Role of cytochrome b-559 in arachidonic acid activation of resting human neutrophils.

Whether or not cytochrome b-559 is a necessary component of NADPH oxidase activity in neutrophils is still controversial. In highly purified plasma membranes isolated from resting neutrophils and lacking cytochrome b, addition of arachidonic acid induced an NADPH oxidase activity. This activity was similar to that of plasma membranes isolated from phorbol myristate acetate (PMA)-stimulated cells which possessed cytochrome b. Addition of arachidonic acid to the latter plasma membranes did not alter the oxidase activity. It can be concluded that plasma membranes isolated from resting neutrophils have, in the presence of arachidonic acid, an NADPH oxidase activity similar to that of PMA-stimulated cells, except that it is independent of cytochrome b-559.

Priming effect of fibronectin on respiratory burst of human neutrophils induced by formyl peptides and platelet-activating factor.

Fibronectin (FN), a glycoprotein present in the plasma and the extracellular matrix, has been shown to enhance adherence-related functions of polymorphonuclear leukocytes (PMNs). In this study we investigated the effects of FN on the activation of human PMNs in suspension by soluble stimuli, as determined by the generation of superoxide radicals (respiratory burst). FN (up to 100 micrograms/ml) did not directly stimulate the PMN respiratory burst assessed using a sensitive assay, luminol-dependent chemiluminescence (CL). Low FN concentrations (Up to 25 micrograms/ml) caused a dose-dependent enhancement of the CL induced by two chemoattractants. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and platelet-activating factor (Paf), and also by phorbol myristate acetate (PMA), a known protein kinase C activator. Higher FN concentrations were less effective. The potentiation involved both initial rate and total CL responses and was more active on extracellular than intracellular generation of oxygen radicals. FN potentiation persisted after cell washing and was abolished by treatment of FN with trypsin. Measurement of the respiratory burst using the cytochrome c reduction assay confirmed that FN enhanced both the initial rate and total amount of superoxide anion generated by FMLP-stimulated PMNs. These data indicate that FN facilitates the respiratory burst of chemoattractant-stimulated PMNs and suggest that FN can prepare PMNs in suspension for amplified biological functions induced by soluble inflammatory stimuli.

Endothelial cationic amino acid transporter-1 overexpression can prevent oxidative stress and increases in arterial pressure in response to superoxide dismutase inhibition in mice.

AIM: Oxidative stress may play an important role in the pathogenesis of hypertension. The aim of our study is to examine whether increased expression of the predominant endothelial l-arginine transporter, cationic amino acid transporter-1 (CAT1), can prevent oxidative stress-induced hypertension. METHODS: Wild-type mice (WT; n = 9) and endothelial CAT1 overexpressing (CAT+) mice (n = 6) had telemetry probes implanted for the measurement of mean arterial pressure (MAP), heart rate (HR) and locomotor activity. Minipumps were implanted for infusion of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETCA; 30 mg kg(-1) day(-1) ; 14 days) or its saline vehicle. Baseline levels of MAP, HR and locomotor activity were determined before and during chronic DETCA administration. Mice were then killed, and their plasma and kidneys collected for analysis of F2 -isoprostane levels. RESULTS: Basal MAP was less in CAT+ (92 ± 2 mmHg; n = 6) than in WT (98 ± 2 mmHg; n = 9; P < 0.001). During DETCA infusion, MAP was increased in WT (by 4.2 ± 0.5%; P < 0.001) but not in CAT+, when compared to appropriate controls (PDETCA*genotype = 0.006). DETCA infusion increased total plasma F2 -isoprostane levels (by 67 ± 11%; P = 0.05) in WT but not in CAT+. Total renal F2 -isoprostane levels were greater during DETCA infusion in WT (by 72%; P < 0.001), but not in CAT+, compared to appropriate controls. CONCLUSION: Augmented endothelial l-arginine transport attenuated the prohypertensive effects of systemic and renal oxidative stress, suggesting that manipulation of endothelial CAT1 may provide a new therapeutic approach for the treatment of cardiovascular disease associated with oxidative stress.

Cardiovascular reactivity and neuronal activation to stress in Schlager genetically hypertensive mice.

Schlager inbred hypertensive mice (BPH/2J) have been suggested to have high blood pressure (BP) due to an overactive sympathetic nervous system (SNS). The brain nuclei associated with the hypertension are also those involved in the integration of the cardiovascular responses to stress. Therefore, in the present study, we hypothesize that BPH/2J mice likely have a greater response to stress that is associated with greater neuronal activation in the limbic system, hypothalamus and medulla in regions known to regulate sympathetic activity. Male hypertensive BPH/2J and normotensive BPN/3J mice were implanted with telemetry devices and exposed to dirty cage-switch, an acute model of aversive stress. Stress exposure caused a 60% greater pressor response in BPH/2J compared with BPN/3J mice and an increase in activity, by contrast the level of tachycardia was less in BPH/2J mice. Stress-induced cardiovascular responses were also associated with greater neuronal activation, as detected by c-Fos expression, in BPH/2J compared with BPN/3J mice in the medial nucleus of the amygdala (MeAm), dorsomedial hypothalamus (DMH) (P<0.001) and marginally in the rostral ventrolateral medulla (RVLM; P=0.7). These findings suggest that hypertension in the BPH/2J mice is associated with greater sympathetic vasomotor responses to central pathways mediating the arousal responses to acute aversive stress in particular the amygdala, hypothalamus and rostral ventrolateral medulla.

Chemotaxis of leucocytes: an improved method of direct microscopic observation.

Leucochemotaxis was evaluated by direct morphological observation of a cell suspension between slide and coverslip. The chemotactic index was obtained by the ratio of the number of moving leucocytes recorded within an area limited on the slide by circles traced, at the beginning and after a time period of exposure to a gradient of chemotactic factor which was placed in the center of the circle in dry state. The optimal conditions (times of exposure, pH of the medium) and the reproducibility of the assay were investigated on normal blood samples.

Luminol assay for microdetermination of superoxide dismutase activity: its application to human fetal blood.

A microtechnique for determining the superoxide dismutase activity in erythrocytes is described. This technique involves the inhibition of luminol-enhanced chemiluminescence of superoxide anion generated by xanthine-xanthine oxidase. Measurements required a steady-state chemiluminescence whether superoxide dismutase was present or absent; the level of luminescence was correlated to enzyme activity. Superoxide dismutase activity measured by this technique was 836 +/- 112 micrograms/g of hemoglobin for whole blood and 834 +/- 109 micrograms/g of hemoglobin for erythrocytes. When the reference technique was applied to larger amounts of blood, the results were 862 +/- 58 and 858 +/- 116 micrograms/g of hemoglobin for whole blood and washed erythrocytes, respectively. The enzymatic activity of superoxide dismutase from fetal blood (obtained by venipuncture in utero and of 19-26 weeks gestational age) was similar to that of adult blood, when measured by the new technique.

Effect of a factor released by K562 malignant cells in culture on human neutrophil bactericidal activity.

We have previously demonstrated that k562 malignant cells in culture contain and release a low-molecular-mass (8-kDa) factor that inhibits adherence-related functions of neutrophils but does not alter fMet-Leu-Phe- or phorbol ester-induced oxidative burst (M. Amar, N. Amit, T. Pham Huu, S. Chollet-Martin, M.T. Labro, M.A. Gougerot-Pocidalo, and J. Hakim, J. Immunol. 144:4749-4756, 1990). In this study, we investigated the effects of this factor, referred to as inhibitory factor 1 (IF1), on the bactericidal activity of human polymorphonuclear cells (PMNs) on Staphylococcus aureus opsonized in various ways. S. aureus was used either nonopsonized or opsonized with heat-inactivated serum or normal serum containing complement factors. The bactericidal activity of PMNs preincubated with IF1-treated or control medium was examined by counting the surviving bacteria. The ability of IF1-treated PMNs to kill bacteria was diminished when they were opsonized with normal serum. When S. aureus was not opsonized or was opsonized with heat-inactivated serum, the bactericidal activity of IF1-treated PMNs was similar to that of controls. Likewise, the phagocytosis of IF1-treated PMNs was diminished when S. aureus was opsonized with normal serum but was not altered when S. aureus was not opsonized or was opsonized with heat-inactivated serum. These results suggest that the decrease in killing might be due to defective ingestion. The chemiluminescence response of IF1-treated PMNs was inhibited when S. aureus was not opsonized or was opsonized with normal serum. No effect on chemiluminescence was observed when S. aureus was opsonized with heat-inactivated serum. These results suggest that IF1 interferes not only with S. aureus stimulation of PMNs via complement receptors but also with oxygen-dependent bactericidal activity.

Plasma membranes of human neutrophils: a one-step isolation procedure by cell disruption in paraffin oil.

Plasma membranes of high purity and good yield have been prepared from human polymorphonuclear neutrophils by a one-step procedure involving disruption of cells suspended in paraffin oil and forced by pressure through an annular slit. This results in a band floating above the oil which is composed of large sheets of plasma membranes. Enrichment values for the plasma membrane marker alkaline phosphatase and 125I-labeled protein after surface labeling performed at the whole cell level were 23-fold and 22-fold, respectively. Contamination of the plasma membrane by other organelles was negligible and approximately 2 mg of membrane protein was obtained from 10(9) neutrophils. The procedure is very fast and the use of paraffin oil avoids lengthy high-speed centrifugation. The technique also allows isolation of granules devoid of plasma membrane and can probably be applied to other cell types.

Prenatal diagnosis of chronic granulomatous disease (CGD) in four high risk male fetuses.

Prenatal diagnosis of chronic granulomatous disease (CGD) was performed in four male high risk fetuses. The male sex was previously determined by an amniotic cell karyotype. Three kinds of test were performed on fetal blood obtained by umbilical venous puncture under fetoscopy at the 20th gestational week: nitroblue tetrazolium reduction (NBT) cytochemical test with phorbol myristate acetate (PMA) as activator; luminol enhanced chemiluminescence with activation by serum opsonized zymosan (STZ) or PMA; superoxide anion (0-2) production by measurement of the superoxide dismutase inhibitable reduction of cytochrome c with PMA as activator. Results were compared to those obtained in six fetuses investigated for other inherited diseases. In one case, absence of granulocyte defects was confirmed at birth. In three other cases, the tests showed deficient metabolic oxidative granulocytes. The pregnancy was terminated and the CGD diagnosis was confirmed on the products of abortion. The use of three different techniques performed on whole blood for CGD prenatal diagnosis is recommended instead of a single isolated test to ensure a higher confidence in the diagnosis.

Opsonic activity of ascitic fluids by a chemiluminescent method.

Cirrhotic ascites are highly susceptible to spontaneous bacterial infection, whereas carcinogenic ascites are seldom infected. This difference may be explained by differences in their chemotactic, bactericidal and/or opsomic activities. We measured the chemotactic and opsonic activity of ascitic fluids from 35 alcoholic cirrhotic ascites and of 12 peritoneal carcinogenic ascites. Chemotactic activity was measured by the under-agarose technique and opsonic activity by a luminol-enhanced method. Ascitic fluids from alcoholic cirrhosis had low chemotactic (62 +/- 24.5% that of N-formylated peptide) and opsonic (67 +/- 50% of normal serum) activities on normal human neutrophils. In contrast, ascitic fluids from peritoneal carcinoma were found to possess high opsonic activity (114 +/- 49% of normal serum) and chemotactic activity similar to that of N-formylated peptide. During a 3-month follow-up, 11 spontaneous bacterial infections were observed among the first group against none in the carcinogenic group.

Chemoattractant and opsonic activity in ascitic fluid. A study in 47 patients with cirrhosis or malignant peritonitis.

We studied prospectively the ascitic fluid of 47 patients. Thirty-five were cirrhotics (group A) and 12 had malignant peritonitis (group B). All ascitic fluid samples were initially uninfected. We measured opsonic activity by a chemiluminescent assay, and chemoattractant activity by the under agarose technique. We also measured ascitic concentrations of C3, C4, fibronectin, C-reactive protein, immunoglobulins G, A and M and total proteins. All patients were followed throughout the presence of ascites. None of the group B patients developed peritoneal infection, nor did 23 of the group A patients (group A2). Twelve group A patients (group A1) developed spontaneous bacterial peritonitis (SBP), four of them with recurrence. All indices except immunoglobulins A and M were significantly different between group A and group B patients. Comparing group A1 and group A2, only chemoattractant activity and concentrations of total proteins and C3 were significantly lower in group A1. Using a multivariate analysis with Cox's model, only C3 concentration had an independent predictive value for occurrence of SBP in cirrhotic patients.

K562 cells produce an anti-inflammatory factor that inhibits neutrophil functions in vivo.

We have previously reported that K562, a chronic myelogenous leukemia cell line, releases a low molecular weight factor (6 to 8 Kd) that inhibits human polymorphonuclear neutrophil (PMN) adherence and adherence-related functions tested in vitro. We now report that this factor, which we have named K562 inhibitory factor (K562-IF), has potent anti-inflammatory activity in mice, associated with an inhibition of PMN functions. Its in vitro actions were less marked with mouse PMN than with human PMN. They included (1) an inhibition of both nonstimulated locomotion and locomotion induced by FMLP or serum; (2) an inhibition of the chemiluminescence induced by opsonized zymosan, but not that induced by phorbol myristate acetate or FMLP; (3) an inhibition of the degranulation stimulated by opsonized zymosan, as reflected by lactoferrin and lysozyme release; and (4) a decrease in arachidonic acid release and leukotriene B4 production by A23187-stimulated PMN. The in vivo actions of K562-IF after intraperitoneal injection included (1) an inhibition of subcutaneous PMN accumulation at the site of injection of opsonized zymosan (PMN accumulated neither outside the vessels nor intravascularly, as shown by means of histochemistry); (2) an inhibition of neutrophil accumulation in the peritoneum of mice having received sodium caseinate or opsonized zymosan intraperitoneally; and (3) lysozyme concentration in neutrophils having reached the peritoneum after opsonized zymosan treatment equal to that in blood, suggesting diminished release. PMN influx and degranulation in the peritoneum were reduced by 50% after 3 hours of treatment with 1 microgram of K562-IF (equivalent to the effect of 120 micrograms of prednisolone). Taken together, these results show that K562-IF is a potent anti-inflammatory agent that acts by inhibiting PMN functions.

Evidence for priming and activation of neutrophils early after coronary angioplasty.

OBJECTIVES: Percutaneous transluminal coronary angioplasty (PTCA) induces deep arterial wall injury and transient ischaemia. The aim of this study was to demonstrate that PTCA could result in priming or activation of the neutrophils and the complement system. METHODS: Blood was drawn from the coronary sinus before and immediately after PTCA in 7 patients and before and immediately after coronary angiography in 7 patients (to ensure that the changes observed after PTCA were not solely related to the angiographic procedure). Neutrophil priming was assessed ex vivo by whole blood chemiluminescence stimulated in vitro by formyl-methionyl-leucyl-phenylalanine, phorbol myristate and opsonized zymosan. Neutrophil activation was assessed by measurement of plasma lactoferrin. RESULTS: Whole blood chemiluminescence increased after PTCA, regardless of the stimulus used, while it did not after arteriography. After PTCA, lactoferrin increased 2-fold (p < 0.02) whereas after arteriography a non-significant increase was observed. Neutrophil count and adherence properties were not modified by either PTCA or arteriography. Total haemolytic complement (CH50), C3, C4 and B factor decreased slightly (7 to 16%) after both PTCA and arteriography. CONCLUSIONS: Early after PTCA, the neutrophil oxidative response, assessed by stimulated whole blood chemiluminescence, increased, suggesting a "priming" effect of PTCA on neutrophils. In addition, plasma lactoferrin levels increased, indicating neutrophil activation. Finally, there was a mild global activation of the complement system, most likely related to the contrast agent, and which may play a role in the "priming" process. Neutrophil priming and activation may participate in several phenomena occurring after angioplasty such as enhanced vasoconstriction and post-ischaemic myocardial dysfunction. In addition, it may participate in triggering local inflammatory processes.

Production by K 562 cells of an inhibitor of adherence-related functions of human neutrophils.

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.