Human PMN Research Articles

Peroxynitrite Formation from Activated Human Leukocytes

We showed direct evidence of peroxynitrite formation from polymorphonuclear cells (PMN) with the nitration of 4-hydroxyphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA). Human PMN from healthy volunteers was stimulated with phorbol-12-myristate-13-acetate (PMA, 10 ng/ml) at 37°C in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid-buffered Hank's balanced salt solution (pH 7.4) with HPA (1 mM). NO2HPA was detected under PMA stimulation only in the presence of myeloperoxidase inhibitor. NO2HPA was eliminated by N-monomethyl-L-arginine (100 μM). The inhibition of myeloperoxidase appears to be essential to demonstrate the production of NO2HPA since myeloperoxidase itself or its product, hypochlorite, reacted with peroxynitrite and hampered the formation of NO2HPA.

Human neutrophil secondary granule exocytosis is independent of protein kinase activation and is modified by calmodulin activity

Exocytosis of the secondary (2°) lysosomal granule is an important process in the activation of human neutrophils. Stored enzymes such as collagenase and gelatinase are released, and adhesion molecules from the granule membrane are inserted in the plasma membrane. This exocytosis is independent of azurophil granule release and respiratory burst activation. We investigated, using kinase and phosphatase inhibitors and activators of adenylate cyclase, common intracellular signalling mechanisms involved in exocytosis (vitamin B12 binding protein release) stimulated by different agonists. Exocytosis in response to tumour necrosis factor α (TNFα), phorbol myristate acetate (PMA) and the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was inhibited by the calmodulin antagonist N-(6-amino hexyl)-5-chloro-1-naphthalene sulphonamide (W7). Neither staurosporine, H7 nor genistein was inhibitory. In contrast, the same doses of W7 synergistically enhanced the exocytosis stimulated by the tyrosine phosphatase inhibitor sodium orthovanadate, while kinase inhibition by staurosporine or genistein dose-dependently inhibited the vanadate response. Furthermore, adenylate cyclase activation with prostaglandin E 2 or dibutyryl cyclic AMP, inhibited exocytosis in response to TNFα and FMLP, while having no effect on the release induced by vanadate or PMA. Thus, 2° granule exocytosis stimulated by receptor-bound ligands is calmodulin-dependent, and is independent of protein kinase activity. In contrast, exocytosis in response to tyrosine phosphatase inhibition is antagonised by calmodulin, since the response to vanadate was enhanced synergistically by W7. Thus, depending on the initial stimulus, calmodulin may promote or inhibit 2° granule exocytosis by human PMN.

Effects of human polymorphonuclear leukocyte elastase upon surfactant proteins in vitro

Recent evidence has suggested that elastase is released by polymorphonuclear leukocytes (PMN) recruited from the pulmonary microcirculation into the alveoli during acute lung injury. This study was undertaken to test the hypothesis that elastase from PMN (PMN elastase) damages or degrades one or more of the surfactant proteins (SP-A, SP-B and SP-C) of the lung, and thereby alters its function. We attempted to use amounts of PMN elastase and quantities of surfactant that would be plausible in the lungs of patients with ARDS. Surfactant from normal dog lungs (2 mg phospholipid, 200 μg protein), and purified SP-A (20 μg), SP-B (10 μg and SP-C (10 μg) from the surfactant (identified by SDS-PAGE and N-terminal amino acid sequences) were incubated for 4–8 h at 37°C with various amounts (0.25–1.0 U) of human PMN elastase purified by affinity chromatography. SDS-PAGE and amino acid composition analysis of the surfactant as well as of the purified SP-A, SP-B, and SP-C showed that degradation of these proteins progressed with incubation time and with the amount of PMN elastase, and was accompanied by decreases in isopycnic density (g/cm 3) and surface adsorption, and increase of surface tension of the surfactant. No effects were observed with heat inactivated PMN elastase (95°C, 30 min) or with PMN elastase in the presence of human α-1 protease inhibitor (2 μg/μg elastase). Phospholipid compositions of the surfactant after exposure to PMN elastase were not significantly different from those of the controls, suggesting that SP-A, SP-B, and SP-C play a major role in altering the surfactant properties. SP-A was also degraded by elastase and trypsin from pancreas whereas SP-B and SP-C remained intact, providing a natural surfactant without SP-A. Surface adsorption rate of the SP-A deficient surfactant was lower than that of the control, but was much higher than that of the surfactant with completely degraded SP-A, SP-B, and SP-C, suggesting that hydrophobic SP-B and SP-C are the essential components in enhancing adsorption. We conclude that proteolytic degradation of SP-A, SP-B, and SP-C causes the decrease of surfactant isopycnic density, and is responsible for retarding adsorption resulting in surfactant dysfunction.

Cloning and expression of human defensin HNP-1 genomic DNA in Escherichia coli

We amplified a 181-bp DNA fragment encoding HNP-1 from genomic DNA of human polymorphonucleated neutrophils by PCR. Sequencing of this fragment revealed that it only contained the mature protein coding region of HNP-1 and no intron was found in the sequences. The PCR product of HNP-1 genomic DNA was then cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (the GST-HNP-1 fusion protein). The GST-HNP-1 fusion protein was expressed as insoluble inclusion bodies, so they were collected and solubilized in 8 M urea. Thus, reasonably pure GST-HNP-1 fusion protein was obtained, with which rabbits were immunized to raise polyclonal antibody against HNP-1. Ouchterlony immunodiffusion and immunoblotting showed that the antiserum reacted with chemically synthesized HNP-1, -2 and human PMN extracts and did not cross react with human GST in these assays. We demonstrated that human defensin HNP-1, a small cationic protein, was expressed in E. coli as an insoluble GST fusion protein, and polyclonal antibody raised by immunizing rabbits with this fusion protein did not cross react with human GST and was specific to defensins.

Azurocidin, a Natural Antibiotic from Human Neutrophils: Expression, Antimicrobial Activity, and Secretion

The azurophil granules of human PMN contain four antibiotic proteins, the serprocidins, which have extensive homology to one another and to serine proteases. Azurocidin, a member of this family, is a 29-kDa glycoprotein with broad spectrum antimicrobial activity and chemotactic activity toward monocytes. Insect cells transfected with a baculovirus vector carrying azurocidin cDNA produced a recombinant azurocidin protein. We purified the recombinant azurocidin protein from the culture medium of the infected cells and showed that it retained the antimicrobial activity of the native neutrophil-derived molecule. In addition, we present evidence that a 49-amino-acid region of the recombinant azurocidin protein is required for its secretion from insect cells.

Involvement of bradykinin B1 and B2 receptors in human PMN elastase release and increase in endothelial cell monolayer permeability

Bradykinin (BK) is a potent inflammatory mediator, which can release other inflammatory mediators by interacting with bradykinin B1 and B2 receptors. The role of kinins in regulating human PMN elastase release was studied. BK induced elastase release 5-fold over basal levels. Elastase release was inhibited by both B1 and B2 receptor antagonists. A specific B1 agonist des-Arg10-KD increased elastase release 4-fold. Since elastase has been implicated in vascular leak, the effect of BK on endothelial cell monolayer (EM) permeability was assessed. BK increased EM leak (I125flux) across the EM, whereas des-Arg10-KD was inactive. When co-cultured with human umbilical vein endothelial cells, des-Arg10-KD-treated PMNs increased EM leak by 35%. The elastase inhibitor AAVPK blocked des-Arg10-KD-induced leak by 80% suggesting that elastase is responsible for the increase in permeability. It is concluded that BK causes increased leak by inducing PMN elastase release via activation of both B1 and B2 receptors. BK blockade and elastase inhibition may be beneficial in inflammatory diseases such as ARDS which is characterized by increased lung permeability and both kinin and PMN activation are thought to participate.

Direct killing of interleukin-2-transfected tumor cells by human neutrophils

We have previously established that human polymorphonuclear cells (PMN) express IL-2R beta- and gamma-chains and that addition of IL-2 maintains the viability of PMN by preventing these cells from undergoing programmed cell death. The purpose of this study was to examine whether IL-2-releasing tumor cells are capable of stimulating PMN tumoricidal activity. We therefore investigated the ability of PMN to kill IL-2-transfected tumor cells using normal human PMN directed against the murine mammary adenocarcinoma TS/A engineered to release high amounts of murine IL-2 (3,600 U, B6) compared with TS/A parental cells and TS/A tumor cells transfected with the neomycin-resistance (NEO) gene only. The potency of PMN as IL-2-induced killer cells was indicated by the low number of cells required for killing (effector cell:target cell ratio 10:1) and the degree of tumor cell lysis (68+/-10%). Evidence for the role of IL-2 as a mediator of tumor cytotoxicity by PMN was substantiated by inhibition of tumor killing with anti-IL-2 and anti-IL-2R beta monoclonal antibodies (MAbs). Furthermore, in vivo depletion of mature granulocytes using MAb RB6-8C5 resulted in B6 adenocarcinoma growth, thereby confirming a direct role for IL-2-activated PMN in tumor cytolysis. Lastly, we suggest that one possible mechanism involved in IL-2-induced PMN cytotoxicity against the B6 clone occurs via the nitric oxide pathway, which could be inhibited upon addition of the arginine analog, N(G)-monomethyl-L-arginine.

Altered Oxygen Tension Modulates Cytokine-Induced Signal Transduction in Polymorphonuclear Leukocytes: Regulation of the GPLCPathway

The purpose of these studies was to examine the sensitivity of the PIP2–PLC-transducing pathway (GPLC) and its relationship to the respiratory burst in human polymorphonuclear leukocytes (PMN) stimulated by IL-8, TNF-α, or IL-1β during sequential changes in buffer oxygen tension from normoxia (pO2= 180–200 mm Hg), to hypoxia (pO2< 30 mm Hg) and then reoxygenation (pO2> 140 mm Hg). Our specific hypothesis was that altered oxygen tensions would regulate the GPLCpathway in human PMN. GPLCactivity was assayed by investigating phospholipase C activity by measuring inositol phosphates and diacylglycerol (DAG) formation. Respiratory burst activity was assayed as O−2production and NADPH oxidase activation in intact PMN and in a cell-free system, respectively, and correlated separately to both early and late DAG production. At 1 min, DAG formation during normoxia was decreased by IL-8 plus fibronectin while hypoxia had no regulatory effect on control of DAG formation by any of the cytokines. In contrast to early DAG formation, hypoxia significantly downregulated late DAG formation induced by buffer without fibronectin, IL-8 plus fibronectin, and IL-1β with or without fibronectin. Hypoxia/reoxygenation in and of itself significantly increased DAG formation vs levels seen in the presence or absence of IL-8, TNF-α, or IL-1β with or without fibronectin. Changes in early DAG production during the alterations in oxygen tension correlated best with corresponding changes in O−2production in intact cells, whereas late DAG production correlated best with NADPH oxidase activation assayed in the cell-free system. Thus, changes in oxygen tension can directly modulate the extent of the PMN response to stimulation by IL-8, TNF-α, or IL-1β and the GPLC-receptor pathway is particularly regulated by physiologically relevant periods of hypoxia/reoxygenation.

5(S),15(S)-dihydroxyeicosatetraenoic acid and lipoxin generation in human polymorphonuclear cells: dual specificity of 5-lipoxygenase towards endogenous and exogenous precursors.

5-Lipoxygenase activation of human blood polymorphonuclear cells (PMN) from asthmatic patients (asthmatics) was studied to investigate whether differences may exist with healthy subjects (controls). The respective cell capacities to produce lipoxins (LXs), leukotrienes, and 5(S), 15(S)-dihydroxyeicosatetraenoic acid [5(S),15(S)-diHETE] were compared under in vitro stimulation by ionophore A23187, with or without exogenous 15(S)-hydroxyeicosatetraenoic acid [15(S)-diHETE]. Eicosanoids were analyzed by elution with an isocratic reverse-phase high performance liquid chromatography system, and their profiles, detected by simultaneous monitoring at 302, 280, and 246 nm, were evaluated on the basis of chromatographic behavior: UV spectral characteristics and coelution with synthetic standards. In the presence of exogenous 15(S)-HETE, human PMN were able to produce LXs and 5(S),15(S)-diHETE, PMN from asthmatics were able to produce 5(S), 5(S),15(S)-diHETE, and LXs from endogenous sources, whereas in the same experimental conditions, no detectable amounts of these compounds were released by PMN from controls. The levels of 5(S),15(S)-diHETE, and LXs biosynthesized from endogenous arachidonic acid were highly correlated. Two different LX patterns were observed involving two possible metabolic pathways: (a) via the intermediate 5,6-epoxytetraene alone for LXs generation from exogenous 15(S)-HETE; and (b) via 5,6- and/or 14,15-epoxytetraenes leading to the formation of an enzyme-bound delocalized carbocation for LXs generation from endogenous arachidonate, respectively. The enhanced 5-lipoxygenase activation of blood PMN from asthmatics and the metabolism of exogenous 15(S)-HETE may reflect a priming induced by various mediators released from environmental cells, and could be considered as a model of transcellular signalization between PMN and endothelial cells.

Transcriptional and post-transcriptional regulation of GM-CSF-induced IL-1 beta gene expression in PMN.

Polymorphonuclear leukocytes (PMN) play an important role in inflammation, immune responses, and tissue repair by secreting interleukin- 1 beta (IL-1 beta). We investigated the regulation of IL-1 beta gene expression in human PMN treated with granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF induced IL-1 beta mRNA accumulation at 0.1 ng/ml and maximal induction was observed at 1 ng/ml. IL- 1 beta mRNA levels reached a maximum with 1-2 h after stimulation with GM-CSF and returned to baseline levels by 4-6 h. The time course of IL-1 beta mRNA induction by GM-CSF was more protracted than previously reported for PMN stimulated with tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml). Nuclear run-on analysis indicated that GM-CSF, like TNF, increases IL-1 beta transcription. Kinetic studies with the RNA synthesis inhibitor, actinomycin D, showed that GM-CSF induces stable IL-1B mRNA. Cycloheximide enhanced the IL-1 beta mRNA accumulation by GM-CSF at the level of mRNA stabilization, but blocked IL-1 beta mRNA expression by TNF. Thus, GM-CSF increases IL-1 beta message accumulation in PMN at both the transcriptional and post-transcriptional levels by mechanisms that are different from TNF induction of IL-1 beta gene expression.

Mechanisms of platelet-activating factor-induced lipid body formation: requisite roles for 5-lipoxygenase and de novo protein synthesis in the compartmentalization of neutrophil lipids.

Lipid bodies, lipid rich cytoplasmic inclusions, are characteristically abundant in vivo in leukocytes associated with inflammation. Because lipid bodies are potential reservoirs of esterified arachidonate and sites at which eicosanoid-forming enzymes may localize, we evaluated mechanisms of lipid body formation in neutrophils (PMN). Among receptor-mediated agonists, platelet activating factor (PAF), but not C5a, formyl-methyl-phenylalanine, interleukin 8, or leukotriene (LT) B4, induced the rapid formation of lipid bodies in PMN. This action of PAF was receptor mediated, as it was dose dependently inhibited by the PAF receptor antagonist WEB 2086 and blocked by pertussis toxin. Lipid body induction by PAF required 5-lipoxygenase (LO) activity and was inhibited by the 5-lipoxygenase-activating protein antagonist MK 886 and the 5-LO inhibitor zileuton, but not by cyclooxygenase inhibitors. Corroborating the dependency of PAF-induced lipid body formation on 5-LO, PMN and macrophages from wild-type mice, but not from 5-LO genetically deficient mice, formed lipid bodies on exposure to PAF both in vitro and in vivo within the pleural cavity. The 5-LO product inducing lipid body formation was not LTB4 but was 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE], which was active at 10-fold lower concentrations than PAF and was also inhibited by pertussis toxin but not by zileuton or WEB 2086. Furthermore, 5-HETE was equally effective in inducing lipid body formation in both wild-type and 5-LO genetically deficient mice. Both PAF- and 5(S)-HETE-induced lipid body formation were inhibited by protein kinase C (PKC) inhibitors staurosporine and chelerythrine, the phospholipase C (PLC) inhibitors D609 and U-73122, and by actinomycin D and cycloheximide. Prior stimulation of human PMN with PAF to form lipid bodies enhanced eicosanoid production in response to submaximal stimulation with the calcium ionophore A23187; and the levels of both prostaglandin (PG) E2 and LTB4 correlated with the number of lipid bodies. Furthermore, pretreatment of cells with actinomycin D or cycloheximide inhibited not only the induction of lipid body formation by PAF, but also the PAF-induced "priming" for enhanced PGE2 and LTB4 in PMN. Thus, the compartmentalization of lipids to form lipid bodies in PMN is dependent on specific cellular responses that can be PAF receptor mediated, involves signaling through 5-LO to form 5-HETE and then through PKC and PLC, and requires new protein synthesis. Since increases in lipid body numbers correlated with priming for enhanced PGE2 and LTB4 production in PMN, the induction of lipid bodies may have a role in the formation of eicosanoid mediators by leukocytes involved in inflammation.

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation.

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, "classical chemoattractants" such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and "pure chemoattractants" such as TGF-beta 1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the "non-classical" group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca2+]i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca(2+)-independent G-protein-coupled pathway for post-receptor signal transduction triggered by "pure chemoattractants." The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds.

Adherence of neutrophils to human peritoneal mesothelial cells: role of intercellular adhesion molecule-1.

Peritonitis is accompanied by a massive influx of polymorphonuclear leukocytes (PMN) into the peritoneal cavity, little is known, however, about the process of neutrophil transmigration across the peritoneal membrane. The study presented here, therefore, investigates the adherence of human PMN to human peritoneal mesothelial cell monolayers and examines the importance of intercellular adhesion molecule-1 (ICAM-1) in the process. Human peritoneal mesothelial cells (HPMC) constitutively expressed ICAM-1 protein and mRNA. Stimulation with IL-1 beta or TNF alpha resulted in time- and dose-dependent upregulation of ICAM-1 mRNA transcript and increased cell-surface immunoreactive protein expression. Peak surface expression of ICAM-1 occurred between 12 and 24 h after cytokine stimulation when the level of expression was increased by on average threefold above control. The adherence of PMN to HPMC after stimulation with either IL-1 beta or TNF alpha was both dose- and time-dependent. Peaks of PMN binding to HPMC occurred at 2 and 12 h after stimulation. After 12 h, the number of PMN binding to HPMC increased from 71.3 +/- 12.5 in control cells to 180 +/- 36.5 and 125 +/- 23.6 (x 10(3) PMN), after IL-1 beta (100 pg/mL) and TNF alpha (1000 pg/mL), respectively (z = 2.52 and 2.38, N = 6, P < 0.02 versus control for both). The roles of HPMC ICAM-1 and the PMN counter receptor LFA-1 (CD11a/CD18) in the adherence of PMN to HPMC were confirmed by using anti-CAM Fab2' fragments, and anti-integrin antibodies, all of which significantly reduced the adherence of PMN to both control and cytokine-treated HPMC. These data suggest that ICAM-1 expression by HPMC may be one mechanism by which neutrophils adhere to the mesothelium during their transmigration into the inflamed peritoneal cavity.

Purification and properties of a Ca(2+)-independent barbed-end actin filament capping protein, CapZ, from human polymorphonuclear leukocytes.

In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL(-1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis.

Regulation by anti‐inflammatory cytokines (IL‐4, IL‐10, IL‐13, TGFβ)of interleukin‐8 production by LPS‐ and/ or TNFα‐activated human polymorphonuclear cells

The capacity to down-regulate the production of IL-8 by LPS-activated human polymorphonuclear cells (PMN) has been demonstrated for IL-4, IL-10, and TGFβ. We compared their relative capacities and further extended this property to IL-13. We report a great heterogeneity among individuals related to the responsiveness of PMN to the IL-4 and IL-13 inhibitory effects while their response to the IL-10 effect was homogenous. The inhibitory activities were observed at the transcriptional level. IL-8 induction by TNFα was, unlike its induction by LPS, resistant to the inhibitory effects of IL-10, IL-4, IL-13 and TGFβ. Furthermore, IL-10 and IL-4 inhibitory activity were less effective when TNFα was acting synergistically with LPS to induce IL-8 production by PMN. LPS-induced cell-associated IL-8, detected in the PMN cultures, could be marginally inhibited by IL-4 and IL-10. Altogether, our data demonstrate that IL-13 is able to inhibit LPS-induced IL-8 production by human PMN, although IL-10 remains the most active anti-inflammatory cytokine. Despite the capacity of IL-4, IL-10, and IL-13 to limit the production of TNFα-induced IL-8 in a whole blood assay, none was able to inhibit this production when studying isolated human polymorphonuclear cells.

マレイン酸プログルメタシンによるヒト好中球スーパーオキシド産生阻害

Polymorphonuclear leukocytes (PMN) play an important role in development of inflammation. In the clinical field, non-steroidal anti-inflammation drugs (NSAID), such as proglumethacin maleate, were used to theraphy with arthritis. In this study, we investigated the effects of NSAIDs on superoxide generation of human PMN from synovial fluid.On the PMNs from synovial fluid of rheumatoid arthritis (RA) patients, proglumethacin maleate significantly inhibited the superoxide generation by phorbol 12-myristate 13-acetate (PMA) . The rate and maximum amount of superoxide generation of PMNs were reduced to 12.72±6.32% and 24.23±9.44%, respectively. The other NSAIDs, Indomethacin farnesil, diclofenac sodium and loxoprofen sodium, were also tended to inhibit the superoxide generation by PMA, but not significantly. Indomethacin farnesil, diclofenac sodium and loxoprofen sodium reduced the rate of superoxide generation to 55.88±5.58%, 41.17±2.36% and 45.24±1.77%, respectively. Proglumethacin maleate also strongly inhibited the superoxide generation of PMNs from patients of osteoarthritis (OA), but other NSAIDs did not. The rate and maximum amount of superoxide generation of PMNs from OA patients were reduced by proglumethacin maleate to 21.20±2.79% and 32.36±10.09%, respectively. Indomethacin which is active metabolite of proglumethacin maleate for NSAID did not inhibit the superoxide generation of PMNs from RA and OA patients.These result indicated that proglumethacin maleate have inhibitory action to superoxide generation of PMNs which is not NSAID's action. This inhibition of superoxide generation of PMNs may be one of the anti-inflammatory mechanisms of proglumethacin maleate.

Identification and molecular characterization of the CalB domain of the cytosolic phospholipase A2 (cPLA2) in human neutrophils.

The receptor-mediated activation of human neutrophils by ligands such as chemotactic peptide N-formyl-methionine-leucine-phenylalanine (fMLP) is coupled with the activation of phospholipases C and A2 (1,2). Phospholipase A2 (PLA2) hydrolyzes arachidonic acid (AA) and lysophospholipid from the sn-2 position of 1-alkyl-2-arachidonoyl-sn-3-glycerophosphocholine of the membrane (3). Currently, it is accepted that the cytosolic PLA2 (cPLA2) and not the secretory PLA2 (sPLA2) is primarily responsible for the release of AA (4) in most of the cell systems. The reasons for this assumption are based on the observations such as low requirements of Ca++ concentrations (micromolar for cPLA2 vs milimolar for sPLA2), fatty acid preference of cPLA2 at the sn-2 position (5), resistance of cPLA2 to disulfide-reducing agents (4) and increased concentrations of cytosolic Ca++ (5,6). In addition, it is stimulated by phosphorylation through protein kinase C (PKC), but inhibited by staurosporine, an inhibitor of PKC (7). Conversely, previous studies from our laboratory have shown that the inhibition of PKC by staurosporine potentiated the activation of PLC and PLA2 in fMLP-challenged human PMN (8–10).KeywordsArachidonic AcidHuman NeutrophilArachidonic Acid ReleaseU937 Cell LineCytosolic PhospholipaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Tissue factor expression in human leukemic cells

Patients with acute leukemia are at increased risk for thrombotic and hemorrhagic complications, particularly those patients with acute promyelocytic leukemia (APL) undergoing induction chemotherapy. These serious complications have been attributed by some authors to the release of tissue factor (TF) procoagulant activity (PCA), particularly during cytotoxic chemotherapy. In previous studies of normal peripheral blood cells, only cells of the monocyte lineage have been found to express TF PCA. Therefore, several questions remain regarding the origin and characterization of the PCA in malignant leukemic cells, particularly those thought to be derived from granulocyte progenitor cells. We utilized a full-length cDNA probe, several monoclonal antibodies (MAbs) and a sensitive one-stage PCA assay to study the expression of TF in the human leukemia cell line, HL-60, in human peripheral blood monocytes/macrophages ( Mo Mø ) and in highly purified populations of human polymorphonuclear leukocytes (PMN). In the HL-60 cells we detected low but significant levels of TF mRNA and TF antigen (TF:Ag). In unstimulated cells, coordinate increased levels of TF mRNA, TF:Ag and TF PCA expression were noted following phorbol-ester-induced macrophage differentiation of the cells, but a decreased level of TF mRNA with no change in the basal level of TF:Ag expression occurred following retinoic acid-induced granulocyte differentiation of this cell line. Long-term cultures of stimulated mature Mo Mø demonstrated initial coordinate expression of TF mRNA, TF:Ag and TF PCA, but TF:Ag expression persisted even after 7 days (when TF PCA was undetectable). No TF PCA, TF:Ag or TF mRNA was demonstrated in highly purified populations of human PMN, regardless of culture conditions. Discordant expression of TF mRNA, TF:Ag and TF PCA in HL-60 cells suggests the possibility of novel, post-synthetic mechanisms for the regulation of TF PCA expression, which might be dependent on the phenotypic differentiation level of the cell. Such mechanisms (yet to be defined) might account for the ability of some leukemic cells, which frequently express characteristics of more than one cell line (e.g. monocytes and granulocytes), to express a TF gene product capable of activating blood coagulation.

Nitric oxide synthase in circulating vs. extravasated polymorphonuclear leukocytes.

It is becoming increasingly apparent that certain forms of acute and chronic inflammation are associated with enhanced production of nitric oxide (NO). Although substantial information has been obtained describing the regulation of NO synthase (NOS) in macrophages, little information is available regarding the biochemistry and molecular biology of NOS in circulating vs. extravasated polymorphonuclear leukocytes (PMNs). The objective of this study was to characterize the molecular and biochemical properties of the inducible NO synthase (iNOS) in circulating vs. extravasated rat and human PMNs. Circulating rat and human PMNs were purified from peripheral blood and extravasated PMNs were elicited in rats by intraperitoneal injection of 1% oyster glycogen or in humans by peritoneal dialysis of patients with peritonitis. Inducible NOS mRNA from circulating and elicited PMNs was quantified using slot blot hybridization analysis with a cDNA probe specific for iNOS. iNOS protein was identified using Western immunoblot analysis, and NOS activity was quantified by measuring the NG-monomethyl-L-arginine (L-NMMA)-inhibitable conversion of 14C-labeled L-arginine to L-[14C]citrulline. In a separate series of experiments, circulating or extravasated PMNs were cultured for 4 h and the accumulation of L-NMMA-inhibitable nitrite (NO2-) in the supernatant was determined and used as a measure of NO production in vitro. We found that circulating PMNs (rat or human) contained no iNOS mRNA, protein, or enzymatic activity. Furthermore, circulating rat or human PMNs (2 x 10(6) cells/well) were unable to generate significant amounts of NO2- when cultured for 4 h in vitro. In contrast, iNOS mRNA levels in 4- and 6-h elicited rat PMNs increased 21- and 42-fold, respectively, when compared with circulating cells. Western blot analysis revealed the presence of iNOS protein in the elicited rat PMNs and iNOS enzymatic activity increased from normally undetectable levels in circulating rat PMNs to 81 and 285 pmol/min/mg for the 4- and 6-h elicited rat PMNs, respectively. Approximately 20-30% of the total iNOS activity was Ca(2+)-dependent. Nitrite formation by elicited rat PMNs in the absence of any exogenous stimuli increased from normally undetectable amounts for circulating PMNs to approximately 8 and 11 microM/10(6) cells for the 4- and 6-h elicited PMNs, respectively. Highly enriched preparations of extravasated human PMNs contained neither message, protein nor iNOS enzymatic activity. Taken together our data demonstrate that inflammation-induced extravasation of rat PMNs upregulates the transcription and translation of iNOS in a time-dependent fashion and that 20-30% of the total inducible NOS is Ca(2+)-dependent. In contrast, neither circulating nor extravasated human PMNs contained iNOS message, protein, or enzymatic activity. These data suggest that the human PMN iNOS gene is under very different regulation than is the rat gene.

Isozyme Analysis of Human Normal Polymorphonuclear Leukocyte Phosphofructokinase

Phosphofructokinase (PFK) from human polymorphonuclear leukocytes (PMN) was characterized by immunological titration with subunit specific antibodies, column chromatography on QAE-Sephadex and SDS-polyacrylamide gel electrophoresis. Two different isozymes, M-type and L-type, were found. The Mr values of the M and L subunits were 79,500 ± 1,914 and 74,250 ± 1,258, respectively. The two isozymes presented different kinetic and regulatory properties. The results suggest that PFK from human normal PMN is a mixture of M-type and L-type homotetramers, mainly, with possible minor heterotetrameric forms.