kinetics with Km(Cl) congruent to 5 mM, Vmax congruent to 0.25 meq/liter X min, and was inhibited by CHC (Ki congruent to 1.7 mM), ethacrynate (Ki congruent to 50 microM), and furosemide (Ki congruent to 50 microM). About 30% of Cl efflux and approximately 8% of Cl influx behaved as electrodiffusion through a low-permeability pathway (PCl congruent to 4 X 10(-9) cm/s; gCl congruent to 1 microsecond/cm2; PK/PNa/PCl congruent to to 10:1:1); these fluxes were linear with concentration and strongly voltage sensitive. The putative Cl channel does not appear to be voltage gated, and gives evidence of single filing.

Effects of baicalein on leukotriene C4 biosynthesis in human leukocyte.

We have studied the effects of baicalein on leukotriene C4 biosynthesis induced by calcium ilonophore A 23187 in human peripheral polymorphonuclear leukocytes. Baicalein inhibited calcium ionophore-induced leukotriene C4 biosynthesis in human polymorphonuclear leukocytes. The concentration of baicalein required for 50% inhibition(IC50) of leukotriene C4 formation was 6.00×10-7 M at a calcium ionophore concentration of 1 μg/ml; at 0.1 μg/ml it was 2.05×10-7 M.

Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.

Increased leukocyte adhesion to the endothelial lining of blood vessels is an essential event in inflammation and the pathogenesis of certain vascular diseases. We have studied the effect of interleukin 1 (IL-1), an inflammatory/immune mediator, on endothelial-leukocyte adhesion using quantitative in vitro assays. Selective pretreatment of cultured human umbilical vein endothelial monolayers with IL-1 (5 U/ml, 4 h) resulted in an 18.3 +/- 2.6-fold increase in human peripheral blood polymorphonuclear leukocyte (PMN) adhesion (mean +/- SEM, n = 16) and a 2.6 +/- 0.3-fold increase in monocyte adhesion (n = 7) over basal levels. IL-1-treated endothelial monolayers also supported increased adhesion of the promyelocytic cell line HL-60 and the monocytelike cell line U937 (33.0 +/- 6.0-fold, n = 6 and 4.9 +/- 0.5-fold, n = 15, respectively). In contrast, selective IL-1 pretreatment of leukocytes, or the addition of IL-1 during the adhesion assay, did not alter endothelial-leukocyte adhesion. Conditioned medium from IL-1-treated endothelial cultures also did not promote leukocyte adhesion to untreated monolayers. IL-1 induction of endothelial adhesivity was concentration dependent (maximum, 10 U/ml), time dependent (peak, 4-6 h), and reversible, was blocked by cycloheximide (10 micrograms/ml) or actinomycin D (5 micrograms/ml) but not by acetylsalicylic acid (100 microM), and occurred without detectable endothelial cell damage. IL-1 treatment of SV40-transformed human endothelial cells and dermal fibroblasts did not increase their adhesivity for leukocytes. These data suggest that IL-1 can act selectively on human vascular endothelium to increase its adhesivity for circulating blood leukocytes, and thus to localize leukocyte-vessel wall interactions at sites of inflammation in vivo.

Dansylcadaverine and rimantadine inhibition of phagocytosis, PAF-acether release, and phosphatidylcholine synthesis in human polymorphonuclear leukocytes

The effect of dansylcadaverine, amantadine, and rimantadine on phagocytosis, release of PAF-acether, phospholipid methylation, and phosphatidylcholine formation by the cholinephosphotransferase pathway was assessed in human peripheral polymorphonuclear leukocytes. All of the drugs induced a dose-dependent and reversible inhibition in the uptake of complement-coated zymosan particles as well as a reduction in the release of PAF-acether. Simultaneously, a marked reduction in the formation of phosphatidylcholine was observed. This was also dose-dependent and reversible, and showed this order of potency: dansylcadaverine > rimantadine > amantadine. Dansylcadaverine reduced phospholipid methylation to a lesser extent than the cholinephosphotransferase pathway. These data show that drugs that block receptor-mediated endocytosis inhibit the response of the human polymorphonuclear cell and suggest that this action may be mediated by their actions on phospholipid metabolism.

SLOW REACTING SUBSTANCE FROM HUMAN PERIPHERAL POLYMORPHONUCLEAR LEUKOCYTES BY INCUBATION WITH FRESH AUTOLOGOUS SERUM AND ZYMOSAN : Part.1 Requirement for SRS Release from PMNs and Comparison of the Behavior of Synthetic Leukotriens and SRS Released from Human PMN on HPLC

SLOW REACTING SUBSTANCE FROM HUMAN PERIPHERAL POLYMORPHONUCLEAR LEUKOCYTES BY INCUBATION WITH FRESH AUTOLOGOUS SERUM AND ZYMOSAN : Part.1 Requirement for SRS Release from PMNs and Comparison of the Behavior of Synthetic Leukotriens and SRS Released from Human PMN on HPLC

LEUKOTRIENE B 4, AN INFLAMMATORY MEDIATOR IN GOUT

Leukotriene B 4 (LTB 4), a metabolite of arachidonic acid and a potent cytotaxin, is generated by human peripheral polymorphonuclear leucocytes (PMNs) exposed to monosodium urate crystals (MSU). The leukotriene is present in gouty effusions in concentrations significantly greater than those found in synovial fluid from patients with active rheumatoid arthritis or osteoarthritis. Further metabolism and biological deactivation of LTB 4 by PMNs are partly inhibited by MSU. It is concluded that LTB 4 is an important chemical mediator in an acute gouty attack.

Interaction of pseudomonas exoproducts with phagocytic cells.

Polymorphonuclear leukocytes play the major role in host defense against infections with Pseudomonas aeruginosa; however, mononuclear cells also may contribute to defense against pulmonary infections with P. aeruginosa. Therefore, we examined the effects of three extracellular products of P. aeruginosa, exotoxin A, alkaline protease, and elastase, on the function of phagocytic cells. Phagocytosis or killing, protein synthesis, and membrane integrity were used as assays of cellular function. Pseudomonas toxin readily inhibited protein synthesis in mouse peritoneal macrophages; in contrast, proteolytic enzymes did not alter protein synthesis, but transiently decreased the sensitivity of macrophages to toxin. High levels of toxin reduced protein synthesis in human peripheral polymorphonuclear leukocytes but did not alter the ability of these cells to kill P. aeruginosa. Elastase and alkaline protease did not cause release of marker enzymes and did not directly inhibit the bactericidal activity of polymorphonuclear leukocytes; killing was reduced due to inactivation of complement components. In conclusion, these potential virulence products do not modify phagocyte function directly and thus do not directly interfere with host response in pseudomonas infections.

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump.

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.

Morphological changes in human polymorphonuclear leukocytes induced by C3a in the presence and absence of cytochalasin B.

Treatment of human peripheral polymorphonuclear leukocytes(PMNs) with C3a leads to profound changes in cell morphology, along with the limited secretion of lysozyme. Simultaneous addition of C3a and cytochalasin B enhances the release of lysosomal enzymes and renders the cell morphology similar to that seen when either C5a or n-formyl peptides are added to PMNs in the presence of cytochalasin B. The morphological response of PMNs treated with C3a is considerably more heterogeneous than that induced by either C5a or n-formyl-methionyl-leucyl-phenylalanine, indicating that some, but not all, PMNs have receptors for C3a.

Degradation of basic protein and Wolfgram protein in central nervous system myelin by soluble enzymes of human peripheral polymorphonuclear leucocytes

The present communication describes the ability of soluble enzymes (SE) of peripheral polymorphonuclear leucocytes of control and multiple sclerosis (MS) patients to degrade major myelin proteins of MS and control myelin. MS and control SE degraded in situ both Wolfgram protein (WP) and basic protein (BP) of isolated myelin. No differences were found between the action of control and MS patients SE on myelin. However, significantly less degradation of BP and WP in control myelin compared to that in MS myelin was found. Only 30% of SE samples (both control and MS) degraded significant amounts of proteolipid protein in myelin. It is postulated that SE associated demyelination in MS may be a factor contributing to the demyelinating process.

Inhibition by sugars of opsonization of Staphylococcus aureus in fetal calf serum.

Phagocytosis of Staphylococcus aureus by human peripheral polymorphonuclear leucocytes in vitro in the presence of fetal calf serum was inhibited by D-galactose, N-acetyl-D-glucosamine and asialofetuin but not by fetuin. Absorption of opsonin in the serum with the bacteria, inhibition of the absorption by sugars and the effect of glycosidase treatment of the bacteria on phagocytosis showed that terminal structures of polysaccharides on the surface of bacteria play an important role in binding of opsonin to the target.