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PubMed1982

Interactions of Neisseria gonorrhoeae with human neutrophils: effects of serum and gonococcal opacity on phagocyte killing and chemiluminescence.

Serum-sensitive strains of Neisseria gonorrhoeae were incubated with suspensions of normal or chronic granulomatous disease human neutrophils in the absence or presence of fresh or heat-inactivated human serum; phagocytosis, gonococcal viability, and chemiluminescence were measured. Nonpiliated opaque or transparent gonococci (colony types 3 and 4, respectively) were used for phagocytic bactericidal assays. In the presence of 2.0% fresh human serum, normal neutrophils killed >90% of types 3 and 4 gonococci by 135 min. Serum alone at this concentration was not bactericidal. In the absence of serum, type 4 gonococci were not killed, whereas type 3 gonococci were killed to the same degree as in the presence of serum. Interestingly, heat-inactivated normal serum slightly inhibited phagocytic killing of type 3 gonococci. Results almost identical to those above were obtained when 5% fresh human serum deficient in complement component 7 was substituted for 2% normal autologous serum. This indicated that the later components of complement were not involved in the observed results. To investigate the mechanisms responsible for the intracellular killing of the gonococci, we used neutrophils from patients with chronic granulomatous disease. These neutrophils are deficient in an activable NADPH oxidase and do not produce bactericidal oxygen products upon phagocytic stimulation. Neutrophils from two unrelated boys with chronic granulomatous disease killed type 3 and 4 gonococci to the same degree as did normal neutrophils. As with normal neutrophils, serum was needed for killing type 4 organisms. As expected, neutrophils from these patients showed absolutely no increased chemiluminescence in the presence of type 3 or 4 gonococci, with or without serum. The effects of serum on gonococcus-induced chemiluminescence by normal neutrophils was also investigated. For these studies, in addition to type 3 and 4 gonococci, we also used transparent colony types of lightly (type 1) and heavily (type 2) piliated organisms. Chemiluminescence induced by type 1, 2, or 3 gonococci (i.e., gonococci possessing either pili or opacity-associated proteins, but not both) was augmented only slightly by serum and then only at low ratios of gonococci to neutrophils. On the other hand, chemiluminescence induced by type 4 gonococci (i.e., gonococci possessing neither pili nor opacity-associated proteins) was substantially increased in the presence of serum. Stimulation of chemiluminescence by type 1, 2, 3, or 4 gonococci was dose dependent in the absence or presence of serum. Heat-killed type 3 gonococci induced chemiluminescence to the same degree as did viable organisms. Since the gonococci used in this research was strongly catalase positive, as are gonococci in general, and since it was killed by chronic granulomatous disease neutrophils, the results indicate that gonococci can be effectively killed within neutrophils, i.e., within phagolysosomes, by nonoxidative bactericidal mechanisms. Whereas type 3 gonococci were phagocytized and killed by neutrophils equally well with or without serum, serum was obligatory for phagocytic killing of type 4 gonococci, i.e., gonococci lacking opacity-associated proteins. In addition, either pili or opacity-associated proteins were apparently necessary for maximal stimulation of neutrophil chemiluminescence. The submaximal stimulation of chemiluminescence by gonococci lacking both pili and opacity-associated proteins, i.e., type 4 gonococci was augmented by low concentrations of nonimmune serum.

Bacterial Proteins↗
PubMed1988

Role of alveolar macrophages in lipopolysaccharide-induced neutrophil accumulation.

B6D2F1/TRU mice were exposed to a lipopolysaccharide (LPS) aerosol that resulted in a 1-h-postexposure lung burden of about 290 ng of LPS. This exposure caused an accumulation of neutrophils in the lung that peaked between 6 and 12 h after exposure. To determine the potential role of alveolar macrophages (AM) in the induction of neutrophil accumulation by LPS, 10(6) AM from normal or LPS-exposed mice were transferred to the lungs of groups of naive recipient mice. A third group of mice was instilled intratracheally with vehicle only. After 5 h, the lungs of the mice were lavaged and the numbers of neutrophils in the lavage fluids were determined. The instillation of AM from unexposed mice did not cause significantly more neutrophils to accumulate than did the instillation of vehicle only, whereas the instillation of AM from LPS-exposed mice caused nearly a threefold increase in the numbers of neutrophils in lavage fluids. Transfer of AM from LPS-exposed mice into cutaneous air pouches of naive mice also caused greater local neutrophil accumulation (10-fold) than did the transfer of AM from normal mice. Repeated freeze-thawing of the suspensions of AM before transfer to recipients significantly reduced the ability of the suspensions to induce neutrophil accumulation. This indicated that AM viability is necessary to cause a maximal neutrophil infiltration upon transfer of the AM. To determine the extent to which LPS-induced neutrophil accumulation depends on the presence of AM, the ability of LPS to elicit neutrophil accumulation when injected alone or together with AM into air pouches was determined. The injection of either AM or LPS alone caused few neutrophils to accumulate, whereas the injection of LPS and AM together caused a large number of neutrophils to accumulate. The results of this study indicate that LPS deposition in the lung can stimulate AM to induce neutrophil accumulation and that this may be the major mechanism by which LPS causes neutrophil accumulation.

Aerosols↗
PubMed1992

Growth of Neisseria gonorrhoeae in CMP-N-acetylneuraminic acid inhibits nonopsonic (opacity-associated outer membrane protein-mediated) interactions with human neutrophils.

Gonococci possessing certain opacity-associated (Opa) outer membrane proteins adhere to and are phagocytosed by human neutrophils in the absence of serum. Recently, it has been shown that serum-sensitive strains of Neisseria gonorrhoeae possessing the appropriate lipooligosaccharide phenotype become serum resistant when grown in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) because of sialylation of their lipooligosaccharide. We investigated whether such sialylation affects nonopsonic (antibody- and complement-independent) interactions of gonococci with human neutrophils in vitro. We grew Opa+ gonococci in the presence of up to 50 micrograms of CMP-NANA per ml, incubated them with neutrophils in vitro, and measured their abilities to adhere to neutrophils, stimulate neutrophil luminol-dependent chemiluminescence (LDCL), and be phagocytically killed by neutrophils. Growth in CMP-NANA dramatically inhibited (in a dose-dependent manner) the ability of Opa+ gonococci to adhere to neutrophils and stimulate neutrophil LDCL. Growth of Opa+ gonococci in 50 micrograms of CMP-NANA per ml appeared to delay, but did not inhibit, their killing by neutrophils. Sialidase treatment of sialylated Opa+ gonococci, i.e., gonococci grown with CMP-NANA, totally restored their abilities to adhere to neutrophils and stimulate neutrophil LDCL. Opa- gonococci grown in the presence of 50 micrograms of CMP-NANA per ml and opsonized with fresh human serum bound to neutrophils only about 30% less efficiently than did Opa- gonococci grown without CMP-NANA and opsonized. The results of our studies show that sialylated Opa+ gonococci have dramatically reduced nonopsonic interactions with neutrophils. Some gonococcal strains may resist killing by human neutrophils in vivo by such a mechanism.

Bacterial Adhesion↗
PubMed2005

Neutrophils interact with adenovirus vectors via Fc receptors and complement receptor 1.

Neutrophils are effectors of the innate immune response to adenovirus vectors. Following the systemic administration of Cy2-labeled AdLuc in mice, flow cytometry and PCR analysis of liver leukocytes revealed that 25% of recruited neutrophils interacted with adenovirus vectors. In vitro, flow cytometry of human neutrophils incubated with Cy2-labeled AdLuc also demonstrated a significant interaction with adenovirus vectors. Fluorescence and electron microscopy confirmed vector internalization by neutrophils. The AdLuc-neutrophil interaction reduced vector transduction efficiency by more than 50% in coincubation assays in epithelium-derived cells. Adenovirus vector uptake by neutrophils occurred independently of coxsackievirus adenovirus receptor (CAR) and capsid RGD motifs, since neutrophils do not express CAR and uptake of the RGD-deleted vector AdL.PB* was similar to that of AdLuc. Furthermore, both AdLuc and AdL.PB* activated neutrophils and induced similar degrees of L-selectin shedding. Neutrophil uptake of AdLuc was dependent on the presence of complement and antibodies, since the interaction between AdLuc and neutrophils was significantly reduced when they were incubated in immunoglobulin G-depleted or heat-inactivated human serum. Blocking of complement receptor 1 (CD35) but not complement receptor 3 (CD11b/CD18) significantly reduced neutrophil uptake of AdLuc. Blocking of Fc gammaRI (CD64), Fc gammaRII (CD32), and Fc gammaRIII (CD16) individually or together also reduced neutrophil uptake of AdLuc, although less than blocking of CD35 alone. Combined CR1 and Fc receptor blockade synergistically inhibited neutrophil-AdLuc interactions close to baseline. These results demonstrate opsonin-dependent adenovirus vector interactions with neutrophils and their corresponding receptors.

Adenoviridae↗
PubMed1988

Neutrophils accentuate ischemia-reperfusion injury in isolated perfused rat kidneys.

The contribution of neutrophils to reperfusion injury after ischemia is not known. To determine the effect of neutrophils on the function of ischemic kidneys, we added purified human neutrophils during perfusion of isolated ischemic or nonischemic rat kidneys. Reperfusion of ischemic kidneys with neutrophils caused a distinct morphological lesion of vascular endothelial and smooth muscle cells and more functional injury than reperfusion with buffered albumin alone; with neutrophils, glomerular filtration rate (GFR) was 113 +/- 7 microliter.min-1.g-1, tubular sodium reabsorption (TNa) was 72 +/- 2%; without neutrophils, GFR was 222 +/- 18 microliter.min-1.g-1; TNa was 90 +/- 2%; both P less than 0.01 vs. reperfusion with neutrophils. In contrast, addition of neutrophils did not injure control kidneys, unless the neutrophil activator, phorbol myristate acetate, was also added. Two experiments suggested that O2 metabolites contributed to neutrophil-mediated injury to ischemic kidneys. First, reperfusion of ischemic kidneys with O2 metabolite-deficient neutrophils from a patient with chronic granulomatous disease did not cause more injury than reperfusion with buffered albumin alone. Second, simultaneous addition of the O2 metabolite scavenger, catalase, prevented the GFR and TNa decreases caused by neutrophils but did not decrease injury in the absence of neutrophils. We conclude that neutrophils by an O2 metabolite-dependent mechanism contribute to ischemia-reperfusion injury in the isolated perfused kidney.

Animals↗
PubMed1990

Activation and decreased deformability of neutrophils after intermittent claudication.

This study investigated local alterations in neutrophil activation and deformability after intermittent claudication. In 17 patients with one-sided peripheral arterial occlusive disease, neutrophil count, proportion of activated neutrophils (by nitro blue tetrazolium test), and neutrophil filterability as a measure of passive deformability were assessed in the femoral arterial and venous blood of the diseased leg and in the femoral venous blood of the healthy leg (n = 10). The values were obtained at rest, immediately after claudication, and 10 minutes after claudication induced by repetitive toe stands. Immediately after exercise, the arterial and venous blood differences in the diseased leg were 1) neutrophil count, 9% (95% confidence interval [CI], 5-14%; relative increase in the venous blood compared with arterial blood); 2) the proportion of activated neutrophils, 26% (CI, 10-42%); and 3) the neutrophil filterability, -10% (CI, -4% to -15%). At rest and 10 minutes after exercise, neutrophil parameters did not differ significantly between the femoral arterial and venous blood. Furthermore, no arterial and venous blood differences in the neutrophil parameters were found in the healthy leg. In addition to local changes, systemic changes occurred immediately after exercise. In the femoral arterial blood, the total neutrophil count had risen by 13% (CI, 8-18%), the proportion of activated neutrophils had risen by 41% (CI, 25-58%), and average neutrophil rigidity had risen 17% (CI, 11-22%) compared with the values obtained before exercise. At 10 minutes after exercise, all neutrophil parameters were still elevated. We conclude that even short periods of ischemia, as in intermittent claudication, cause local alterations in neutrophil function and distribution.

Arteries↗
PubMed1993

Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart.

BACKGROUND: Based on recent evidence showing that endothelin-1 stimulates several activation mechanisms on neutrophils, the aim of the present study was to analyze the effects of endothelin-1 on neutrophil adhesion to endothelial cells and neutrophil accumulation in the heart. METHODS AND RESULTS: The experiments included (1) adhesion of 51Cr-labeled human neutrophils to bovine endothelial cells in culture both in the presence and absence of monoclonal antibodies against the alpha- and beta-subunits of integrins; (2) surface expression of the alpha- and beta-integrin antigens; (3) accumulation of 51Cr-labeled neutrophils on the isolated perfused rabbit heart; (4) in vivo accumulation of autologous neutrophils in the heart, as assessed by myeloperoxidase activity. Endothelin-1 stimulated neutrophil adhesion to endothelial cells (increase of 1 x 10(5) +/- 1 x 10(4) neutrophils per well). The endothelin-1-induced adhesion was blocked (83 +/- 6%) by the anti-CD18 antibody TS1/18 and by several anti-alpha-subunit antibodies. The expression of CD18 and CD11b on the neutrophil surface was also increased by endothelin-1. Endothelin-1 enhanced neutrophil accumulation in the isolated rabbit heart by 4.2 times throughout a TS1/18-inhibitable mechanism. Myeloperoxidase activity increased by 4.2 times in hearts infused in vivo with endothelin-1. CONCLUSIONS: Endothelin-1 stimulates neutrophil adhesion to endothelial cells by an effect on the expression of adhesive molecules on the neutrophil surface. Endothelin-1 stimulates neutrophil accumulation in vivo and in vitro in the heart. Antibodies against the integrin complex block the endothelin-1-dependent neutrophil adhesion. These findings have potential importance in the pathophysiology of endothelin-1-increased states.

Animals↗
PubMed1995

Paradoxic effect of ibuprofen on neutrophil accumulation in pulmonary and cutaneous inflammation.

Evidence exists to suggest that the local accumulation of neutrophils in pulmonary inflammation occurs by a different mechanism than in other microvascular beds. In the present study, this suggestion was investigated using the nonsteroidal antiinflammatory drug ibuprofen. This drug was used because in addition to inhibiting cyclooxygenase, it prevents several aspects of neutrophil function, including adhesion. Local inflammation in the lung and skin of the same rabbits was induced by the administration of C5a, and the effect of intravenous injection of ibuprofen on the accumulation of neutrophils at these two sites was examined. In the skin, neutrophil accumulation was inhibited by ibuprofen, and this appeared to be independent of cyclooxygenase blockade. A possible mechanism was prevention of neutrophil adherence to endothelium in postcapillary venules, and in vitro experiments showed that ibuprofen could entirely prevent neutrophil adherence, in addition to suppressing azurophil granule secretion and superoxide anion generation. However, the effect on adherence appeared to be independent of expression of the CD11/CD18 adhesion complex on the neutrophil. By contrast, in the pulmonary circulation of the same rabbit, C5a-induced neutrophil accumulation was enhanced by ibuprofen treatment. This was suggested to result from the prevention of thromboxane production, which normally serves to decrease local pulmonary blood flow to diminish delivery of neutrophils to the inflammatory site. Ibuprofen had no effect on retention of neutrophils in filters (models of pulmonary capillaries), suggesting that the drug was not enhancing the retention of neutrophils in capillaries on the first pass through the lung. Thus, it was possible that enhancement in the lung was caused by an increase in neutrophil supply. Because the accumulation of neutrophils in the skin is known to be dependent on the adherence phenomena, the paradoxic effects of ibuprofen on inflammation suggest that mechanisms responsible for the accumulation of neutrophils in cutaneous and pulmonary microcirculations are different.

Animals↗
PubMed1984

The injurious effect of neutrophils on pneumocytes in vitro.

Neutrophils have been implicated in the pathogenesis of pulmonary injury in many clinical entities, but in vitro studies of neutrophil-mediated pneumocyte damage are limited. To study the role of neutrophils in mediating pulmonary injury, we cocultured these cells with monolayers of human A549 pneumocytes and rat type II alveolar cells. As indexes of injury, we measured cell detachment from monolayers, frank cytolysis, and the effect on pneumocyte protein and DNA synthesis. Unstimulated neutrophils effected minimal lysis or detachment of both pneumocyte targets, but neutrophils stimulated with phorbal myristate acetate and other secretogogues produced marked target cell detachment without lysis, which was time- and dose-dependent. Supernatants of activated neutrophils were similarly effective, suggesting that the mediator was a stable, soluble substance. Catalase and superoxide dismutase were minimally inhibitory to neutrophil-mediated detachment, and neutrophils from patients with chronic granulomatous disease produced detachment comparable to that produced by normal neutrophils. Furthermore, target cells were quite resistant to reagent H2O2 and non-neutrophil-derived toxic oxygen species, further suggesting that oxidative injury was not a major factor in causing detachment. Target cells were susceptible to detachment by the neutral proteases, elastase and collagenase, whereas neutrophil-mediated detachment was markedly inhibited by neutral protease and elastase inhibitors. Human and bovine serum were also inhibitory, but not albumin or pepstatin A, an acid protease inhibitor. Furthermore, we found that activated neutrophils inhibited protein and DNA synthesis of pneumocyte targets, providing additional evidence that neutrophils cause nonlytic injury to pneumocytes. These studies indicate that stimulated neutrophils cause nonlethal injury to pneumocytes, as measured by detachment from monolayers, and inhibition of vital intracellular synthetic functions. The mechanism of detachment is through the action of granule neutral proteases, rather than toxic oxygen metabolites, and is probably due to degradation of the extracellular matrix of the pneumocytes. In vivo, detachment could lead to desquamation of alveolar cells and increased permeability of the epithelial barrier of the lung. Similarly, inhibition of protein and DNA synthesis could have profound effects on the normal function and replication of alveolar epithelium.

Animals↗
PubMed1993

Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils.

Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.

Adult↗
PubMed2003

The in vitro production and characterization of neutrophils from embryonic stem cells.

An embryonic stem (ES) cell/OP9 coculture system for the effective production of functional neutrophils is described. A 3-step differentiation strategy was developed that uses liquid culture, enabling reliable and abundant production of neutrophils at high purity without the need of sorting for isolation of mature neutrophils. Use of the OP9 stromal cell line significantly enhances the number, percentage, and duration of differentiated neutrophils produced from embryonic stem cells. Effective and sustained differentiation of ES cells into neutrophils provides a useful model system for studying neutrophil differentiation and function and the factors that regulate them. Morphologic and functional evaluation of these ES-derived neutrophils indicates that large numbers of mature neutrophils can be produced from pluripotent ES cells in vitro. Specifically, their morphology, ability to produce superoxides, flux calcium, undergo chemotaxis in response to macrophage inflammatory protein 2 (MIP-2), stain for the granulocyte-specific marker-specific chloroacetate esterase, and contain the neutrophil-specific markers Gr-1 and the mouse neutrophil-specific antigen indicates that they are comparable with purified mouse bone marrow neutrophils. They also express gelatinase and lactoferrin granule proteins. During the differentiation of these ES-derived neutrophils, regional areas of neutrophil production can be identified that have been designated as neutrophil generating regions (NGRs).

Animals↗
PubMed2006

Neutrophil cannibalism--a back up when the macrophage clearance system is insufficient.

BACKGROUND: During a lipopolysaccharide-induced lung inflammation, a massive accumulation of neutrophils occurs, which is normally cleared by macrophage phagocytosis following neutrophil apoptosis. However, in cases of extensive apoptosis the normal clearance system may fail, resulting in extensive neutrophil secondary necrosis. The aim of this study was to explore the hypothesis that neutrophils, in areas of the lung with extensive cellular infiltration, contribute to clearance by phagocytosing apoptotic cells and/or cell debris derived from secondary necrosis. METHODS: Intranasal lipopolysaccharide administration was used to induce lung inflammation in mice. The animals were sacrificed at seven time points following administration, bronchoalveolar lavage was performed and tissue samples obtained. Electron microscopy and histochemistry was used to assess neutrophil phagocytosis. RESULTS: Electron microscopic studies revealed that phagocytosing neutrophils was common, at 24 h after LPS administration almost 50% of the total number of neutrophils contained phagosomes, and the engulfed material was mainly derived from other neutrophils. Histochemistry on bronchoalvolar lavage cells further showed phagocytosing neutrophils to be frequently occurring. CONCLUSION: Neutrophils are previously known to phagocytose invading pathogens and harmful particles. However, this study demonstrates that neutrophils are also able to engulf apoptotic neutrophils or cell debris resulting from secondary necrosis of neutrophils. Neutrophils may thereby contribute to clearance and resolution of inflammation, thus acting as a back up system in situations when the macrophage clearance system is insufficient and/or overwhelmed.

Animals↗
PubMed2000

Neutrophils die in the gingival crevice, periodontal pocket, and oral cavity by necrosis and not apoptosis.

BACKGROUND: Neutrophils play important roles in the homeostasis of periodontal tissues. However, remarkably little is known about how these cells live and die in the gingival crevice and periodontal pocket. We have examined whether subgingival neutrophils die by necrosis or apoptosis and have begun to study the mechanisms controlling the functional lifespan of these cells. METHODS: Neutrophils collected from clinically healthy gingival crevices, periodontal pockets, and the oral cavity (saliva) were examined with Hoechst dye 33342, propidium iodide/eithidium bromide, and FITC-annexin V to identify live, dead, and apoptotic cells. Blood neutrophils were cultured for 17 to 20 hours in the presence or absence of gingival crevice washings (GC-w) to study the effect of GC-w on neutrophil apoptosis. In addition, endotoxin was removed from GC-w by affinity resin to investigate the contribution of LPS to the inhibitory effect of GC-w on blood neutrophils. RESULTS: The percentage of dead neutrophils in all subgingival sites and in all oral samples far exceeded the percentage of apoptotic neutrophils. In all 3 locations, approximately 30% of neutrophils were dead, whereas less than 1% of neutrophils were apoptotic. We conclude that the majority of neutrophils which lose their viability within gingival crevices, periodontal pockets and the oral cavity die by necrosis and not by apoptosis. Washings obtained from clinically healthy gingival crevices (GC-w) variably, but significantly, delayed apoptosis of peripheral blood neutrophils (mean suppression 45.7% +/- SD 22.3). Removal of endotoxin from GC-w significantly reduced this inhibitory effect. CONCLUSIONS: Our findings provide insights into the mechanisms of neutrophil death and the control of the functional lifespan of neutrophils in gingival crevices and periodontal pockets and therefore into the pathogenesis of periodontal diseases.

Adult↗
PubMed2006

Modulation of phagocytosis, chemotaxis, and adhesion of neutrophils by areca nut extracts.

BACKGROUND: A higher prevalence of periodontal diseases among areca chewers than non-areca chewers has been demonstrated. Neutrophils, representing the first line of the host defense mechanism against microbial infection, play important roles in maintaining periodontal health. This study determined the possible effects of areca nut on phagocytosis, chemotaxis, and adhesion of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE) and fresh and tender areca nut with husk (tANE) were examined for their effects on neutrophil phagocytosis using flow cytometry and confocal laser scanning microscopy. The effects of rANE and tANE on chemotaxis and adhesion of neutrophils to human aortic endothelial cells were examined using fluorescence-labeled neutrophils. RESULTS: Both rANE and tANE inhibited the phagocytic activity of neutrophils in a dose-dependent manner. The levels of internalized fluorescent bacteria in neutrophils decreased after ANE treatment. However, exposure of neutrophils to rANE and tANE stimulated the chemotaxis activity of neutrophils to N-formyl-Met-Leu-Phe (fMLP) and enhanced adhesion of neutrophils to human aortic endothelial cells in a dose-dependent manner. Moreover, treatment of neutrophils with rANE was more effective than incubation with tANE. CONCLUSIONS: Components of areca nut inhibited phagocytosis activity of neutrophils but enhanced chemotaxis and adhesion of neutrophils. Alterations in functions of neutrophils may lead to signs of clinical diseases associated with areca chewing. The components in ANEs that are responsible for these observations remain to be elucidated.

Areca↗
PubMed2000

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils.

OBJECTIVE: To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. SAMPLE POPULATION: Blood collected from 5 healthy adult Holstein cows. PROCEDURE: Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. RESULTS: Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.

Alkaline Phosphatase↗
PubMed2001

[Effects of estradiol and isoflavoid on the expression of adhesion molecules on neutrophils].

OBJECTIVE: To elucidate the effects of estradiol and isoflavoid on the expression of adhesion molecules on neutrophils. METHODS: Neutrophils of healthy subjects and ischemic stroke patients were treated with tumor necrosis factor alpha (TNF alpha), in the presence or absence of different concentrations of isoflavoid (WZ1, WZ2) and estradiol (WZ3, WZ4). Flow cytometry was used to detect the expression of CD18 and CD62L on neutrophil surface. RESULTS: 1. 10 ng/ml TNF alpha could activate neutrophils of healthy subjects; it increased the expression of CD18 by 10% on neutrophil surface and shed CD62L from the surface as shown by a 15% decrease of the fluorescence intensity and a 30% decrease of the percentage of positive cell. 2. Isoflavoid (WZ1, WZ2) had no significant effect on the expression of CD18 and CD62L on neutrophils. 3. Pretreatment of neutrophils with estradiol (WZ3, WZ4) could inhibit the activation of neutrophils by TNF alpha, which decreased the fluorescence intensity of CD18 by 8%, increased the fluorescence intensity of CD62L by 15% and increased the percentage of CD62L positive cell by 20%. 4. TNF alpha could activate the neutrophils of ischemic stroke patients strikingly; it increased the fluorescence intensity of CD18 by 20% and decreased the fluorescence intensity and percentage of positive cell of CD62L by 30%, and there was a significant difference when the patients were compared with the healthy subjects. Estradiol had the same effect on the expression of CD18 and CD62L as on those of healthy subjects. CONCLUSION: 1. TNF alpha is a strong activator of neutrophils; it plays an important role in the occurrence and development of ischemic stroke. 2. Isoflavoid has no obvious effect on the expression of adhesion molecules on neutrophils, so its role in protecting the cardiovascular system may come into play not by the way of affecting adhesion molecules expression. 3. Pretreatment of neutrophils with estradiol could protect them from activation by TNF alpha, thus decreasing the expression of adhesion molecules, the adhesion of neutrophils -endothelial cells, and hence the risk of ischemic stroke. However, in case that the neutrophils have been activated by TNF alpha, estradiol has no effect on the expression of adhesion molecules; this implies that estradiol possibly has no valid anti-adhesion therapeutic effect on ischemic stroke.

Adult↗
PubMed1992

Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells. Role of intracellular calcium changes and fusion of specific granules with the plasma membrane.

Chemoattractants, used at concentrations to invoke optimal neutrophil chemotaxis, induce rapid changes in neutrophils such as a transient increase in intracellular Ca2+ ([Ca2+]i). We have previously observed that neutrophils adhering to cytokine-activated endothelial cells (EC) also respond with a rapid rise in [Ca2+]i caused by an endothelial membrane-bound form of platelet-activating factor. After preloading with the intracellular Ca(2+)-chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), neutrophils were no longer able to respond with a rapid rise in [Ca2+]i toward the chemoattractant FMLP or to rIL-1 beta-pretreated EC. These neutrophils were still able to adhere and migrate under the conditions tested. The only difference was that the BAPTA/AM-treated neutrophils migrated a little slower than untreated control neutrophils. This discrepancy was not observed at later time points. The BAPTA/AM-preloaded neutrophils did not differ from unloaded neutrophils in actin polymerization responses. Whereas untreated neutrophils demonstrated an up-regulation of the specific granule markers CD11b, CD45, and CD67 during migration (without any release from the azurophil granules), the BAPTA/AM pretreatment completely prevented this process. The BAPTA/AM-preloaded neutrophils did not release vitamin B12-binding protein from the specific granules upon treatment with FMLP. The down-modulation of the selectin member LAM-1, as seen upon neutrophil activation, was not affected by BAPTA/AM pretreatment of the neutrophils. Thus, neither the rapid rise in [Ca2+]i nor specific granule fusion with the plasma membrane constitute a prerequisite for neutrophil migration across resting or cytokine-activated EC.

Antigens, CD↗
PubMed1992

Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties.

Intravascular LPS rapidly induces neutrophil sequestration in pulmonary capillaries by mechanisms that, although currently unknown, must take into account the size difference between the neutrophil and capillary diameter. To determine whether LPS alters neutrophil stiffness, and hence the ability of neutrophils to traverse capillaries, neutrophil passage through pulmonary capillaries was modeled by passage through filters with 6.5-microns pores. LPS increased retention in the pores in a concentration-dependent fashion that required the presence of heat-inactivated platelet-poor plasma, and was evident as early as 10 min after stimulation. The effect of LPS on the structural properties of the neutrophil was then studied. LPS induced f-actin reorganization in neutrophils in the presence of plasma. Disruption of actin organization and assembly with cytochalasin D completely inhibited early LPS-induced retention and attenuated retention at later timepoints, indicating that LPS-stimulated retention depends on filament organization. LPS-induced actin assembly and retention were abrogated by an antibody directed against CD14, a putative LPS receptor. CD18-dependent adherence of neutrophils contributed significantly to retention only at later timepoints with no significant contribution to retention at 20 min as determined by inhibition of adherence with the mAb 60.3. Morphometric assessment of neutrophil accumulation in the lungs of rabbits given 1 microgram LPS showed a marked increase in apparent neutrophil number, which was unaltered by antibodies to CD18, suggesting that mechanisms other than adhesion may account for accumulation in vivo. Direct measurements showed that neutrophil stiffness increased with exposure to LPS in a fashion similar to LPS-induced retention and actin organization. Pretreatment of neutrophils with cytochalasin D attenuated the increased stiffness. These data suggest that reorganization of filamentous-actin induced by LPS leads to cell stiffening and retention in capillary-sized pores. Although the organization of f-actin continues to be important in retention at later time points, adherence of cells also contributes significantly to cell retention. The changes in mechanical properties of the neutrophil may be important in the sequestration of neutrophils in pulmonary capillaries noted in endotoxemia.

Actin Cytoskeleton↗