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PubMed1993

Dextran sulphate inhibits neutrophil emigration and neutrophil-dependent plasma leakage in rabbit skin.

Rolling of neutrophils in postcapillary venules is believed to be an obligatory step in the adhesion of neutrophils to endothelial cells, preceding firm attachment and emigration. This rolling on the endothelial surface can be blocked by sulphated polysaccharides. Therefore, dextran sulphate or dextran T500 was injected intravenously (i.v., 25 mg/kg) and neutrophil infiltration and plasma leakage in skin were measured in response to intradermal (i.d.) injection of inflammatory mediators in the presence of calcitonin gene-related peptide (CGRP). Neutrophil accumulation and oedema formation induced by the neutrophil chemoattractants, f-Met-Leu-Phe (FMLP) or C5adesArg, were completely suppressed in the presence of dextran sulphate. Neutrophil emigration in response to interleukin-1 (IL-1) was not affected by dextran sulphate. These results suggest that neutrophil recruitment induced by FMLP and C5adesArg, but not by IL-1, is mediated via sulphated glycans, perhaps on endothelial cells.

Animals↗

PubMed1998

The ratio of neutrophils to lymphocytes and the phenotypes of neutrophils in patients with early gastric cancer.

We examined 55 patients (40 male, 15 female) who were diagnosed from 1987 to 1991 as having early gastric cancer (EGC) stage I according to the general rules of classification of the Japanese Research Society for Gastric Cancer. Of the 55 patients, 42 (30 male, 12 female) were alive in April 1992. The prognosis correlated well with the ratio of neutrophils to lymphocytes (N/L ratio) but not with the total number of white blood cells in the peripheral blood. The patients were divided into two groups according to their N/L ratio. Of the 29 patients with an N/L ratio less than 2, 27 were alive in 1992, whereas only 15 of the 26 patients with an N/L ratio of 2 or more were alive (chi2 analysis, P=0.0022). We further examined the phenotypes of neutrophils from 29 other patients with EGC at the time of diagnosis before surgical operation. These patients were divided into two groups: 17 patients with a low N/L ratio (less than 2) and 12 patients with a high N/L ratio (2 or more). CD10 and CD35 expressions on neutrophils from the patients with a low N/L ratio were lower than those from the patients with a high N/L ratio. The N/L ratio correlated well with both CD10 and CD35 expression, whereas no correlation was observed between the numbers of neutrophils and the expression of these phenotypes. The respiratory burst of neutrophils from the patients with a high N/L ratio was higher than that of neutrophils from the patients with a low N/L ratio, though there was no correlation in the phagocytic activity between both groups. It was thus suggested that the heterogeneity of neutrophils is, at least partly, related to the prognosis of patients with EGC.

Adult↗

PubMed1986

Isolation of human neutrophil plasma membranes employing neutrophil cytoplasts and changes in the cell-surface proteins upon cell activation.

A plasma membrane fraction, highly enriched in 5'-nucleotidase activity, was prepared from human neutrophils by disruption of previously formed neutrophil cytoplasts (enucleated neutrophils), which were devoid of intracellular organelles. This plasma membrane fraction shows an extremely low contamination by specific and azurophilic granule markers as compared to previous reported preparations. Nevertheless, a novel tertiary granule (Mollinedo, F. and Schneider, D.L. (1984) J. Biol. Chem. 259, 7143-7150), unlike specific and azurophilic granules, fuses partially with the cell surface under the experimental conditions used for cytoplast preparation. Comparison between the external cell-surface proteins in resting neutrophils and neutrophil cytoplasts by lactoperoxidase-catalyzed iodination showed some differences both in deletion and in addition of proteins. In resting cells, iodine was incorporated into at least 13 proteins ranging in size from over 200 to 30 kDa. A 140 kDa polypeptide, representing the major labeled surface component in resting neutrophils, was absent from cytoplasts. Furthermore, high-molecular-weight proteins (110 and over 160 kDa were more exposed to iodination after cytoplast preparation. Activation of human neutrophils by N-formylmethionylleucylphenylalanine induced some alterations in the pattern of labeled cell-surface proteins, which correlated to a certain degree with those observed during cytoplast preparation.

5'-Nucleotidase↗

PubMed1992

Time-dependent utilization of platelet arachidonic acid by the neutrophil in formation of 5-lipoxygenase products in platelet-neutrophil co-incubations.

The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

Arachidonate 5-Lipoxygenase↗

PubMed1996

Effects of salvianolic acid A on oxygen radicals released by rat neutrophils and on neutrophil function.

Salvianolic acid A (Sai A) has demonstrated potent antioxidant activity in previous studies. In the present study, the effects of Sai A on oxygen radicals released by rat neutrophils and on neutrophil function were investigated. Superoxide anion assayed by the nitroblue tetrazolium test and hydrogen peroxide detected with redox of scopoletin were scavenged concentration dependently by Sai A in n-formyl-methionyl-leucylphenylalanine (fMLP)- and phorbol myristate acetate (PMA)-stimulated rat neutrophils. Hydroxyl radicals generated in PMA-stimulated neutrophils, measured by HPLC, also were scavenged significantly by Sai A, whereas Sai A showed no significant effects on chemotaxis toward fMLP and phagocytosis of latex beads by rat neutrophils. In addition, the intracellular free calcium and cyclic nucleotide levels of neutrophils, when stimulated by fMLP, were not affected by Sai A. These results suggest that Sai A could significantly scavenge oxygen radicals released by activated neutrophils without affecting their functional ability.

Animals↗

PubMed1994

Lipopolysaccharide-induced pleural neutrophil accumulation depends on marrow neutrophils and platelet-activating factor.

The involvement of platelet-activating factor (PAF) in lipopolysaccharide (LPS)-induced leukocyte accumulation in the rat pleural cavity was investigated. Intrathoracic (i.t.) injection of LPS (250 ng/cavity) induced a marked increase in the number of neutrophils at 1 h, which was maximum within 6-12 h, reducing after 24 h. In parallel, an increase in blood neutrophil counts within 1-6 h, concomitantly with a reduction in the number of these cells in the bone marrow, was observed. The number of eosinophils recovered from LPS-injected pleural cavity increased at 12 h and was maximum within 24-48 h. No change in blood or bone marrow eosinophil counts was detected. The pretreatment with WEB 2086 or PCA 4248 (20 mg/kg) significantly inhibited pleural neutrophil accumulation, blood neutrophilia and the decrease in the marrow neutrophil content, but not eosinophil accumulation. The blood neutrophilia and the decrease in marrow neutrophil counts induced by the intravenous (i.v.) injection of LPS (250 ng) were significantly lower than those observed after i.t. injection. Furthermore, WEB 2086 and PCA 4248 were ineffective against the systemic alteration induced by i.v. LPS. it was concluded that LPS-induced neutrophil, but not eosinophil, accumulation in the pleural cavity is related to the mobilization of neutrophils from the bone marrow and involves PAF dependent mechanisms.

Animals↗

PubMed2006

From acute febrile neutrophilic dermatosis to neutrophilic disease: forty years of clinical research.

In 1964, Sweet described an acute febrile neutrophilic dermatosis. It is now widely accepted that Sweet's syndrome belongs to a group of associated neutrophilic dermatoses. Although clinically dissimilar, Sweet's syndrome, pyoderma gangrenosum, subcorneal pustular dermatosis, erythema elevatum diutinum, and a few other conditions can be considered a part of this same pathologic spectrum of inflammatory disorders because of (1) the existence of transitional and overlap forms; (2) the similar histopathologic feature of an infiltrate by normal polymorphonuclear leukocytes; (3) the possible occurrence of extracutaneous neutrophilic infiltrates, defining the neutrophilic disease; and (4) the frequent association with systemic diseases. According to the localization of the neutrophilic infiltrate, we describe neutrophilic dermatoses en plaques (dermal), superficial (epidermal), and deep (dermal and hypodermal). Almost every organ of the body may be involved by a neutrophilic aseptic inflammation. The main systemic diseases associated with neutrophlic dermatoses are hematologic, gastrointestinal, and rheumatologic diseases. Although the pathophysiology of these conditions is still poorly understood, treatment with systemic anti-inflammatory agents is usually efficacious.

Abscess↗

PubMed2004

An IgM class anti-neutrophil cytoplasm antibody inhibits neutrophil adhesion and apoptosis via a Syk dependent signaling cascade.

Anti-neutrophil cytoplasm antibodies (ANCA) with specificity for myeloperoxidase (MPO) are implicated as pathogenic agents in pauci-immune systemic vasculitis. In agreement with previously published observations we show that human neutrophils incubated with an MPO-specific IgG class monoclonal antibody are pro-adhesive and undergo apoptosis more readily in vitro. If apoptotic neutrophils are incubated with this antibody they are readily phagocytosed by macrophages and we show that 'blocking' antibodies to FcgammaRIIa (CD32) on the macrophage inhibit this process. We also examined the effect of E3MPO, a monoclonal anti-MPO antibody derived from a patient with severe systemic vasculitis. E3MPO is closely related to the cold-agglutinins and bears an epitope recognized by the monoclonal antibody 9G4 which is expressed on antibodies derived from the V4-34 germ-line immunoglobulin gene. In previous studies, we have shown that anti-MPO antibodies present in sera from patients with vasculitis often bear this epitope. In contrast to the IgG-class antibody, incubation of neutrophils with E3MPO inhibited neutrophil adhesion and apoptosis. Apoptotic neutrophils however were phagocytosed more readily by macrophages in the presence of E3MPO. The effects of E3MPO on neutrophil adhesion and apoptosis were inhibited by piceatannol, an inhibitor of Syk-family kinases; activation of which is associated with cross-linking of the beta(2)-integrins. We show that surface-expressed MPO co-localizes with these beta(2)-integrins and suggest that cross-linking of beta(2)-integrin-bound MPO by polyvalent antibodies could result in signaling through these receptors. We have demonstrated that there are different consequences of Fcgamma-receptor-dependent and -independent signaling mediated by ANCA.

Adenosine↗

PubMed2006

A novel flow cytometric assay of human whole blood neutrophil and monocyte CD11b levels: upregulation by chemokines is related to receptor expression, comparison with neutrophil shape change, and effects of a chemokine receptor (CXCR2) antagonist.

RATIONALE: Smokers who develop chronic obstructive pulmonary disease (COPD) have amplified inflammation within their lungs, involving selective tissue accumulation of neutrophils, macrophages and CD8+ T cells. CD11b (Mac-1, alphaMbeta(2)-integrin) is both a complement receptor (CR3) and a cell adhesion molecule present on the surface of peripheral blood leukocytes, and undergoes rapid surface upregulation from preformed cytoplasmic stores on activation. Cellular activation can also trigger chemotaxis and shape change, the activation itself being caused by the binding of chemokines to cell surface receptors. METHODS: We developed a method of whole blood flow cytometry to measure neutrophil and monocyte CD11b upregulation on CD16+ and CD14+ cells, employing staining with the nuclear dye LDS-751 immediately before flow cytometry. In addition we assessed neutrophil shape change by modified gated autofluorescence with forward scatter (GAFS), this being correlated with chemotactic responses. RESULTS: In smokers with COPD there was a lower maximal shape change for neutrophils in response to CXCL8 (IL-8) in comparison to healthy smokers (p=0.025), and a trend for lower expression of CD11b and shape change in response to CXCL1 (GRO-alpha). Neutrophils were found to predominantly express chemokine receptors CXCR1 and CXCR2 and respond to CXCL8 with CD11b upregulation, while monocytes express more CCR2 and upregulate CD11b preferentially to CCL2 (MCP-1). A CXCR2 antagonist (SB-656933) was found to inhibit neutrophil CD11b upregulation (IC50=260.7nM) and shape change (IC50=310.5nM) in COPD patients. CONCLUSIONS: Neutrophils and monocytes participate in inflammatory processes in a range of diseases. These whole blood assays can be employed to monitor activity in disease and perform in vitro and ex vivo assessment of chemokine receptor (CXCR) antagonists.

Antigens, CD↗

PubMed2002

CXCL8((3-73))K11R/G31P antagonizes the neutrophil chemoattractants present in pasteurellosis and mastitis lesions and abrogates neutrophil influx into intradermal endotoxin challenge sites in vivo.

The ELR(+) CXC chemokines are critical for protective neutrophil responses to most bacterial infections, but nevertheless can contribute importantly to the pathogenic effects of many inflammatory responses. We recently engineered a series of high affinity CXCL8/IL-8 antagonists, one of which, CXCL8((3-73))K11R/G31P, binds very strongly to neutrophils via the CXCR1 and CXCR2. Herein we show in competitive 125I-ligand binding assays that bovine CXCL8((3-73))K11R/G31P has an affinity for neutrophils that is 2-3 orders of magnitude higher than that of CXCL8/IL-8. Furthermore, when used at approximately 0.5 nM, CXCL8((3-73))K11R/G31P inhibited by 50% the chemotactic responses of neutrophils to 129 nM CXCL8/IL-8, but it also blocked chemotactic responses to the alternate ELR-CXC chemokines CXCL1/GRO alpha and CXCL5/ENA-78. Furthermore, CXCL8((3-73))K11R/G31P could inhibit by 93-97% the spectrum of neutrophil chemotactic activities present within wash fluids from clinical bacterial pneumonia or experimental endotoxin-induced mastitis lesions. Finally, intramuscular or subcutaneous application of CXCL8((3-73))K11R/G31P (75 micro g/kg) reduced by up to 97% neutrophil infiltration into intradermal endotoxin challenge sites in cattle, and prevented their circulating neutrophils from responding to CXCL8/IL-8 or ENA-78 in vitro. This data thus encourages further investigation of the potential impact of this novel antagonist on ELR-CXC chemokine-driven inflammatory disorders.

Animals↗

PubMed1999

The effects of a neutrophil elastase inhibitor (ONO-5046.Na) and neutrophil depletion using a granulotrap (G-1) column on lung reperfusion injury in dogs.

BACKGROUND: Activated neutrophils are reported to be closely involved in ischemia-reperfusion injury after lung transplantation. We investigated the beneficial effects of a new recombinant specific neutrophil elastase inhibitor, ONO-5046.Na, and an extracorporeal-type granulotrap (G-1) column on ischemia-reperfusion lung injury, by using an in situ warm lung ischemia model in dogs. METHODS: Warm ischemia was induced for 3 hours by clamping the pulmonary arteries and veins. The left main bronchus was bisected and reanastomosed prior to reperfusion. The left lung was collapsed for 3 hours. A total of 27 adult mongrel dogs were divided into three groups: the control group (n = 9) treated with a saline vehicle; the ONO group (n = 9), in which ONO-5046.Na was continuously administrated from before induced ischemia and to ending 2 hours after reperfusion; and the G-1 group (n = 9), in which a G-1 column was applied for 90 minutes starting 30 minutes before reperfusion under passive bypass support. RESULTS: Circulating neutrophils in the G-1 group decreased significantly (p<.05) compared to preischemia, and significantly decreased compared with the other groups after reperfusion. Oxygenation was improved actually and pulmonary vascular resistance was kept lower level after the administration of ONO-5046.Na. The increase of lung weight was significantly ameliorated in both the G-1 and ONO groups. In the histopathological study, lungs from the control group demonstrated diffuse alveolar edema, neutrophil infiltration, massive alveolar exudate and hemorrhage, and thickening of the interstitium. Lungs from the G-1 group showed mild swelling of the alveolar wall and neutrophil infiltration. Lungs from the ONO group showed virtually no abnormalities. CONCLUSION: This study demonstrated that a neutrophil elastase inhibitor and neutrophil depletion prevented lung reperfusion injury. These treatments may prevent ischemia and reperfusion injury in lung transplantation.

Animals↗

PubMed1992

High- and low-affinity binding of GRO alpha and neutrophil-activating peptide 2 to interleukin 8 receptors on human neutrophils.

GRO alpha and neutrophil-activating peptide 2 (NAP-2), like their analog interleukin 8 (IL-8), are considered to be inflammatory mediators since they recruit and activate neutrophil leukocytes. After introduction of tyrosines by substitution for other residues at the C terminus, GRO alpha and NAP-2 were labeled with 125I and used for binding studies. A total of 60,000-90,000 receptors per neutrophil were found for either ligand. Of these 30-45% were of high affinity with a mean Kd value of 0.3 and 0.7 nM for GRO alpha and NAP-2, respectively, and 55-70% of low affinity (Kd = 30 nM). Two proteins of approximately 70 kDa and 44 kDa (p70 and p44) were specifically cross-linked with labeled GRO alpha, NAP-2, and IL-8. Unlabeled IL-8 fully inhibited this cross-linking and the binding of labeled GRO alpha or NAP-2 to the high-affinity sites on neutrophils or neutrophil membranes. Treatment of membranes with digitonin resulted in the preferential solubilization of a single receptor species, corresponding to p44, that bound GRO alpha and NAP-2 with low affinity (Kd = 30 nM) and IL-8 with high affinity (Kd = 0.4 nM). Exposure of neutrophil membranes to 100 microM guanosine 5'-[gamma-thio]triphosphate led to a 75-fold increase of the Kd in approximately 60% of the IL-8 receptors. High-affinity receptors for GRO alpha and NAP-2 were similarly affected. In contrast, guanosine 5'-[gamma-thio]triphosphate had no effect on the binding of IL-8 to p44 solubilized by digitonin. These results demonstrate that human neutrophils bear two classes of receptors for GRO alpha, NAP-2, and IL-8 (p70 and p44) that may differ in their mode of interaction with GTP regulatory proteins.

Cell Membrane↗

PubMed1995

Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.

Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface-bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.

Cathepsin G↗

PubMed1981

Neutrophil bipolar shape formation in whole blood. A simple and rapid method for the assessment of neutrophil leukocyte responsiveness.

The described procedure permits assessment of the neutrophil shape change response to formyl-methionyl peptides added directly to EDTA anticoagulated whole blood. Response is monitored by counting the resultant number of "bipolar" neutrophil forms present on stained blood films. The preparation of monolayer blood films with a blood "spinner" facilitates uniform sample analysis. Time- and dose-response relationships for neutrophil bipolar shape formation (BSF) agree with data reported for other neutrophil response measurements. No significant change in leukocyte or platelet counts occurred after the addition of control or stimulatory agents to whole blood, nor did 90 min aging of freshly collected blood samples appear to influence the BSF response. In a study of hospitalized patients, BSF values were generally similar in those patients with normal or elevated leukocyte counts, while significantly decreased BSF was observed in leukopenic patients. A significant rise in BSF was detected as patient absolute neutrophil counts increased from a normal to an elevated range. When compared with a control group, the distribution of BSF response values for patients was significantly different. The simple and rapid measurement of BSF in whole blood might facilitate clinical assessment of neutrophil responsiveness, by eliminating many of the difficulties associated with neutrophil isolation.

Cell Separation↗

PubMed1991

The effect of anti-neutrophil cytoplasm autoantibodies on the signal transduction in human neutrophils.

The effect of anti-neutrophil cytoplasm autoantibodies (ANCA) on neutrophil activation was studied by pre-incubating neutrophils with IgG and F(ab')2 prepared from ANCA patients with Wegener's granulomatosis or microscopic polyarteritis. We measured the generation of inositol triphosphate (IP3) (a product of hydrolysis of membrane phospholipid which acts as a second intracellular messenger), and the translocation of protein kinase C (PKC) upon stimulation by a chemotactic peptide, fMet-Leu-Phe (fMLP). ANCA+ F(ab')2 did not induce a significant increase in IP3 generation. Nonetheless, ANCA+ F(ab')2 and ANCA+ IgG pretreatment of human neutrophils reduced the production of inositol phosphates upon subsequent fMLP stimulation compared with experiments performed when cells were pretreated with F(ab')2 and IgG prepared from ANCA- healthy subjects. A significantly reduced generation of IP3 and inositol biphosphate (IP2) was observed. ANCA+ F(ab')2 pretreatment of neutrophils inhibited fMLP-stimulated IP3 generation in a dose-dependent manner. The membrane-bound PKC activity upon stimulation by FMLP and PMA was reduced in neutrophils pretreated with ANCA+ F(ab')2 and IgG. These results indicate that ANCA affect in vitro signal transduction (IP3 generation, and translocation of PKC) in human neutrophils. Apparently, further activation of signal transduction by chemotactic peptide is significantly blunted in cells pre-incubated with ANCA+ F(ab')2 but not with F(ab')2 from healthy controls.

Arteritis↗

PubMed1984

Reduced infectivity of Nematospiroides dubius larvae after incubation in vitro with neutrophils or eosinophils from infected mice and a lack of effect by neutrophils from normal mice.

Neutrophils and eosinophils, isolated from the blood of mice infected with Nematospiroides dubius, were tested for their capacity to damage exsheathed third stage N. dubius larvae in vitro. In the presence of fresh serum from infected mice, both types of granulocyte caused a significant reduction in larval infectivity (up to 40-50%) whereas lymphocytes/monocytes prepared from the same blood samples were inactive. Neutrophils were at least as active as eosinophils, on a cell for cell basis. None of the cells exhibited larvicidal activity in the absence of serum and serum alone had no effect. The reduction in larval infectivity caused by neutrophils in the presence of fresh normal mouse serum (NMS) was only marginally less than that obtained using immune mouse serum (IMS), suggesting that complement, which is activated by the larvae via the alternative pathway and mediates the adherence of both cell types, was able to promote the larvicidal effect of these cells in vitro. In contrast to neutrophils, eosinophils were considerably less effective in NMS than in IMS. Both NMS and IMS were ineffective if they had been heat-inactivated or incubated with methylamine at pH 8.0 to destroy complement activity. The immunoglobulin fraction of IMS was also ineffective in promoting neutrophil or eosinophil-mediated larval damage. These results indicate that in this in vitro system antibodies are incapable of directing the activity of either cell type in the absence of complement. A novel finding of this study was that neutrophils from uninfected mice were unable to reduce larval infectivity in the presence of fresh NMS or IMS. 'Altered' neutrophils possessing larvicidal activity appeared in the blood of mice within 4 days of infection with N. dubius.

Animals↗

PubMed1993

Nanomolar arachidonic acid influences the respiratory burst in eosinophils and neutrophils induced by GTP-binding protein. A comparative study of the respiratory burst in bovine eosinophils and neutrophils.

To investigate a possible role of phospholipase A2 (PLA2) in the respiratory burst in bovine eosinophilic and neutrophilic leukocytes dependent on GTP-binding protein (G-protein), we permeabilized these cells with Staphylococcus aureus alpha-toxin and induced NADPH oxidase activity with the non-hydrolysable GTP analogue GTP[S] or the aluminium tetrafluoro complex AlF4-. Under same experimental conditions, cells responded with different onset times. The onset time for eosinophils was 50-200 s, for neutrophils it was only a few seconds. GTP[S] stimulated in neutrophils only 5% of the respiratory burst compared to eosinophils, whereas AlF4(-)-induced comparable responses (neutrophils 120% of eosinophils). GDP inhibited these responses with an IC50 value of 2.4 mM. Arachidonic acid showed, with the exception of AlF4- stimulated neutrophils, on both stimuli and cell types an enhancing effect (150%) that reached its maximum at 0.1-1 microM. The PLA2 inhibitor 4-bromophenacylbromide reduced the GTP[S]- and AlF4(-)-induced response almost completely (10 microM) and the inhibition was not significantly different for eosinophils and neutrophils (IC50 1-3 microM). If the respiratory burst was reduced with 4-bromophenacylbromide to 1-4% of the original value, 10% of the basal NADPH oxidase activity could be restored by addition of only 20-100 nM arachidonic acid. In addition, the PLA2 activator adriamycin enhanced the response in a dose-dependent manner and in the same order as arachidonic acid did. The results presented above suggest that the respiratory burst may be regulated by different low-molecular-mass and/or heterotrimeric G-proteins and an active role for arachidonic acid or its metabolites in the activation and the maintenance of the direct G-protein-stimulated respiratory burst in bovine eosinophils and neutrophils.

Aluminum Compounds↗

PubMed1998

Effects of a neutrophil elastase inhibitor (ONO-5046) on acute pulmonary injury induced by tumor necrosis factor alpha (TNFalpha) and activated neutrophils in isolated perfused rabbit lungs.

The aim of this study was to examine the effect of ONO-5046, a neutrophil elastase (NE) inhibitor, on a model of acute lung injury induced by tumor necrosis factor alpha (TNFalpha) and phorbol myristate acetate (PMA)-activated neutrophils in isolated perfused rabbit lungs. 120 min after TNFalpha (4,000 JRU/ml) was injected into the pulmonary artery (PA), 5 x 10(7) PMA-stimulated neutrophils were infused into the PA together with 1251-rabbit serum albumin (RSA). In the ONO-5046-treated group (ONO), ONO-5046 (20 mg/kg/h) was continuously infused during the experimental period from 30 min prior to neutrophil administration. Saline, the ONO-5046 vehicle, was infused instead of ONO-5046 in the positive control group (ALD) and nonactivated neutrophils were infused without TNFalpha in the negative control group (Cont). PA pressure was monitored over a 240 min period, and bronchoalveolar lavage (BAL) was performed at the end of the experiment. Lung tissues were examined immunohistochemically for the expression of thrombomodulin (TM). The levels of TM in the perfusate were also measured by ELISA and the radioactivities in the BAL fluid, lung tissue and perfusate were determined to calculate the permeability index (PI) as an indicator of alveolar septal or vascular endothelial damage. The rabbit lungs infused with ONO-5046 showed slower and less increases in PA pressure compared with ALD group. The PI was significantly higher in ALD group (PI[BAL] = 0.028 +/- 0.014, PI[LUNG] = 0.04 +/- 0.003) than Cont (PI[BAL] = 0.002 +/- 0.001, PI[LUNG] = 0.015 +/- 0.003) and ONO group (PI[BAL] = 0.004 +/- 0.003, PI[LUNG] = 0.028 +/- 0.003 (p < 0.05). ALD group had higher TM levels in the perfusate and showed decreased expression of TM on the vascular endothelium compared to Cont and ONO group, suggesting that there was shedding of TM on endothelium and ONO-5046 attenuated a shedding of TM. In conclusion, ONO-5046 attenuated acute lung injury by inhibiting the alveolar epithelial and vascular endothelial injury triggered by activated neutrophils. NE appears to play an important role in the neutrophil-induced increase of pulmonary epithelial and microvascular permeability observed in acute lung injury.

Animals↗