Burst Of Human Polymorphonuclear Leukocytes Research Articles

Aldosterone Stimulates Neutrophils Leading To Increased β-Glucuronidase, Protein Disulfide Isomerase and Myeloperoxidase Secretion

Aldosterone (ALDO) has been shown to play an important role in inflammatory responses in addition to its well described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). However, its effects on polymorphonuclear leukocytes (PMNC) are not well described. We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for the myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses. We incubated PMNC with ALDO (10–9–10–7M) for 30 min and observed a dose-dependent rise in β–glucuronidase release with an EC50 of 6.11 nM (P<0.001, n=3), an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). In addition, our results show that incubation of human PMNC with 10-8M ALDO likewise led to increases in myeloperoxidase ([MPO], P<0.05, n=3) and protein disulfide isomerase ([PDI], P<0.01, n=4), a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions (Prado, 2013 FASEB J). We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. Our results likewise show an increase in MPO responses upon 10–8M ALDO stimulation as compared to vehicle (AUC: 1090±147 to 505±48, P<0.02, n=3). We have recently reported that aldosterone stimulates increases of striatin, a scaffolding protein that interacts with caveolin-1, and co-precipitates with striatin and as such may facilitate cross talk of signaling complexes. As there are no pharmacological inhibitors of striatin we used a molecular approach to reduce striatin levels. In differentiated HL-60 cells, siRNA against striatin led to reduced MPO responses (AUC: 590±14 to 528±13, P<0.05, n=3) that were associated with significantly reduced striatin mRNA levels but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and β-microglobulin used as an endogenous control (P<0.01, n=3). These results suggest that striatin plays an important role in ALDO-stimulated degranulation responses. Of importance we also observed that incubation with ALDO (10–9–10–7M) in differentiated HL60 cells led to increases in the oxidative-respiratory burst [superoxide production] in a dose- and time-dependent manner (P<0.01, n=4). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). As there is evidence that activated neutrophils, MPO and PDI are elevated in Sickle Cell Disease, we studied the in vivo effects of MR blockade in BERK sickle transgenic mice, a model of increased oxidative stress. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and blood collected. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units [RFU]; P<0.005, n=6 and 9) and lower MPO levels (AUC: 214±11 to 73±20, P<0.03, n=3); events that were associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). Thus, our results suggest that MR activation by ALDO is a novel mechanism for neutrophil stimulation and as such represents a novel therapeutic target aimed at ameliorating the vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 (AR) and R01HL096518 (JRR). Disclosures:No relevant conflicts of interest to declare.

Aldosterone Stimulates a Degranulation Response in Human Neutrophils: Role of Protein Disulfide Isomerase

AbstractAbstract 1034There is growing evidence for an important role of aldosterone (ALDO) in inflammatory responses in addition to its well-described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). We studied the effects of ALDO on activation of ex vivo human polymorphonuclear leukocytes (PMNC). We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses and when incubated with ALDO (10−9 −10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 2 min using FURA-2AM fluorescence. We then studied the effect of ALDO on PMNC degranulation following incubations with ALDO (10−9 −10−7 M) for 30 min and observed a significant increase in β–glucuronidase release (P<0.001, n=3) by established fluorescent detection methods, an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). PMA and N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) were used as positive controls for PMNC activation. We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcripts by quantitative RT-PCR using TaqMan detection probes in these cells and as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence. To assess the degranulation response of these cells we quantified the in vitro release of myeloperoxidase (MPO) and observed that 10−8M ALDO was likewise associated with increased degranulation when compared to vehicle treated cells (AUC: 590±14 to 185±11, P<0.01, n=6). To characterize the mechanisms by which ALDO regulates the degranulation responses of these cells we studied the effects of Protein Disulfide Isomerase (PDI) on ALDO-stimulated cells. PDI catalyzes the oxidation or reduction of thiol/disulfide groups and modulates leukocyte function. Our results show that blockade of PDI, by bacitracin, led to a blunted ALDO-stimulated degranulation response in both cell types. Consistent with these observations, we show that in differentiated HL-60 cells, siRNA against PDI likewise led to reduced MPO responses (AUC: 590±14 to 290±13, P<0.01, n=6) that were associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls (0.55 ± 0.02, ΔΔCT of PDI siRNA relative to scrambled transfected cells, P<0.01, n=6). These results suggest that ALDO stimulates MPO release. MPO has been shown to be one of the predominant granule proteins associated with Neutrophil Extracelullar Traps (NETs), extracellular structures that contain chromatin (DNA and histones) that can also trap microorganisms. We studied the effects of ALDO following digestion of the NETs by DNAse, and observed that 30–35% of the total cellular MPO was NET-associated. We also observed that incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3), a responses that was blocked by pre-incubation of cells with 1 uM CA (P<0.03, n=3). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). Thus our results suggest that activation of MR by ALDO leads to degranulation and NET production in neutrophils that may contribute to the inflammatory responses associated with MR activation in vivo. Furthermore, the association between degranulation and NET release implicates PDI as a novel regulator of MPO generated NET production. Disclosures:No relevant conflicts of interest to declare.

Effect of progesterone on gonococci‐induced apoptosis and respiratory burst of human polymorphonuclear leukocytes in vitro

Progesterone may have clinical relevance in females with asymptomatic gonococcal infections. To investigate the regulatory effect of progesterone on apoptosis and oxidative burst activity of polymorphonuclear leukocytes (PMNs) challenged by Neisseria gonorrhoeae in vitro. Isolated PMNs were incubated with progesterone or staurosporine. Staurosporine was used as a positive control for our in vitro model. Expression levels of inhibitory apoptosis proteins (IAPs), cellular IAP2 (cIAP2), and X-linked IAP (XIAP) were determined by real-time polymerase chain reaction (PCR). In addition, PMN apoptosis at various time points (3, 8, 12, and 24 h) was assayed by flow cytometry. Luminol amplified chemiluminescence methods were used to quantify the oxidative burst function of PMNs challenged with N. gonorrhoeae ST2951. cIAP2 was upregulated significantly in PMNs with progesterone treatment in 3 h, and XIAP was upregulated slightly compared with the medium + ST2951 group, whereas cIAP2 was downregulated in staurosporine-challenged PMNs. In addition, we found that progesterone delayed the onset of apoptosis activity in N. gonorrhoeae ST2951-challenged PMNs, notably at 12 h. No statistically significant changes in PMN oxidative burst activity were observed at 10 ng/mL of progesterone. Staurosporine enhanced the production of superoxide anion (respiratory burst) of human PMNs stimulated by N. gonorrhoeae. Progesterone plays an important regulatory role in the interaction of PMNs and N. gonorrhoeae. Delayed PMN apoptosis induced by progesterone presumably acts as a mechanism for N. gonorrhoeae to avoid the innate immune response and establish long-term, low-level infection in the female reproductive tract.

Manno(Rhamno)Biose-Containing Capsular Polysaccharides of Klebsiella pneumoniae Enhance Opsono-Stimulation of Human Polymorphonuclear Leukocytes

We tested the relationship between the capsular and the O-antigen structures and the ability of bacteria to trigger respiratory burst in human polymorphonuclear leukocytes (PMNL).Capsulated and non-capsulated variants as well as capsule-switched derivatives of Klebsiella serotypes bearing or lacking manno(rhamno)biose repeats in their capsular polysaccharides and expressing either mannose-rich or mannose-poor O antigens were tested for their ability to induce respiratory burst and survive in human PMNL. Luminol-enhanced chemiluminescence (CL) was measured to quantify respiratory burst. Intracellular survival was quantified by determining the viable counts of intracellular bacteria. K serotypes and the capsule-switched derivative lacking manno(rhamno)biose induced significantly lower CL than those expressing manno(rhamno)biose. Manno(rhamno)biose-lacking serotypes survived in the cells significantly better than serotypes expressing these repeats. C1q depletion did not affect CL induced by the manno(rhamno)biose-containing serotype, whereas factor B depletion revealed a significantly reduced CL. Likewise, EGTA in the presence of Mg²⁺ significantly decreased CL, but the values were higher than those induced by the bacterium opsonized with factor B-depleted serum. In the presence of EGTA, Mg²⁺-treated factor B-depleted serum revealed a significant reduction in the CL response compared with the responses induced by opsonization with factor B-depleted serum alone. These results indicate, in addition to the alternative pathway, a manno(rhamno)biose pattern recognition of Klebsiella by PMNL probably by the complement lectin pathway.

Effect of combination of heavy metals on respiratory burst of isolated human polymorphonuclear leukocyte

Objective: The aim of the study is to evaluate the effect of various combination of mercuric chloride, cobalt chloride and cadmium chloride on respiratory burst of human polymorphonuclear leukocytes (PMNs). Method: Fresh blood was collected by venipuncture from volunteers in sterile containers with heparin (10 IU/ml, Fisher Scientific Co., NJ). Neutrophils were separate from the whole blood using the isolation medium PMN (Robbins Scientific Corporation, Sunnyvale, CA). Isolated PMNs were pre-treated with various concentration of the heavy metals alone and in combination. Chemiluminescence (CL) response was carried out using the principle of oxidation of luminol (a cyclic hydrazide), by reactive oxygen species produced during phagocytosis, and stimulated by soluble agent, phobol myristate acetate (PMA) (Sigma Chemical Co., St. Louis, MO). Measurement of luminol-dependent CL begun immediately, using an Autolumat Plus (Berthold, Germany). Results: Results will be presented in the conference.

T cell contact-mediated activation of respiratory burst in human polymorphonuclear leukocytes is inhibited by high-density lipoproteins and involves CD18

Polymorphonuclear neutrophils (PMN) are recruited to sites of inflammation, where they are in close vicinity with other immune cell types. The present study demonstrates that direct cell-cell contact with stimulated T cells activates PMN respiratory burst. To discard interferences with soluble products, membranes isolated from human T lymphocytes (msT) or the monocytic cell line HUT-78 (msHUT) were used to mimic cellular contact. msT and msHUT induced a dose-dependent production of radical oxygen species (ROS) in PMN, as detected by chemiluminescence. Similar results were obtained with fixed, stimulated T cells, confirming that ROS production was a result of cell-surface molecules and not to soluble products of T cells. ROS production was mainly intracellular, suggesting that ROS may take part in intracellular processes. High-density lipoproteins (HDL), which had previously been shown to inhibit T cell contact-induced cytokine production in monocyte-macrophages, potently reduced ROS production induced in PMN upon contact with stimulated T cells. This supports the emerging role of HDL as immunomodulators in inflammatory diseases. Furthermore, monoclonal antibodies to CD18 inhibited 60% of the PMN respiratory burst induced by msT, suggesting that CD18 contributed to PMN activation. The present results emphasize the importance of direct cell-cell contact with stimulated T cells in inflammatory processes.

Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) increases the adherence to epithelia and the oxidative burst of human polymorphonuclear leukocytes but decreases bacteria phagocytosis

Recruitment of polymorphonuclear leukocytes (PMNL) is a hallmark of both urinary and digestive infections caused by Escherichia coli. Cytotoxic necrotizing factor 1 (CNF-1) is a toxin produced by uropathogenic E. coli strains that mediates its effects via the activation of small GTP-binding proteins. However, the role and the consequences of CNF-1 on PMNL physiology remain largely unknown. In this study, we provide evidence that CNF-1 dramatically affects the PMNL cytoskeleton architecture by inducing an increased content of F-actin. Furthermore, we demonstrate that CNF-1 increases functional features of PMNL, such as superoxide generation and adherence on epithelial T84 monolayers, but significantly decreases their phagocytic function. Our results suggest that CNF-1 may behave as a virulence factor in urinary or digestive infection by stimulating PMNL cytotoxicity as a result of its enhancing effect on their adherence to epithelial cells as well as the production of radical oxygen products. Moreover, the decreased phagocytosis of PMNL induced by CNF-1 likely facilitates growth of bacteria. In these conditions, CNF-1 would intervene in the initiation and in the perpetuation of the inflammatory process.

Effects of 2-ethylhexanoic acid on the production of reactive oxygen species in human polymorphonuclear leukocytes in vitro

2-Ethylhexanoic acid (2-EHA), is an industrial chemical and a toxic biotransformation product of the plasticizer di(2-ethylhexyl)phthalate. Its immunological effects are unknown. 2-EHA resembles structurally C18 fatty acids, which are known activators of respiratory burst in human polymorphonuclear leukocytes (PMNL). Therefore, we exposed PMNL to 2-EHA in vitro and measured the production of reactive oxygen species (ROS) and explored the associated cellular mechanisms. 2-EHA (10–2000 μM) inhibited dose-dependently formyl-methionyl-leucyl-phenylalanine (FMLP)-induced respiratory burst in PMNL. Moreover, 2-EHA decreased oxidative burst evoked by the protein kinase C (PKC) activators, phorbol myristate acetate (PMA) and dioctanoyl-s,n-glycerol (DIC 8). 2-EHA affected neither the levels of free intracellular calcium nor inhibited PKC. The results indicate that 2-EHA inhibits activation of PMNL to produce ROS, i.e. has an immunosuppressive effect in vitro. The site of action in the PKC is after activation of this enzyme.

Pharmacological evidence for the putative existence of two different subtypes of PAF receptors on platelets and leukocytes; studies with yangambin

Yangambin, a new naturally-occuring platelet activating receptor (PAF) receptor antagonist competitively displaced [ 3H]-PAF from its high affinity binding sites on washed human platelets with a Ki value of 1.1 ± 0.3 μM ( n = 3). Studies carried out in parallel demonstrated that SR 27417, a newly-developed PAF receptor antagonist also antagonized [ 3H]-PAF binding to these cells with a Ki value of 51 ± 2 pM. SR 27 417 ( N-(2-dimethylamino ethyl)- N-(3-pyridinyl methyl) [4-(2,4,6-triisopropyl phenyl) thiazol-2-yl] amine) selectively and competitively inhibited the specific binding of [ 3H]-PAF on human polymorphonuclear leukocytes (Ki = 65 ± 5.2 pM) whereas high doses of yangambin remained ineffective. Yangambin inhibited PAF-induced aggregation of human platelets in vitro (IC 50 = 1.0 ± 0.2 μM) but had no effect PAF-induced oxidative burst in human polymorphonuclear leukocytes. In guinea pigs, yangambin inhibited PAF-induced thrombocytopenia but did not affect leukocytopenia whereas SR 27 417 afforded complete protection against both PAF-induced thrombocytopenia and leukocytopenia. In conclusion, yangambin discriminates between two different types of PAF receptors on platelets and polymorphonuclear leukocytes and can be considered as the first PAF receptor antagonist described to date exhibiting such an effect.

Effect of amphotericin B associated with a lipid emulsion on the oxidative burst of human polymorphonuclear leukocytes

1. 1. Despite its toxicity, amphotericin B (AB) continues to be the drug of choice for the treatment of systemic fungal infection. The drug acts on several cell types, including polymorphonuclear leukocytes (PMN), where it inhibits the oxidative burst of cells submitted to several stimuli. 2. 2. It was previously shown that the association of AB with a triglyceride-rich emulsion that physiologically mimics chylomicrons reduces toxicity. 3. 3. We found that the association of AB with a triglyceride-rich emulsion reduces the loss of PMN viability produced by the drug. 4. 4. The inhibition of the PMN oxidative burst triggered by phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) also was decreased by the association of the drug with this lipid emulsion. 5. 5. Delivery of AB in a lipid emulsion may be of advantage in the treatment of immunosuppressed patients.

Erucic Acid and Erucic Acid Anilide-induced Oxidative Burst in Human Polymorphonuclear Leukocytes

Human polymorphonuclear leukocytes (PMNL) were exposed to erucic acid or erucic acid anilide to explore their effects on the production of reactive oxygen species (ROS) and the levels of free intracellular calcium. The compounds did not change the levels of intracellular calcium, but both dose-dependently induced respiratory burst in PMNL. Maximal production of ROS by erucic acid exceeded that induced by its anilide 13-fold. A protein kinase C inhibitor, Ro 31–8220, completely inhibited erucic acid and erucic acid anilide-induced production of ROS. Neither erucic acid nor erucic, acid anilide modified FMLP-induced production of ROS. However, erucic acid (500 μM) amplified 5 nM PMA-induced ROS production 1.8-fold, but did not have this effect at a lower PMA concentration. On the contrary, erucic acid anilide inhibited PMA-induced oxidative burst, and shifted the peak ROS production induced by PMA to a later time-point. The present results show that aniline moiety modifies the effects of erucic acid on the activation of PMNL, and suggest that both erucic acid and erucic acid anilide may activate PMNL through a protein kinase C-dependent mechanism.

Palmitic Acid Anilide-Induced Respiratory Burst In Human Polymorphonuclear Leukocytes is Inhibited by a Protein Kinase C Inhibitor, Ro 31-8220

Human polymorphonuclear leukocytes (PMNL) were exposed to palmitic acid anilide, an impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981, and to the corresponding fatty acid, palmitic acid. The effects of these compounds were studied on the production of reactive oxygen metabolites (ROM) and changes in the levels of free intracellular calcium. Palmitic acid anilide induced the production of reactive oxygen metabolites in PMNL. Interestingly, the palmitic acid anilide-induced respiratory burst was completely blocked by a protein kinase C inhibitor, Ro 31-8220. Moreover, palmitic acid anilide additively amplified the production of ROM caused by a chemotactic peptide, formyl-Methionyl-Leucyl-Phenylalanine (FMLP). In contrast, palmitic acid anilide did not have any effect on the production of ROM induced by a tumor promoter, phorbol myristate acetate (PMA). Palmitic acid, in turn, did not markedly induce the production of ROM nor did it amplify the agonist-induced respiratory burst. Neither of the compounds, alone or in combination with FMLP, affected the levels of intracellular calcium in PMNL. These results indicate that the aniline moiety in palmitic acid modifies its effects on the activation of human PMNL, and the subsequent oxidative burst. The present results also suggest that palmitic acid anilide may activate PMNL through a protein kinase C-dependent mechanism. © 1997 Elsevier Science Inc.

Intestinal epithelial cytoskeleton selectively constrains lumen-to-tissue migration of neutrophils.

Migration of neutrophils (polymorphonuclear leukocytes; PMN) across polarized epithelia is asymmetrical: basolateral-to-apical (physiologically directed) migration is far more efficient than migration in the reverse direction, suggesting the presence of luminal retention signal(s). Following pilot observations, we used polarized intestinal epithelial monolayers (T84) to examine whether asymmetrical constraint of migration afforded by the epithelial cytoskeleton might underlie such retention signals. Rearrangement of epithelial cortical F-actin accompanied PMN transepithelial migration (in either direction) and was prevented by preloading monolayers with the F-actin stabilizing agent phallacidin. Although phallacidin preloading did not influence physiologically directed PMN transepithelial migration, such treatment greatly enhanced migration in the reverse direction (i.e., effective loss of luminal retention signal). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) preloading also prevented epithelial cortical actin rearrangements and selectively resulted in loss of luminal retention signal(s). BAPTA preloading did not influence resistance or forskolin-induced Cl- secretion, and phallacidin preloading did not influence resistance or carbachol-induced Cl- secretion, suggesting that barrier function and surface polarity were maintained under these conditions. These and supplementary data suggest that epithelial actin (but not microtubule) cytoskeletal reordering asymmetrically influences PMN migration and underlies, at least in part, the observed signal that biases for retention of PMN in the luminal space.

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.

Respiratory Burst of Human Polymorphonuclear Leukocytes in Response to the Galactoside-specific Mistletoe Lectin

The phagocytic activity of human polymorphonuclear leukocytes (PMNLs) towards Staphylococcus aureus Cowan 1 was evaluated in chemiluminescence assays. As to check its activating ability, galactoside-specific mistletoe lectin (ML-1) was coincubated with PMNLs which were then challenged with S. aureus. Statistically significant (p < 0.001) chemiluminescent response (correlating with phagocytic activity) could be demonstrated at optimal experimental condition, viz: 1 x 10(6) PMNLs incubated with 0.005 ng ML-1 for 30 and 60 minutes before S. aureus challenge. Other experimental schedules (different timing and PMNL/ML-1 concentrations) did not present with statistically relevant changes in chemiluminescent response. These studies suggest that optimal ML-1 concentrations enhance the phagocytic activity of PMNLs which might be of benefit in thus treated patients as to prevent (or lower the rate of) infections under antineoplastic therapy.

Adhesion to and invasion of HeLa cells by Helicobacter pylori

Eight clinical isolates and two reference strains of Helicobacter pylori were studied with regard to their interactions with HeLa cells. All the isolates adhered poorly to HeLa cells and the number of invasive bacteria was very low. No correlation was found between the adherence and invasiveness of the isolates on one hand, and the corresponding patients having ulcer or non-ulcer disease, or the ability of the strains to produce cyrotoxin and to induce an oxidative burst of human polymorphonuclear leukocytes without opsonins, on the other. These results indicate that invasion of epithelial cells would play no important role in the pathogenesis of infections caused by H. pylori.

Effect of Insulin on Hydrogen Peroxide Production by Human Polymorphonuclear Leukocytes

This study evaluated the effect of insulin on the respiratory burst of human polymorphonuclear leukocytes (PMNLs) and the signalling pathways involved in this process, especially the involvement of protein kinase C (PKC). Isolated human PMNLs from healthy volunteers were incubated with different concentrations of insulin (10-10-10-7 mol/l) and for different durations of incubation (5-90 min). The intracellular production of hydrogen peroxide (H2O2) was detected employing a previously validated flow cytometric assay using 2',7'-dichlorofluorescein-diacetate (DCFH-DA) as a marker for H202 production. Specificity of insulin action was verified using an insulin antagonist (the monoclonal antibody MA-10). To identify the signalling pathway involved, we used; (a) monoclonal antibody MA-5, directed against the alpha-subunit of the insulin receptor, that partially mimics insulin without activating tyrosine kinase; (b) H7, an inhibitor of PKC involved in O2- production in PMNLs, and (c) phorbol myristate acetate (PMA) that binds and stimulates PKC. Insulin caused a dose- and time-dependent stimulation of H202 release by human PMNLs. The effect of insulin was blocked MA-10. The actions of insulin and PMA on H2O2 release were additive, whereas the actions of MA-5 and PMA were not. H7 partially inhibited the H2O2 production stimulated by insulin and completely inhibited MA-5 action. We conclude that insulin stimulates, in a dose- and time-related manner, the respiratory burst of human PMNLs. PKC activation can only partially account for the intracellular mechanisms involved in this process.

Nonopsonic activation of neutrophils and cytotoxin production by Helicobacter pylori: ulcerogenic markers.

Fifty-four clinical isolates of Helicobacter pylori from human gastric biopsy specimens were tested for cytotoxin production, as determined by intracellular vacuolization of HeLa cells, and for the induction of oxidative burst in human polymorphonuclear leukocytes (PMNLs), as measured by luminol-enhanced chemiluminescence (CL). Nonopsonized, 20 of the H. pylori strains induced a rapid and strong CL response, in contrast to 30 other strains, which showed only weak and slow responses. Another four strains gave inconclusive results. Cytotoxin production was demonstrated in 10 of the 20 strains with rapid responses but only in 3 of the 30 strains with slow and low responses (p = 0.0027, Fisher's exact test, two-tailed). Eleven of the 15 cytotoxin-producing strains (p = 0.0135) and 13 of the 20 strains with strong CL responses (p = 0.0209) were from 22 patients with peptic ulcer disease (PUD). The ability of some nonopsonized H. pylori to activate PMNLs showed co-variation with their ability to produce cytotoxin, but these two properties seem to be independent markers of PUD.

Polymorphonuclear leukocyte chemiluminescence induced by Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus in serum and saliva.

The ability of different strains of Actinobacillus actinomycetemcomitans (A.a.) and Haemophilus aphrophilus (H.a.) to trigger activation of an oxidative burst in human polymorphonuclear leukocytes (PMNL) was examined by measuring the luminol-amplified light emission--chemiluminescence (CL)--from these cells. Bacterial cells were incubated with PMNL from one healthy subject, in the presence of either active serum, heat-inactivated serum, saliva, or saliva and active serum. In the presence of active serum, all five H.a. strains and two out of five A.a. strains triggered a CL response. The CL induced in the presence of heat-inactivated serum was considerably less than that achieved with fresh serum. In the presence of only saliva, all strains induced considerably weaker CL responses than those induced in the presence of saliva with active serum. In the presence of serum, intracellular reactions appeared to be the main source of CL, while addition of saliva and active serum increased the extracellular CL. The results indicate that strain-dependent differences exist among A.a. strains in their ability to trigger the oxygen-dependent bactericidal mechanisms of human PNML. In contrast, the CL patterns of H.a. strains were equivalent. Various factors in the environment, such as activated complement and salivary compounds, affect the interaction of these species with neutrophils.

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila serogroup 1.

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.